key: cord-0880581-oitqxuyd
authors: Sahoo, Ankit; Mandal, Ashok Kumar; Dwivedi, Khusbu; Kumar, Vikas
title: A cross talk between the immunization and edible vaccine from natural origin: Current challenges and future prospects
date: 2020-08-26
journal: Life Sci
DOI: 10.1016/j.lfs.2020.118343
sha: ebc8273a2b8aa4ead087fd99c7ced1227397b752
doc_id: 880581
cord_uid: oitqxuyd

INTRODUCTION: It is well known that immune system is highly specific to protect the body against various environmental pathogens. The concept of conventional vaccination has overcome the pandemic situation of several infectious diseases outbreak. AREA COVERED: The recent idea of immunization through oral route (edible vaccine) is vital alternatives over conventional vaccines. Edible vaccines are composed of antigenic protein introduced into the plant cells which induce these altered plants to produce the encoded protein. Edible vaccine has no way of forming infection and safety is assured as it only composed of antigenic protein and is devoid of pathogenic genes. Edible vaccines have significant role in stimulating mucosal immunity as they come in contact with digestive tract lining. They are safe, cost-effective, easy-to-administer and have reduced manufacturing cost hence have a dramatic impact on health care in developing countries. EXPERT OPINION: The edible vaccine might be the solution for the potential hazard associated with the parenteral vaccines. In this review we discuss the detailed study of pros, cons, mechanism of immune stimulation, various outbreaks that might be controlled by edible vaccines with the possible future research and applied application of edible vaccine.

The immune system is a dynamic structure in our body that protects us from various pathogens. Immune system continuous tracking of molecules which circulate within the body to detect substances which negatively affect our health. Once the foreign body (pathogens) are identified, the immune system attacks to neutralize them with the help of antibodies 1 .

Human immune cells are extremely complex and quickly adjusted to overcome every day's challenges. Literarily, the immune system can be described as a complex collection of cells, tissues, organs and process working together to prevent disease. The immune system targets microorganism like a virus, bacteria, pathogen; parasitic worm, toxins, allergens and even sometimes own cell that show unusual characteristics. As microorganism rapidly evolves in a very short period, the immune system has to be prepared to handle massive diversity of antigens (which are commonly identified as an agent that activates immune system) 2 .

Generally, each molecule may be an antigen, but researchers have found that carbohydrate and proteins offer the best response, whereas lipids and nucleic acids are poor antigens. The sensitivity and the specificity of the immune system should be taken in to account for the developments of highly specific chemical and cellular tools as it is known that immune system is complex but well organized. As a result, this destroys or kills the invading pathogens, ensuring long-term protection against pathogens and immunological memory for the body that immediately reacts to subsequent encounters with the same antigen 3 .Sometimes body immune system is unable to combat with these pathogens or microorganisms may be due to the resistance causes in the microbe over the long period in which they modified their internal, external structure (modify receptors that present on their surface) or due to the new strain that does not attack before to the human body. To overcome this problem scientists develops vaccines usually, contains whole microbe (either killed or as a live form), microbe Mason et. al. was the first person who conducts the vaccine-based assay produced in potato (Solanum tuberosum) to fight against LT-B stain produced by E. coli in mice 14 . In that same year, in rats and human volunteers, In the same year, the effectiveness of the antigens produced from potatoes (Solanum tuberosum) towards the non-toxic subunit of Vibrio cholera endotoxin and the Norwalk virus capsid pathogen was identified in rats and human volunteers 12, 15 .Thanavalas'group proposed in 2005 that potatoes could play a role in human hepatitis B as an oral reinforcement since injectable vaccine cause redness, swelling, or itching at the site of administration. Also, the edible vaccine for the animal has now been developed to replace the injectable vaccine for animal protection 12 .

