Edible vaccines a patent driven exploration of immunization technologies

This review states the technologies, host plants, current status, recent patents, the future of this new preventive modality, and dif-ferent regulatory issues concerning edible vaccines.

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Edible Vaccines: A Patent-Driven Exploration of Immunization Technologies

Sahil Kashyap1, Shikha Kamboj1, Rohit Kamboj1, Kumar Guarve1 and Sweta Kamboj1,*

1Guru Gobind Singh College of Pharmacy, Yamuna Nagar, Haryana

Abstract: Vaccines are biological preparations that improve immunity to particular

dis-eases Particularly for poor developing nations, edible vaccines show significant potential as a financially advantageous, simple to administer, straightforward to store, fail-safe, and socially and culturally acceptable vaccine delivery system A vaccine incorporates the gene-encoding bacterial or viral disease-causing agent in plants without losing its immunogenic property Potatoes, tomatoes, rice, soybeans, and bananas are the primary plants for edible vaccines It activates the systemic and mucosal immunity responses against a foreign disease-causing organism It offers exciting possibilities to reduce dis-eases like hepatitis B, rabies, HIV/AIDS (human immunodeficiency virus infection and

acquired immune deficiency syndrome), etc These vaccines provide many benefits, like

being convenient to administer, efficiently storing, and readily acceptable drug delivery systems for patients of different age groups So, an edible vaccine may be the most con-venient vaccine to improve immunity However, there are a lot of technical and regulato-ry challenges to overcome in the way of edible vaccine technology Though all seem surmountable, various technical obstacles and regulatory and non-scientific challenges need to be overcome Moreover, edible vaccine patents represent a cutting-edge area of biotechnology, where the integration of genetic material into edible substances holds great promise for revolutionizing vaccination methods These patents aim to harness the potential of plants and other edibles to stimulate immune responses, offering a potential alternative to traditional injectable vaccines This review states the technologies, host plants, current status, recent patents, the future of this new preventive modality, and dif-ferent regulatory issues concerning edible vaccines

As man has progressed in his development, he has abused this environment so much in his strug-gle for survival that microorganism infection is rising In this way, to be safe from illness, a person needs to have good health [1] The higher our im-munity, the better we can keep these diseases away and protect ourselves from these dreaded infec-tions Therefore, we will be protected from the vi-rus only when our immunity is good, so first of all,

*Address correspondence to this author at the Guru Gobind Singh College of Pharmacy, Yamuna Nagar, Haryana; E-mail: swetakamboj20@gmail.com

we have to increase our immunity, and for this, we should take vaccines to protect against different infections so that our bodies can get ready for the coming dangerous virus infection by building up the immunity beforehand [2] A vaccine is a bio-logical preparation that stimulates our immune system to generate more antibodies toward a par-ticular disease-causing antigen The vaccine con-tains a specific disease-causing virus antigen's killed or attenuated (inactive) form When these inert antigens enter our bodies, our immune sys-tem recognizes them as harmful foreign bodies and starts the production of antibodies against that par-ticular antigen [3] These antibodies are stored in

our body, and when the disease-causing virus en-ters our body, our immune system releases the an-tibodies to neutralize the disease-causing antigen and normalize the body [4] In 1796, Edward Jen-ner proposed the first vaccination against small-pox But since then, needles have been used for the vaccine, so the immunization process has become painful and complicated, and it is not possible to take the vaccine without an experienced doctor [5,6] The storage of vaccines is also a difficult task under proper conditions Also, reusing nee-dles can transmit serious infections like Hepatitis B, HIV/AIDS (human immunodeficiency virus infection and acquired immune deficiency

syn-drome), syphilis, etc

WHO (World Health Organization) estimates that 10 million children die in developing coun-tries yearly from infectious diseases that vaccines could prevent However, conventional vaccines are expensive and inconvenient for the poor people of developing nations [7,8] In such a situation, find-ing a few new cheap vaccination methods that people can take conveniently without any pain or the help of a professional becomes even more es-sential That's why food or oral vaccines are the only medium by which we can increase our im-munity by taking them ourselves without profes-sional help [9] The American biologist Dr Charles Arntzen was the first scientist to introduce us to the concept of an edible vaccine [10, 11] These are the subunit vaccine types prepared by a plant's genetic modification [12] Various transgenic techniques incorporate The virus antigen into a suitable host plant to produce edible vaccines [2] The person himself eats these oral vaccines with-out any professional help to improve or boost his immunity against a particular virus infection Alt-hough the orally administered food vaccines di-rectly go to the gastrointestinal tract, they now strengthen the mucosal layer of the GIT and make a protective shield against the virus antigen [13]

1.1 Genetically Modified Plants

Transgenic plants are genetically modified plants created through DNA recombinant tech-niques It is a modified plant in which different genes are combined by genetic engineering tech-niques to form a new species or plant Plant genet-ic engineering has long been used for various pur-poses, including improving fruit quality,

increas-ing yield, and producincreas-ing pest-resistant plants [14]

