Engineering Plants as Platforms for Production of Vaccines

Microbial pathogens have 
always posed serious threats and challenges to human existence. Pathogenic 
microbes causing epidemic and pandemic outbreaks have the potential of effacing 
life on earth. Vaccines are used as prophylactic as well as treatment measures 
against diseases and are effective in eradicating deadly pathogens. 
Conventional vaccines though effective, have high production costs, involve 
tedious purification processes and have biosafety issues, requiring 
time-consuming biosafety tests for commercial production. Plant-based vaccines 
offer several advantages over the conventional systems such as ease of 
production, storage, higher yields, stability and safety. The review discusses 
significance, advantages, comparisons, prospects and challenges or constraints 
in the production of plant-based vaccines and antibodies.


Introduction
"Let food be thy medicine"-Hippocrates Man has evolved in his continuous journey of "struggle for survival" on the earth. Microbial diseases not only had adverse effects on human health and life due to the fatality rate, but also had dwindled the economies of nations world-wide. Pandemics caused by known as well as newly evolved pathogens, which are more frequent during recent times, have sent alarm signals to mankind, to prepare against more deadly pandemics in future and promote development of vaccine platforms for handling the worst outbreaks. The ongoing global catastrophe of massive scale, the COVID-19 is wreaking havoc, killing causes a disease. They provide strong long-term immune response as they are similar to natural infection. Attenuated vaccine may have small amount of the live virus which may be risky for people with weak immune system. Also, they require cold storage facilities. Live attenuated vaccines are used against smallpox, chickenpox, Measles, Mumps and Rubella (MMR vaccine) etc. [4]. Inactivated vaccines use killed or inactive pathogen and require booster doses of vaccine for immunity [5]. Flu, pertussis, polio, rabies vaccines etc. are inactivated vaccines.
Subunit, recombinant, polysaccharide and conjugate vaccines use part of the pathogen such as protein, capsid (viral coat protein), sugar moiety etc. Subunit vaccines comprise of purified antigen(s) derived from the pathogen, while conjugate vaccines, consist of a polysaccharide component of the pathogen that is poorly immunogenic, so that it is chemically linked to a protein. Recombinant subunit vaccines are safer since they do not have a pathogen and can also be scaled up. Since subunit vaccines consist of small fractions of the pathogen, immunogenicity is greatly decreased with respect to those derived from whole cells, generating the need for co-administering adjuvants to attain immune-protection [6]. They can be used in patients with weak immune systems and require booster doses to maintain immunity. These vaccines are used against Hepatitis B, Whooping cough, Human Papilloma Virus (HPV) etc.
Toxoid vaccines use toxins from the pathogen that causes a disease. Diphtheria and tetanus vaccines are toxoid vaccines [7]. Viral vector-based vaccines such as Adenovirus (Ad) or measles virus vectors are highly versatile platforms for vaccine development. Viral vector-based vaccines can be used for different viruses, delivered without additional adjuvants and can be administered as intramuscular, intranasal, intradermal and oral vaccination. High yield production processes and means of upscaling have been established for these vaccines so that they can be used immediately in case of a pandemic outbreak. But viral vectors are genetically modified organisms (GMOs) considered as potential risks to human health and environment and unsafe due to persistent replication of attenuated vaccines. Viral vectors can integrate into the host genome, or undergo recombination during production, leading to emergence of uncharacterised or novel pathogens. These safety concerns might also delay clinical studies in case of a pandemic. Viral vector-based vaccines are highly complex and comparatively cost-intensive [3] [8].
Nucleic acid-based vaccines employ antigen-encoding plasmid DNA or RNA or messenger RNA or viral replicons. Due to the ease of antigen manipulation they are also versatile. Vaccine can be developed against various pathogens such as virus, bacteria or parasite and administered as intramuscular or intradermal injections. A eukaryotic expression cassette carrying the antigen is inserted into a bacterial plasmid for propagation in E. coli. Minimal DNA constructs devoid of a bacterial backbone, such as the semi-synthetic minicircle DNA and the fully synthetic Doggybone TM , have been developed to avoid safety issues related with selectable marker [9]. DNA vector vaccine provides relatively low immuno-genicity, since DNA vaccines must cross both plasma and nuclear membranes for protein expression, unlike the RNA vaccines which upon crossing plasma membrane are translated. Encapsulation of DNA vaccines in lipid nanoparticles, adsorption to polymers and use of molecular adjuvants like cytokines can enhance the uptake of DNA vaccines and enhance the immune response. DNA vaccines have long-term persistence, however, potential risk of genomic integration of exogenous DNA into the host genome or chromosomes may result in mutagenesis and oncogenesis or new diseases. Molecular adjuvants like cytokines may also have undesirable, side-effects such as inflammation or autoimmunity. DNA vector-based antigen expression is the first effective vaccine against Ebola virus, Zika virus etc. and used against human pathogens such as HIV, influenza virus, malaria, hepatitis B virus, respiratory syncytial and herpes simplex virus [3] [10].
RNA vaccines use either non-replicating mRNA and/or self-amplifying mRNA as vaccine. Non-replicating mRNA contains antigen sequence flanked by 5' and 3' untranslated regions (UTRs). The mRNA with a protein-encoding open reading frame (ORF) flanked by a 5' cap structure, poly(A) tail at the 3' end, as well as 5' and a 3' untranslated regions are obtained by in vitro transcription of a cDNA template, typically plasmid DNA (pDNA) produced in E. coli, which is linearized using restriction enzyme and transcribed using recombinant phage DNA-dependent RNA polymerase. Self-amplifying mRNA vaccines are based on the alphavirus genome, where the genes for structural proteins are deleted and replaced with the antigen of the pathogen. Large size of these vaccines, lower yields and increased occurrence of abortive constructs are challenges to vaccine production. Extracellular ribonucleases can catalytically hydrolyze unprotected "naked" mRNA, which is also highly unstable under physiological conditions. Hydrophilicity and strong net negative charge of RNA prevents its uptake by cells after application in vivo. This can be overcome by complexing of mRNA with highly efficient carriers to form protamine-complexed mRNA or with complexing agents such as lipid-and polymer-based nanoparticles. mRNA can be administered as intradermal, intra venous or intra-muscular injections. RNA vaccines are used against influenza, Zika and Ebola virus infections [10] [11].