Tomato (Solanum Lycopersicum) an appropriate candidate for vaccine development for coronavirus that causes a highly acute respiratory syndrome (SARS). For the development of recombinant SARS-coronavirus (CoV) vaccine, S-spike protein (S-protein) and its truncated fragment are considered as the best choice 9, 12 . The genome of tomato and tobacco is incorporated with N-terminal fragment of SARS-CoV protein (S1) used to develop the safe, effective and inexpensive vaccine. When these plant-based vaccines for SARS give to mice, shows significant increase level of SARS-CoV-specific IgA after oral ingestion of tomato, expressing S1 protein. whereas tobacco-derived S1-protein indicate the presence of SARS-CoV-specific IgG detect by ELISA analysis and Western blot 16 .Tobacco is not an edible plant but play a major role in the development of the vaccine as it is used as a proof-of-concept model species for the edible vaccine 1 .

For HBsAg gene of hepatitis B, lines of transgenic cherry tomatillos have been grown.The expression of genes was seen through the whole plant, but it was maximum in the fresh leaves weight of 300 ng/g and with fresh fruit weight of 10 ng/g) 12 .

Lactuca sativa express the B-subunit of the thermolabile protein of E. coli, responsible for both human and animal enteric disease, show the possibility of this vegetable as an edible vaccine. In 2005, the typical swine fear hog pest virus glycoprotein E2 was expressed by lettuce. In Poland, the transgenic lettuce that shows effect against hepatitis B virus is in the development stage 17 .

In the study, E. coli bacteria B-subunit of thermolabile toxin, expression was performed in the endoplasmic reticulum (ER) of soybean (Glycine max), which yielded a total antigen level J o u r n a l P r e -p r o o f of up to 2.4% of the total soybean seed protein without any problem during drying for further processing. Moreover, when this protein is given orally to rats leads to a rise in systemic IgG and IgA 18,19 .

Chlamydomonas reinhardtii (green algae) has been used as a tool to achieve a large number of proteins specific to both animal and humans for therapeutic purpose 18, 19 .The use of algae for the production of vaccines is optimistic, as algae have a very high growth rate, the entire system can be used as a raw material for the development of edible vaccines. Besides, to facilitate the already rapidly growing algae can be cultivated in bioreactors. C. reinhardtii contains one chloroplast which facilitates the stability of the desired antigens in the algal line.

Notably, the effectiveness of algal vaccines after lyophilization is unchanged, which might promote global delivery of edible algae vaccine 12 .

Based on the expression of the capsid protein Norwalk virus, the transgenic plant was developed. Protein deposition in the un-ripened fruit with a lower accumulation in red fruit was reported up to 8% of soluble protein. Expression in seeds allowed the storage of antigenic peptides, thus creating a plant with a high yield of proteins, with an average protein content of about 20%-40%, which would preclude intensive purification procedure by pharmaceutical industries 12, 20 .In addition to the expression of hemagglutinin protein (H), a PA against rinder pest virus (RPV), pea plants were used. The total soluble protein level of expression in leaves was observed to be 0.12%-49% determined by Western Blot. Even more, studies are also required to improve the expression of a protein in transgenic peas.

In banana plants, HBsAg expression was reported with four distinct cassettes (PHER, PHB, pEFEHER and pEFEHBS). At the different level expression of HbsAg were studied with PCR, reverse transcription PCR, and Southern hybridization method. Expression levels reached a height of 19.92 ng/g in the plant, and the antigen was found in banana leaves.

However, because of the long period required the shrub needs to grow, the use of this vaccine was denied 21 .

In 2007, a papaya (Carica papaya) vaccine was developed to counter cysticercosis caused by Taenia solium by expressing synthetic peptides in 19 Transgenic Papaya clones. Vaccine was tested in rats, with an immunogenic response of 90 per cent in vaccinated animals. These J o u r n a l P r e -p r o o f edible vaccines may offer good relief both in humans and in pigs, which are the two major carriers of the disease 22-24 .