Vaccine Production Systems
In conventional method of vaccine development, a pathogen is inactivated or attenuated, concentrated and purified to develop a vaccine. The vaccine production systems can be broadly categorised into three viz., the egg-based vaccines, cell-based vaccines, and vaccines produced using investigational-manufacturing systems [12]. Each vaccine technology has its own advantages and disadvantages related to its ability to induce certain immune responses, manufacturing capacity and safety for human use. Embryonated eggs are used for vaccine production in egg-based vaccines, which is a commonly used system for production of Influenza vaccine. The virus particles are injected into eggs, incubated for virus replication and the viral antigens or vaccines are isolated and purified from eggs. But this method cannot be used for all strains of the virus, large number of eggs are required and involves time-consuming regulatory processes. Cell-based production systems such as mammalian cell culture systems could be used for production of viral antigens of subunit vaccine because they can produce high titres (1 -5 g/L) of complex proteins with mammalian glycan structures, but require costly infrastructure for production and monitoring for safety, since mammalian cultures are prone to contamination with mammalian pathogens and oncogenic agents and are poor in scalability [13]. Recombinant subunit vaccines produced in genetically modified cells have better safety, less antigenic competition, specificity and the ability to differentiate between infected and vaccinated animals. The gene encoding a protective antigen is expressed in a heterologous system and the resulting protein is purified and administered as a vaccine [14]. Investigational manufacturing systems such as bacteria, yeast or insect cells and plants are used for production of recombinant vaccines. Escherichia coli was the bacterial system which was used earlier for production of recombinant subunit vaccines [15]. But protein folding and post-translational modifications do not occur correctly in bacterial system. Hence eukaryotic cell systems like yeast which were simple, use inexpensive culture media for growth and carry out folding and N glycosylation of proteins, were used. Saccharomyces cerevisiae was used for production of hepatitis B virus surface antigen particles or vaccine [16]. However, the glycan structures in yeast system differ from that of mammals. Insect cell cultures though less expensive also have low scalability.
The conventional vaccine production approaches such as egg-based and cell-based production systems were followed for eradication of smallpox and for controlling polio, tetanus, measles etc. But the conventional method of whole pathogen cultivation for vaccine production may not be feasible during a disease outbreak because of low producibility, requirement of in vitro conditions, high biosafety level and specialised labs for cultivation. Also, there is a risk of reversion of the attenuated inactive form of the pathogen to a highly pathogenic form, no protective responses as in Ebola or undesirable side effects as in case of formalin-inactivated respiratory syncytial virus. Currently, the average development time for conventional vaccines from preclinical phase is more than 10 years [3]. In case of an outbreak, time is a major constraint, requiring development of vaccine at a fast pace, in large quantities and with nominal side-effects. Other challenges during an outbreak are unpredictablility in pathogenicity, mutation rate and adaptability of the novel pathogen. Already licensed vaccines would take 3 -5 months between identification of a pandemic influenza and vaccine distribution, which would cause wide global spread of the pandemic virus.
The genes encoding the antigen protein of the pathogen causing a specific disease are integrated into the plant genome through artificial methods, where the plant produces the antigen protein which confers immunity, when purified from plant and administered as vaccine, or directly consumed as an edible vaccine. The plants act as bioreactors for these pharmaceuticals or therapeutically important proteins, that can be used for humans as well as animals. Plant-based vaccines are, biologically active and produced inexpensively as well as in substantial amounts to elicit an immune response [18]. Plants thus offer a less-expensive production system and an effective and efficient delivery system. Plant vaccines are effective, feasible alternatives for resource-poor or low-income countries which do not have powerful healthcare infrastructure to produce their own vaccines nor have benefited from the current vaccination programs due to the expensive vaccine development technologies [2].

Strategies of Production of Plant Vaccines
Production of plant vaccines involves two components 1) Research and development and 2) Commercial production. Generally, vaccine development has 6-phases according to the Center for Disease Control and Prevention (CDC), USA. These are exploratory, preclinical, clinical development, regulatory review and approval and finally manufacturing and quality control [19].
In exploratory phase, research and development on synthetic or natural antigens or weakened strains of the pathogenic virus are carried out to treat or prevent a disease. In the pre-clinical phase, tissue culture or cell culture systems and animal testing are undertaken, to verify the effectiveness of the candidate vaccine to provide immunity. In the third phase of clinical development, a proposal or application describing the research findings and for conducting clinical trials, is submitted by the vaccine manufacturing firm to the sanctioning authority. Once proposal to conduct clinical trials are approved, human testing or trials are conducted in 3 stages. In Phase I, the candidate vaccine is administered to a small group of people (<100) to know the safety. Phase II involves larger group of subjects in hundreds to know about safety immunogenicity, immunization schedule, dosage etc. Still larger subject group of thousands are covered in Phase III trials where side-effects, safety and effectiveness of the candidate vaccine is assessed. This is followed by regulatory review and approval where application for licence for manufacturing by the firm is scrutinised for approval. Next step is manufacturing the vaccines and then quality control to monitor the perform- These are discussed below.