In an experiment, Carrot along with A. thaliana was utilized to develop an edible vaccine for surface HIV antigen expression, and in the study, it was reported that rats showed more positive effect compared to those non-treated animals 25 . Carrot (Daucus carota) has a positive effect in the treatment of HIV not only because carrots are nutritious and tasty, but because of carrot main chemical constituent carotenoids which on consumption by rats increases monocytes, lymphocytes, and other immune defence. Thus, people with a weakened immune system might benefit from the use of this potential edible anti-HIV vaccine 26 . The efficacy of this anti-HIV vaccine must be confirmed by a clinical trial. In 2010 it has been reported that the UreB subunit of helicobacter pylori was used in transgenic carrots as a potential vaccine. Transgenic carrot expressing the B subunit from E. coli thermolabile toxin-induced IgA and IgG production and occurred at the intestinal and systemic level in the rat 27,28 .

A research in 2007 found that transgenic rice (Oryza sativa) plants expressing the B subunit of E. coli induces significant number of antibodies to this subunit. In the same year, an immune response was found to be caused in chicken by transgenic rice that is a result of the VP2 antigenic protein from infectious bursitis. In 2008, PCR and Southern blot analysis confirmed the functional expression of HBsAg in rice seeds 29, 30 .

In addition, transgenic rice was developed in 2008 in parallel to express the subunit B of the E. coli thermolabile toxin used to convert plant cells using bio-ballistic approach; PCR verified the expression. India and China both are the world two biggest rice producer and have the capability to export these modified rice (vaccine plant) all over the globe 31 . Agrobacterium, a soil bacterium naturally occurring, has been used to transfer a small fragment of DNA into the plant genome and is called transformation 50, 51 . In this method, the appropriate recombinant DNA is inserted into the T-region of disarmed Ti-plasmid of Agrobacterium tumefaciens (plant pathogens), which is co-cultured with the plant cells and/or the tissue that will be transformed. The insertion of the exogenous genes and the infection of such a plant tissue into sufficiently modified

Agrobacterium T-DNA cell contributed to studies of the stable gene incorporation in the genome of the plant, as well as transgenic protein production 32 . However, this technique is sluggish and yield are lower; but the application of this transformation is first limited to tobacco plant and too few other plant species, which extend to most vegetable species including Leguminose and Graminae.

This is a sophisticated method, based on the micro-projectile bombardment. The selected DNA sequence is precipitated on microparticles of metals (e.g. tungsten, gold) and bombarded by a particle gun at an accelerated rate toward selected plant tissue 52, 53 . These metallic microparticles penetrate the cell walls, and the exogenous DNA is emitted into the cell, where it is integrated into the nuclear genome through a process known for the photosynthetic role of cytoplasmic organelles called chloroplast comprising chlorophyll. Particle gun shoots adequately charged metallic particles with selected and processed DNA, which penetrate chloroplast and merge with its genome 32 . Transformation of the chloroplast is an effective alternative to nuclear transformation 54,55 .

PDs in chloroplast are more stable when plant cells lyophilized and when preserved at ambient temperature. Therefore, the freeze-drying method improves PDs concentration and prevents bacterial contamination 11 .

In this method, DNA is introduced into the cells to which the electrical pulses of high voltage are released, which are intended to create transitory pores in the 

Immune response to the vaccine is affected by the route of immunization. The form of antigen and the active content of vaccine mediate specific tissue tropism. There is now substantial evidence supporting the existence of at least two immune systems, a "peripheral" immune system and a "mucosal" immune system 20 . These systems operate separately and simultaneously in most species including human. Protective immunity acquired during recuperation is usually referred to as "systemic immunity," but the fact is that it might be dominated by an incomplete form of immunity dictated by a specific pathogen as the Systemic immunity might be a combination of mucosal and peripheral immunity.

Lymphocyte traffic patterns, regulated by selective expression of adhesion proteins in peripheral or mucosal lymphatic tissues affects the outcome of an immune response. For example, the same antigen may produce qualitatively different immune responses in lymph nodes, spleen or Peyer's patches. The antigens in the lymph are presented over the fixed antigen-presenting cells in lymph nodes results in "peripheral immunity" characterized by the appearance of specific IgG in the blood. The antigen in the blood is presented in strategic tissue interface in the spleen. This also results in "Peripheral Immunity". However, the microenvironment of the spleen is somewhat more complicated as it also accommodates circulating antigen-presenting cells and immunoreactive T-and B cells from other tissues committed to either peripheral or mucosal immunity. Triggers of antigen in the lumens of enteric organs presented on Payer's patches commitment to "Mucosal immunity" characterized by the release of specific IgA into the secretions 20,56 .

The mucosal surfaces are a popular site for delivering therapeutic small molecules due to the ease of administration and speed of uptake across the large surface areas. efficacy of the mucosal route of immunization is largely based on the fact that mucous membranes constitute the largest immunogenic organ of the body. This interface is endowed with the well-organized lymphatic structure called MALT (mucosa-associated lymphoid tissue) which constitute T and B cells (Innate and Adaptive arms of the immune system). Oral vaccines stimulate the generation of immunity in gut-associated lymphoid tissue (GALT), which includes lymph nodes, Payer's patches (in which lymphocytes are the major component: J o u r n a l P r e -p r o o f ~75% are B cells, while ~20% are T cells), and isolated lymphoid follicles in the gastrointestinal tract (GIT). A significant hurdle impacting protein delivery to the GIT that the antigens are rapidly degraded within the harsh environment of the digestive tract is the remarkable challenge for vaccine development. It will also be important to consider the characteristics of the GIT, in which several factors, including proteolytic enzymes, acidic pH, bile salts, and limited permeability that may hinder the induction of a protective immune response 20 . 

Antigen loaded in the specific plant tissue is the principle of edible vaccines. Thus, the efficacy and the potency of the vaccines are significantly affected by the nature of "adjuvants". "Adjuvants" are the biomolecules (lectins, saponins) that do not exhibit immunogenic response but potentiate the immune response when co-administered with an antigen. Adjuvants can improve immune and potentiate responses by acting as a depot to guide antigens to relevant sites, protect them from degradation, control release and activate APCs 20 . Immunogenically inert biodegradable adjuvants like lipids, proteins, starch, polysaccharides, or polyesters act as the delivery vehicles for efficient availability at Antigen Presenting cells (APCs). There are major two methods to associate antigen and particles i.e. 

Due to the use of the oral route, the administration of the edible vaccine is less complicated than the conventional methods that are given through IM, SC, and intra-dermal. Thus, it removes the needs of trained medical personnel and decreases the risk of infection as there is no need for the sterilization of premises and the manufacturing areas.

 Such oral immunization will become a practical key strategy for effective disease prevention in low-income countries, in general.

 Processing, purification, sterilization, packaging or delivery does not require rigorous structure in edible vaccines, minimizing long-term costs in relation to conventional vaccines 32 .

 Maintenance and distribution of edible vaccine are easier than the conventional vaccines as it enabling the preservation without the constant cold chain storage.

 Improved storage possibilities for edible vaccines become possible as transgenic plant seeds have lesser moisture content, heat stable and can quickly be dried 60, 32 .

 A plant containing therapeutically active edible vaccine protein is free of toxins, pathogens and do not have the risk of proteins to reform into the infectious organism 8 .

 Improved compliance is particularly related to children's who refuse to take injections of the vaccine.

 Most specifically, in this case, the immunity is activated on the mucosal surfaces of the GI, including those which are the first line of defence on the mouth (Mucosal immunity).

 Sophisticated equipment and machinery are not required in edible vaccines since they can easily be cultivated on rich soils and are cost-effective relative to fermenters where the recombinant cell is cultured in a controlled manner 32 .

Beyond the several advantages and the convenience over the conventional vaccines, Edible vaccines have certain challenges that have to be overcome for efficient and pure beneficial edible vaccination. Some of them are pointed below: J o u r n a l P r e -p r o o f  Uncertainty in calculating the appropriate oral dosage which might take multiple rounds for a patient to get the effective therapeutic action, and raises the final expense of its application 32 .