1) Selection and design of antigen/ vaccine
The first step in vaccine production involves selection of the protective antigen from the pathogen and designing immunogen using bioinformatics, genomics and proteomics tools. Immuno-protective epitopes can be identified by assays such as phage display technology and requires fully annotated genome sequence of the pathogen, a heterologous protein expression system and a model that mimics human immunological mechanisms. A design based on highly immunogenic carriers for the elicitation of effective immune responses to unrelated antigens is important. After design of immunogen, the transgene encoding the antigen must be designed and synthesised using recombinant DNA technology.
Flanking restriction sites have to be included to facilitate the molecular cloning construct expression vectors, codon bias should be matched with that of the expression host, and undesired introns or unstable RNA motifs should be removed to optimise gene expression in the specific host [20].
2) Selection and design of vector A specific expression vector for plant-based expression of antigen has to be selected or designed. Expression cassettes can be driven by constitutive promoters or, alternatively, by inducible or specific promoters such as seed-specific promoters, for selective expression of the antigen protein in a particular tissue or organ, in order to maximize accumulation or for higher yield, long-term storage at ambient temperature and to avoid harmful deleterious effects on the plant host [21]. Also, transcription machinery such as T7 RNA polymerase expressed from the nuclear plant genome to enhance the expression of a transgene, 5' UTR for translational efficiency, 3' UTR for transcript polyadenylation and mRNA stability, expression cassette-flanking regions, which mediate homologous recombination events for site-specific integration of the expression cassette etc. can be engineered [22]. Many plant expression vectors are commercially available.
3) Choice of plants The plants to be chosen depend on the amenability to transformation and regeneration in vitro. Availability of or development of a stable transformation as well as regeneration protocol is pre-requisite for choosing a species or plant for production of plant vaccines. The expression strategy, life cycle, biomass yield, containment, and scale-up cost are the factors to be considered while choosing a plant system [23]. They should also have fast growth and high biomass production. Nicotiana benthamiana and N. tabacum are widely used for molecular farming for these reasons. Non-food crops or model systems such as tobacco, duck weed, Arabidopsis etc do not accumulate high amount of protein and increase extraction costs of vaccines due to high proportion of phenolic compounds. Earlier, tobacco and potato were the systems of choice for production of many plant-based recombinant proteins, due to the ease of genetic modifications. Food crops have higher proportion of stored protein, are safe for human consumption, can be directly ingested as oral vaccines, but raises concerns on potential of contamination of food and feed crops. However, for edible vaccines, fruit crops are preferred since they can be consumed directly without cooking, else heat during cooking may destroy the protein antigen. Many plant species including maize, carrot, tomato, soybean, lettuce, potato, and alfalfa are used now, since they offer better yields with no toxic compounds, making possible oral immunization using raw plant materials. Papaya and banana are good candidates for rapidly producing cheap vaccines in developing nations since they have high quantity of vitamin "A" and have sterile condition as in banana. Also, the genes are not transferred from one plant to another [2] [24]. culture conditions such as light, temperature etc. that direct the regeneration processes or morphogenetic response of the tissue is to be optimized for the selected plant. Somatic embryogenesis and organogenesis are the two pathways for plant regeneration. In direct somatic embryogenesis, the embryo is formed directly from a cell or group of cells without the production of an intervening callus, while in the indirect somatic embryogenesis, callus is first produced from the explants. In organogenesis, organs are produced from callus or explant. Regeneration steps are avoided in transient expression systems, where whole plants are used and the transgene or DNA is not stably integrated in the genome nor inherited, but expressed temporarily in the host. 6) Evaluation and characterisation of plant vaccine or immunogen Enzyme-linked immunosorbent assay (ELISA) and western blot assays are used to quantify the foreign protein or antigen in the plant. Immunogenicity of the plant vaccine is assessed in the preclinical level, when test animals are subjected to a defined immunization scheme and antibody levels and proliferation of specific immune cells are often evaluated by ELISA and splenocyte proliferation assays. Immuno-protective potential of the vaccine is evaluated by scoring of deaths in vaccinated and unvaccinated test animal groups or by measuring humoral or cellular immune responses. A small number of vaccines or candidates have been tested for immunogenicity in humans. Clinical trials utilizing transgenic plants for vaccines mostly consist of either the leaves or fruits from the plants [6].
Generation Plant-based systems still face one major bottleneck that needs to be overcome-their lower yields compared to mammalian cell cultures [13].

Plant Virus-Based Expression Systems
The

Edible Vaccines
Edible vaccines include all vaccines produced in an edible format (i.e., part of a plant, its fruit, or sub products derived from that plant which, upon oral ingestion, stimulates the immune system. Edible does not necessarily mean nutritious, tasty, or organoleptically pleasing since edible vaccines need only be safe (non-toxic) for human consumption [32]. The term edible vaccine was coined in 1990 by Charles Arntzen [33].

Plantibodies
Antibodies constitute the humoral adaptive immune system which specifically recognize and bind to target antigens or toxins of pathogens. An antibody has a binding region or paratope which binds to the epitope or antigenic determinant on the antigen and varies depending on the conformation of the epitope. The constant region of antibody (located on the heavy chain) determines the class and subclass of antibody. For binding of antigen and antibody, the shape of the paratope must fit the epitope, so that several non-covalent bonds can form simultaneously. Antibody-coding genes from mammals/humans can be engineered into plants to make antibodies and antibody fragments called the plant-derived antibodies or plantibodies [36]. In 1990, plants were first considered as a potential host for producing antibodies and the word "plantibody" was coined.
Plants are capable of synthesizing and assembling virtually any kind of anti-  [37]. The first plantibody produced in tobacco, CaroRx®, is a clinically advanced anti-Streptococcus mutans secretory immunoglobulin that binds to the bacterium, thus protecting humans from dental caries [38]. Later a humanised antibody against herpes simplex virus glycoprotein B was expressed in soybean [39]. Plantibodies have also been produced against anthrax, Ebola and various forms of cancer in humans.
The drug, called ZMapp, contains a cocktail of three humanized anti-Ebola virus mAbs and was developed by Mapp Biopharmaceutical Incorporated, San Diego.
360 million doses of plantibodies against anthrax can be produced from a single acre of tobacco while 1.5 kg of antibodies is provided per acre of corn, compared to vaccines. However, the introduced plantibodies are flushed through a person's system relatively quickly, in a matter of hours or days, which necessitates the patient to take doses indefinitely.

Human Interferons
Higher levels of accumulation of human interferons were obtained by targeting the hIFN-γ protein to endoplasmic reticulum (ER) or apoplastic space than in cytoplasm of tobacco. The protein was biologically active and protected from infection generated by vesicular stomatitis virus (VSV) [40].

Other Pharmaceuticals
Plants are natural reservoirs of compounds or metabolites which have antimicrobial (antiviral, antibacterial, antifungal, anti-parasitic) properties. However, the production of such compounds may be low in their natural source. The plants that synthesize these compounds do so in low concentrations and grow slowly resulting in only minute quantities of the desired compound [41]. Engineering the biosynthetic pathways for these compounds into heterologous plants optimized for molecular farming could boost supplies and reduce costs [42].
Anti-cancer drug Taxol (paclitaxel) and artemisinin, a crucial anti-malarial compound are few examples of such pharmaceutical compounds [43].

Status of Production of Plant Vaccines
The attempt to produce vaccines in plants was made by Hiatt and co-workers in   [14]. Bovine trypsin derived from maize has been commercialized since 2002 [49]. Neutralizing antibody responses were elicited against homologous and heterologous Newcastle Disease virus by inoculating plant-produced fusion protein (F) antigen (transmembrane glycoprotein), into Specific Pathogen Free (SPF) chickens [50]. Newcastle disease vaccine derived from tobacco cells was first approved for poultry use by United States Department of Agriculture [49].
In addition to expression of antigen for vaccine production (Table 1), pharmaceuticals such as antibodies, enzymes, therapeutically important proteins or peptides and growth hormones are produced in plants [49] [76].

Low Cost of Production
The vaccines used for immunisation against contagious disease are mostly costly and not easily accessible. On the contrary, the plant bioreactors are cost-effective and cheaper. Plants are most economical and feasible production systems for vaccines or recombinant products. Replacement of fermenters and bioreactors with contained plant growth rooms or greenhouses with appropriate biological containment reduces manufacturing cost [6]. Production costs of a recombinant protein in transgenic plants are 10 -50-fold lower than that by E. coli fermentation [77]. Plant vaccines can also be delivered orally, overcoming the cost and inconvenience of purification and injections [78].

High Yield
Use of plants as source of therapeutic proteins has a major advantage that production in large quantities is possible. Feasibility for scaling up and high expression level of recombinant genes/proteins are also high in plant systems. Also, large scale cultivation is possible, and this can be adopted in less developed or resource-poor nations which lack sophisticated facilities or infrastructure for production of life-saving drugs.