 The concentration of peptide or protein of edible vaccine varies from generation-togeneration, plant-to-plant, and fruit-to -fruit 32 .

 Patient-related factor such as patient age and weight also affected the dose to be administered.

 Repeated intakes of these antigen-bearing plants which stimulate the immune system might over stimulate the immune system itself leads to immune-tolerance to peptide or protein of edible vaccine 61 . Administration of a single potato a week for one month with periodic boosters was seen providing immunity. Mutant cholera toxin subunit A (mCT-A) and LT-B in crop seed, when expressed together, has shown to be effective by nasal administration and is practical 66 .

When human trials of the potato-based vaccine of Hepatitis B were performed, it has shown remarkable result. Specific antibodies were induced in mice by VLPs. In a single potato expression of a single dose of HBsAg can be achieved 66 . Protective level of 10 mIU/mL of specific antibodies exceeded significantly. Higher expression of plasmid HBsAg subtype ayw was seen in roots as compared to leaves tissue of transgenic potato when cloned with CaMv (cauliflower mosaic virus) 66 . Further studies need to be done for increasing the antigenic expression in potato by using promoter like patatin promoter. A superior plant material then yeast-derived antigen for both priming and boosting immunity in mice was observed. When mice were given a single parenteral dose of yeast-derived recombinant HBsAg followed by transgenic potatoes lead to the development of antibodies that immediately peaked at >1,000 mIU/mL and were maintained at >200 mIU/mL for five months. This was a prime-boost strategy in mice which useful in developing countries. The guarded greenhouse was used to grow plants of transgenic tomatoes and around 4000 vaccine doses were obtained from just 30 tomato plants. Lettuce plant is also being developed.

When transgenic potatoes expressing Norwalk virus antigen were fed to 20 people, out which 19 people were observed developing antibodies. Bananas and tomatoes expressing Norwalk virus are also being engineered 58,66 .

When tobacco expressing MV-H (measles virus haemagglutinin from Edmonston strain) was fed to the mice antibody were developed which were five times of the levels considered protective for humans and they secreted IgA in their faeces. The titers were increased 20 times the level protective for human by using a prime booster strategy of combining parenteral and subsequent oral MV-H boosters 66 

When BLB/c mice were given Human papillomavirus type-11 (HPV-11) recombinant VLPs produced in insect cells, it was found immunogenic to them. The response is dose-dependent, conformationally-dependent and genotype-restricted 66 . Thus, VLPs may be effective oral immunogens for the prevention of anogenital HPV disease.

A protein identical to the major protein present in existing vaccines could be expressed when tobacco leaves bombarded with pag gene (anthrax protective antigen -PA) using a gene gun 66 . Oedema factor and a lethal factor which were responsible for toxic effects were absent in these vaccines. A numerous anthrax antigen could be produced. Tomato plants are being used to put the same anthrax antigen. Transformation of spinach by inoculating it with TMVexpressing PA are also being developed by scientists as spinach is safer as a vaccine 58 .

Research works of developing a various vaccine is undergoing and are being tested regularly for their safety and efficacy. Clinical trials of all the vaccines are done so that a safer and effective drug can be obtained. Due to the complex multistage life cycle of the parasite, its inaccessibility to study and by its large genome advances in the vaccine of malaria are hindered. However chimeric coat proteins of CPMV expressing malarial and foot-and-mouth disease epitopes have been reported 66 To overcome this problem in collaboration with U. S. Army, developed a vaccine to address bioterrorism of Ebola virus with its surface glycoproteins which can be transiently expressed in plants 12 .

Unlike the conventional vaccine, the edible vaccine is capable to bring mucosal immunity.