Easy to Prepare and Ease of Administration
Designing a recombinant vector, introduction and integration into plant system for production of antibodies, or other proteins of therapeutic value is relatively easy. The edible vaccines are easy to handle as well. When a new microbe or its antigen is evolved posing a threat to human health, it is easy to modify the synthesis of plant-based vaccines than animal-based ones. Edible vaccines are easy to deliver through oral administration and can be directly consumed without need for any injection. Edible vaccine is a needle-less vaccination method or a substitute of painful immunization procedures that require sophistication or trained manpower. It is also inexpensive, attractive to children, can be stored nearby the place of usage, harmless, and offers systemic and mucosal immunity. American Journal of Plant Sciences Edible vaccines are safe oral-delivery vehicles wherein specific plant tissues such as grains, fruits, or leaves can be used as formulation of vaccines, without extensive purification and processing.

Post-Translational Modifications
Post-translational modifications such as glycosylation, folding and assembly are significant for a protein to be biologically active and function as a vaccine. Plants have machinery for expression, folding, assembly, and glycosylation, necessary for preservation of immunogenic activity of vaccines. In plants, the foreign or recombinant proteins of therapeutic value are glycosylated, accurately folded and the multimeric proteins assembled properly, to have structural integrity and biological activity for functioning as a vaccine [18]. Protein synthesis as well as post-translational modifications of proteins in plants is similar to that of animal cells, making it possible to use plants as bioreactors for animal proteins/pharmaceuticals.

Safety
The plantibodies or plant vaccines produced using plant-based systems are mostly safe and devoid of any toxic components [79].

Stability, Storage and Transport
The plant products can be stored safely for long duration at room temperature, unlike the need for refrigeration in case of other animal-based vaccines. Edible or plant-based vaccines can also be easily produced by a freeze-dried process, leading to formulations with high stability under a cold chain-free distribution [80]. Proteins produced in plants such as seeds remain stable for years at ambient temperatures, without loss of activity.

Challenges or Constraints of Plant-Based Vaccines
Plant expression system has several advantages for human as well as veterinary vaccine production, however, only few of vaccine candidates are under clinical trials. Commercial human vaccines are not available due to low level of expression, relatively weak efficacy, and comparatively shallow knowledge on the characteristics of plant-made antigen and production system [49]. Some of the challenges or constraints in the plant-based vaccines are discussed.

Poor Immunogenicity
Immunogenic response depends on nature of the vaccine, route of administra-American Journal of Plant Sciences tion and the delivery system. Many antigens are poor immunogens, recognized poorly by the immune system and are prone to degradation in the harsh environment of the digestive tract. Plant cells protect vaccine antigen and prevent degradation as it passes through the gut. Immunogen such as Cholera toxin B subunit (CTB), which can modify the cellular environment to present the antigen, can act as an efficient transmucosal carrier molecule and delivery system for plant-derived subunit vaccines and can overcome this problem.

Variability in Dosage
It is difficult to measure the effective dose for a mucosally delivered vaccine as it is exposed to the complex environment of the gastrointestinal tract. Further, oral vaccines may require co-administration with specific adjuvants to reach sufficient immunogenic activity [81]. An insufficient amount of antigen would not produce the immune response needed for protection against disease and inappropriate dosage could lead to tolerance to vaccine and ineffectiveness of vaccine.

Alteration in Glycosylation and Allergenicity
Many therapeutic proteins or N glycoproteins synthesised in plants differ in their glycosylation patterns from those derived from the mammalian systems.
This may also induce increased allergenicity or reduced immunogenicity. The glycosylation pathways in plants can be altered for humanising the plant-derived vaccines or therapeutic proteins.

Degradation
Antigens delivered to the intestinal immune system is rapidly degraded within the harsh environment of the digestive tract, though plant cells provide protection and prevent degradation of the vaccine antigen, as it passes through the gut.

Spoilage
Edible vaccines such as fruits are perishable and cannot be stored for long time.
They quickly spoil after ripening and the protein content is also very less.

Generation Time for Transgenics
Stable plant transformation to generate transgenics that express vaccine proteins takes much time, from months to years, depending on the plant species. Long time required for development or transformation, analysis of transgenics, selection and bulking up of producer line are some constraints.

Risks to the Environment and Human Health
Environmental issues of plant vaccines include gene transfer and exposure to antigens or selectable marker proteins, while 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 [45]. American Journal of Plant Sciences

Transgene Escape and Containment
The potential and prospects of plant made pharmaceuticals is restricted by the potential of transgene spread from crops through outcross, challenges in transgene biocontainment, unpredictable impact of epigenetic events on transgene expression etc [14] [17]. Escape of foreign genes to weedy relatives through outcrossing is a concern. Plant cell culture bioreactors or greenhouses and use of plant virus expression systems to produce vaccine proteins in large quantities can be thought of as safer alternatives. The infamous escape of transgene in case of Prodigene and Starlink corn are examples. ProdiGene produced a transgenic corn that expressed a vaccine for preventing bacteria-induced diarrhoea in pigs, but in 2002, ProdiGene failed to eradicate plants that had seeded from their previous season's transgenic corn crop which contaminated non-transgenic soybeans. In 2003, the Animal and Plant Health Inspection Service (APHIS) of the US Department of Agriculture (USDA) made it mandatory for engineered plants producing pharmaceuticals to be grown under permit. Inefficient transgene biocontainment is a serious hurdle to commercialisation of molecular pharming using plants [82].

Current Status of Regulations on Plant Vaccines
Regulatory hurdles remain a barrier to molecular farming, further increasing the cost and time, which otherwise are major advantages of plant-made vaccines. Purification, quality controls for vaccine approval are major cost factors in (human) vaccine production [25]. Containment of the recombinant material is a concern which needs to be carefully monitored, to prevent these from entering the food chain and environment. The recombinant plant-based vaccines produced in transgenic plants must undergo a tight regulatory process before commercialisation. The paradigm of plant-made vaccines (PMVs) has evolved from vaccines consumed by world's poorest populations through fresh produce derived from their local farm, to eating engineered fruit or vegetables prescribed by a health care worker, to a plant product derived from batch processed, freeze-dried plant tissues prescribed by a health care worker to current paradigm that PMVs are not food materials that need to meet still-evolving regulations of national regulatory authorities for drug administration(FDA) and Department of Agriculture (USDA) [44]. Plants producing pharmaceuticals are regulated by USDA and the regulatory framework is developed by the FDA and Centre for Veterinary Medicine (CVM) [78]. The antigen present in edible vaccines is considered as a chemical, that does not comply with FDA rules concerning nutritional additives, but is recognized as non-GRAS (Generally Recognized As Safe). These vaccines, under the category of food, would be included as a genetically modified food and thus are not considered a high health risk. Due to this ambiguity, a legal void currently exists with respect to regulations for standardizing edible vaccine commercialization. It is not yet clear what part of the vaccine discharges the antigen itself, the transgenic, modified fruits or the transgenic seeds [49]. In the presence of this legal uncertainty, every country is expected to evaluate whether the entrance of edible vaccines (or the plants producing them) is permitted [32]. In 2005, the World Health Organization (WHO) delivered a report on the implementation of good agricultural practices for the development of biopharmaceuticals. This report includes detailed information about methods of quality control for medicinal plants, testing to assess identity and purity, and recommended materials for plants in biopharmaceuticals