But there is a remarkable limitation of the conventional vaccine over the edible vaccine. The latest novel approach for vaccination has several benefits and the formulation technique, transportation and mode of administration is so convenient that it can be practised without sophisticated technology and specialized medical expertise. It also doesn't require subsidiary elements to stimulate the immune system 32 . The conventional vaccines are safe as it contains attenuated and heat-killed pathogens hence do not present any risk of proteins to reform into the infectious organism 32 . Regarding the economy for production and the quantity to be manufactured, edible vaccines are much more convenient as it minimum cost and its process for production can be scaled rapidly by breeding 61 , but the concentration of the protein depends on the weather condition (sunlight, rainfall, altitude etc.), whereas conventional vaccine is produced in the controlled environment. The route of administration and delivery of the vaccine improves compliance than conventional vaccination.

The basic concept that is involved in the development of vaccines is to select desired genes and then incorporation of these genes into plants and then encoded proteins are produced by (glutamic acid decarboxylase), linked to CTB subunit. In this study, delayed onset of high blood sugar level and immune attack suppression was proved successful 7,32,58 .

For effective cancer therapy agents, many plants have been engineered to generate monoclonal antibodies. Soybean (BR-96) is an effective agent in curing breast cancer, ovarian cancer, colon cancer and lung tumours 32 .

When TMV is administered, a protein is produced which is found in Mousezona pellucida (ZB3). Due to resulting antibodies, the protein can prevent fertilization of eggs in mice 32,72 .

Apart from the production of vaccines and antibodies, plant compositions are altered by engineered viral inoculations to produce enzymes; drugs (serum protease, albumin and interferon). For example-tobacco plants produce glucocerebrosidase (hGC) to treat

Goucher's disease, interleukin-10 to treat Crohn's disease. This method is cost-effective 32 .

The causative agent of rabies is a single-stranded negative-sense RNA virus. The name of the virus is lyssavirus of the family Rhabdoviridae, which is having a cylindrical shape with bullet-shaped virus particles. In concern of rabies, the transgenic tomato was noticed with stable expression of the rabies surface protein but it lacked immune-protective ability. A synthetic gene coding for the surface glycoprotein(G-protein) of rabies virus identifies the major antigen that induces protective immunity was strategically designed to achieve highlevel expression in transgenic plants for immune-suppression by rabies vaccines glycosylation of G-protein is required 73 . 

Hepatitis B virus is the causative agent for the infectious disease Hepatitis B. In this the liver of the humans are affected and inflammation occurs called serum hepatitis. The gene encoding Hepatitis B surface antigen (HBsAg) that was linked with a constitutive promoter transformed the tobacco plant genetically 75 . The presence of HBsAg in extracts of transformed leaves was seen by the Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg at levels that correlated with mRNA abundances. This led to a conclusion that there were no major inherent of transcription or translation of this foreign gene in plants were found. Purification of this recombinant HBsAg from the transgenic plant was done by immune-affinity chromatography and then is examined by electron microscopy. When negatively stained preparation applied a spherical particle of 22nm was observed. Similar physical properties were observed in recombinant HBsAg and human serum-derived HBsAg due to sedimentation of transgenic plants in sucrose and caesium chloride density gradients 76, 77 . As the similarity was observed the conclusion from it can be drawn that transgenic plants hold promise as low-cost vaccine production systems.

Rota viral disease is a common disease occurring in infants and young children. At least once by the age of five, nearly every child in the world is infected with rotavirus. This causes diarrheal disease amongst children. It is very less likely to occur in adults. There are 10 species of the genus, referred to as A, B, C, D, E, F, G, H, I and J 6 . More than 90% of rotavirus infections are caused by most common rotavirus namely Rotavirus A. Synthesis and insertion of a codon-optimized gene (sVP6) encoding the VP6 protein of human group A rotavirus was done into the alfalfa genome using Agrobacterium-mediated transformation 6 .

When oral immunization with pBsVP6-transgenic alfalfa was done provides a potential means of protecting children and young animals from severe acute rotavirus-induced diarrhoea 61 .

Cholera is a diarrheal disease caused by bacteria Vibrio cholera, a bacteria of comma shape.