Future Prospects
Bio-farming or molecular farming is attractive because of its flexibility, scalability, low manufacturing cost, no toxicity or pathogenic contamination, but many projects are at various developmental stages and not many are yet available to the pharmaceutical industry. Optimization of lab protocols for up-scaling the production of therapeutics at commercial level is important for clinical use. Plant metabolic engineering is a highly significant technology for production of high-value pharmaceutical compounds. Fusion proteins for multicomponent vaccines against multiple diseases are a potential tool to incorporate into immunisation programmes. Unlike genetically engineered microbial systems such as viruses, which pose more risks to the environment and humans, and have more chances of escape, difficulty in controlling and monitoring such escapes or unintended presence, plants are immobile. Control, containment and monitoring of genetically engineered plants are easier and containment can be achieved by regulating pollen transmission. Transgenic mitigation through linking the transgene to genes that confer a selective disadvantage such as a dwarfing mitigator gene, or other mechanisms like cleistogamy in rice, total sterility in tubers and bulb-propagated crops, synthetic auxotrophy etc. could contain gene flow.
Low-cost technologies for the production of biopharmaceuticals using plant systems should be used in cases of unprecedented public health crisis such as that caused by COVID-19. Since the production processes and systems of production or sophisticated expression platforms for vaccines and therapeutics are already established, it is possible to rapidly generate the vaccines with higher yield under cGMP practices. N. benthamiana plants can be transiently transformed with target vaccine gene and the leaf biomass harvested and processed to purify the antigen or vaccine. In addition to transient expression systems, plant biomass propagation with plant cell suspension cultures should be refined.
The Canada-based biopharmaceutical company Medicago Inc. is into transient expression of SARS-CoV-2 virus S protein, using a virus-like particle (VLP) grown in Nicotiana benthamiana to develop a potential vaccine against the coronavirus disease that has now reached a global pandemic level. Universities and Institutes from several countries including the US, Germany, UK, South Africa, South Korea, Mexico and Thailand are working in the molecular farming field, investing efforts and establishing partnerships and collaborations for treatment or vaccine for COVID-19. Transient transformation approaches are rapid (expression within a week) while regeneration of stably transformed plant takes up to 3 months, which is not suitable for addressing a fatal, exponen-tially-growing pandemic such as COVID-19.Though concept and methodology are concise, there are only limited number of edible vaccines which are approved, tested and commercialised. The COVID-19 pandemic outbreak warrants a deep investigation and introspection into the technology status, application and progress to address the problem [83]. Transient expression in plant cell suspension cultures seem to be a feasible strategy to produce plant-based vaccine for deadly pandemic outbreak of COVID-19.

Conclusion
Plant-based vaccine production systems can highly reduce the manufacturing costs involved in conventional vaccine production systems and can rapidly increase the scale of production. These two major advantages are a boon to vaccine development in developing or resource-poor countries, to reduce morbidity and mortality due to infectious diseases, and other orphan diseases which are poorlyfunded in terms of research and development. Also, the plant-based vaccines can be administered orally, skilled administration is not required, can be produced in food and non-food crops, and easily commercialised due to possibility of growth in controlled environmental conditions as in cell suspension culture.
There is also an element of flexibility of production in plant-based systems. But a major drawback is the tight regulatory systems which takes a long while to release the product. A vaccine or curative drug is the only way and means to prevent the spread and mounting death toll caused by novel deadly pandemics such as the novel coronavirus SARS-CoV-2. The anticipated cure or vaccine can be developed by 2021 by 12-18 months, when the solution to the problem of pandemic will lose its significance due to the alarming fatality rate. In the wake of the global calamity of the pandemic COVID-19 caused by SARS-CoV-2, can the regulatory processes be revisited and amended or reframed for development of vaccines and approving the strategies of vaccine production, which are in the pipeline? Mechanisms and strategies already existing to prevent them from entering food chain should be incorporated. The production of the vaccines can be stopped after achieving a tight grip or control over the disease or cure for the disease. In the long run, we should be better equipped and prepared to face the deadly epidemic or pandemic outbreaks with vaccines, which are the weapons in the war against the pathogens. Beyond COVID-19 as well, there will always be a need to immediately respond to new strains of viruses and pandemics that may emerge in the future. In an emergency or crisis, what is required is solution to the problem than solution unrealised by further problems of regulations. Regulatory processes are for risk management and for maintaining quality standards of production, for the successful use of vaccines and not to completely freeze the use of the most economical and feasible promising vaccine development technology for resource-poor nations. Plant-based vaccines are one of the affordable, powerful ammunitions for developing nations, against pandemic outbreaks and can be used as a potential weapon to save mankind.