It colonizes the intestine and produces enterotoxin Cholera toxin B (CTB) and causes acute watery diarrhoea 78, 79 . When taken orally CTB acts as a mucosal immunogen. A mucosal response to pathogens was obtained due to the CTB binding to the eukaryotic cell surfaces via the GM1 ganglioside receptors present on the epithelial surface of the intestines. An Nicotiana tobacco leaves was carried out. In a medium containing a selection marker, in this case, streptomycin, the transformed1 leaves were cut and grown. It was analysed using PCR analysis followed by southern blot analysis. Quantification of the CTB protein produced was carried out using Western blot analysis and ELISA. A strong affinity for GM1 ganglioside was demonstrated by both the chlorophyll-synthesized CTB and the bacterial CTB in GM1 ganglioside assay 78 . The growth rate, flowering, and seeding in transgenic tobacco are not expressed due to high levels of constitutive expressions, unlike when expressed in the nuclear genome.

Malaria is a disease caused by plasmodium parasite and is spread by female Anopheles 

Measles is a disease caused by a virus and is highly contagious. It occurs mainly in children < Table 5> < Table 6>

Stable colonization and survival in vivo, particularly in the harsh gut environment 88 .

Improve the efficacy of the vaccine.

Other beneficial effects include anti-inflammatory activity and antibacterial activity 88 .

The major limitation of probiotic vaccine is oral tolerance and difficult to monitor the immune complexity 88 . Another major factor innate low immunogenicity of probiotics. 

The conventional vaccine previously developed have a great role in reducing the annual death toll from several infectious diseases, yet these discoveries mask the tragic gaps in drug delivery process and fail to achieve the required therapeutic effect especially in the remote and improvised part of globe due to lack of availability of vaccine. Due to the nonexistence, unreliable or too costly immunization technique and non-effective drug delivery system, millions of people are still dying from infectious diseases. This is worrisome for the entire globe.

The 

It is important to introduce the genes encoding mucosal adjuvants into transgenic plants used for creation of edible vaccines and/or use plants containing the secondary metabolites capable of playing the role of mucosal adjuvants.

Edible vaccines have significant role in stimulating mucosal immunity as they come in contact with digestive tract lining.

plant virus-based vectors can be an alternative for production of antigens for an inexpensive and safe subunit vaccine.

provide the most reliable protection against this viral infection.

The major limitation of this manuscript is that as in date, neither the allergy nor the tolerance has been studied in humans. The development of an edible vaccine starts with the identification of desired genes or proteins which is biotechnologically modified with plant-bacteria or virus plasmid and introduced into it. Then the modified plasmid containing bacteria or virus is introduced to the desired plant cell and cultured in the lab with controlled environment. When plant successfully grew then moves for mass production in the crop field, from where these edible vaccines can be distributed to the whole world. After an edible vaccine has been consumed orally, it will trigger a response to B-cell and T-helper cell and induce an individual immune system as they are the main factors. Figure 2 : -The steps that stimulates mucosal immunity in a very fine way which is elucidated by this diagram. Figure 3 : -Edible vaccine containing antigen induces immunity response from the intestine. 

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Adjuvant Strategies for Lactic Acid Bacterial Mucosal Vaccines

Resistance of Young Mice to Pneumococcal Infection can be Improved by Oral Vaccination with Recombinant Lactococcus lactis

Oral immunization with live Lactococcus lactis expressing rotavirus VP8* subunit induces specific immune response in mice

Oral vaccination of mice against Helicobacter pylori with recombinant Lactococcus lactis expressing urease subunit B

Development of a tripartite vector system for live oral immunization using a Gram-negative probiotic carrier

Oral colonization and immune responses to Streptococcus gordonii expressing a pertussis toxin S1 fragment in mice

Evaluation of different promoter sequences and antigen sorting signals on the immunogenicity of Bacillus subtilis vaccine vehicles

Prodigene Production and transfer of recombinant antigen to plant cells through the plasmidvector system; production of vaccine for hepatitis and transmissible gastroenteritis virus