For example, Bacillus thuringiensis corn is a

transgenic corn that contains a toxic protein against insects and pests but is entirely safe for humans Therefore, genetic modification offers a wide range of benefits [15] Thus, genetic engi-neering makes edible vaccine preparation possible, stimulating our immune response without harming us conveniently However, different types of plants have different abilities because their nutri-tion varies As a result, selecting a host plant is an

essential factor in producing edible vaccines via

genetic engineering The host plant is the plant that is best suited for the production of edible vaccines [9] It is a challenging task to choose the best host plant Several factors may affect the host plant

se-lection, which are shown in Fig (1)

1.2 Plants Commonly Used as Host

The host plant is the plant that is best suited for the production of edible vaccines It is a challeng-ing task to choose the best host plant Several fac-tors may affect the host plant selection, such as antigen stability with plant genes, the degradation time of fruit, the effect of physical characteristics

on antigen stability with plants, etc [16,17,18]

Various types of plants are used for edible vaccine production or clinical trials Generally, tobacco, potato, tomato, maze, rice, and carrots are used for the production of edible vaccines due to their vari-ous characteristics, which are explained as follows [4, 5, 16, 19-22] Now, let us look at some of the specific plants commonly used for the production of edible vaccines and the characteristics that make them suitable:

1.2.1 Tobacco

Tobacco is often used for producing edible vac-cines due to its relatively simple genetic makeup and well-established transformation techniques It has been used in research for many years and is particularly suitable for antigens that can be pro-duced in the leaves

1.2.2 Potato

Potatoes are a preferred choice because they store well, have a relatively long shelf life, and their tubers can be used as an edible vaccine deliv-ery system They are also genetically accessible and can be engineered to express vaccine antigens effectively

Edible Vaccines Recent Patents on Biotechnology, XXXX, Vol XX, No XX 3

1.2.3 Tomato

Tomatoes are another popular choice as they are widely consumed, and the fruit can be used as a delivery system Their fruit structure is relatively durable, making them suitable for vaccine stabil-ity

1.2.4 Maize (corn)

Maize is used for producing edible vaccines, primarily for antigens that can be expressed in the corn kernels It is a staple crop in many regions, making it an accessible choice for vaccination ef-forts

1.2.5 Rice

Rice is consumed globally and is used for vac-cine production, particularly when targeting dis-eases prevalent in regions where rice is a dietary staple The seeds of rice plants are utilized to ex-press antigens

1.2.6 Carrots

Carrots are a choice for antigens that can be ex-pressed in the root, which is edible and has a rela-tively long shelf life They are especially useful for

vaccines targeting diseases that can be combated

via the mucosal immune system Researchers

care-fully evaluate these factors to select the most suit-able plant for their edible vaccine production, with the goal of creating an effective and accessible

means of immunization Table 1 shows the list of

plants that are used as hosts for edible vaccines

1.3 Mechanism of Action for Edible Vaccine

As we know, the mucosal lining of the Gastro-intestinal Tract (GIT) is the primary site for the risk of viral infection Most of the pathogens, bac-teria, and viruses enter the oral region That is why the mechanism of action of the edible vaccine stimulates both the systemic and mucosal systems of our body [10, 16] First, this mucosal-targeted vaccine enters our body through the oral region, which is edible and reaches our gastrointestinal area, and the outer cell wall of the plant protects the antigen from enzymatic degradation [12] When this edible antigen reaches our intestinal re-gion, the digestive enzymes or the intestinal bacte-ria break down the outer capsule of the antigen (plant cells) and releases the antigen near the Pey-er patches, which consist of 30-40 lymphoid nod-ule which contains the follicle from which the germinal centre develops, and these follicles helps for the penetration of the antigen toward the epi-thelium lining of the intestine Then, it is taken up

Fig (1) Factors affecting the host plant selection (A higher resolution / colour version of this figure is available in the

elec-tronic copy of the article)

by the microfold cells (M cells) These antigens containing M cells activate the B-cells, which causes B-cells to leave lymphoid follicles and reach the mucosal-associated lymphoid tissue (MALT) It causes the degradation of plasma cells, and thus, Ig-A antibody production occurs These antibodies are then transported and stored in the lumen When the virus antigen enters our body, these antibodies get released from the lumen and

neutralize the antigen [23-27] Fig (2) represents

the mechanism of action for edible vaccines

2 EDIBLE VS TRADITIONAL VACCINES

One of the most significant benefits is the con-venience they provide Administered orally, these vaccines eliminate the need for injections, making them easier to distribute and use, especially in are-as with limited access to healthcare facilities

Moreover, the safety profile of edible vaccines is considered superior, as they eliminate the risk of contamination with harmful substances or patho-gens during production and administration In ad-dition to convenience and safety, edible vaccines offer remarkable cost-effectiveness By using plants as bioreactors, they can be produced at a fraction of the cost of traditional vaccines, making immunization more accessible and affordable for a broader population This cost advantage is crucial in low-income regions where traditional vaccines might be financially burdensome Edible vaccines hold immense promise in transforming vaccination strategies globally Their convenience, safety, in-creased acceptance, versatility, and stability make them an appealing alternative to traditional vac-cines, potentially revolutionizing immunization efforts and improving healthcare access for all [28, 29], as we discussed the various advantages and

Table 1 List of plants used as Host for edible vaccine

Sr.no Host

i Tobacco Charles Arntzen Abundant material for protein

Not grow well in regions where vac-cines are needed Quickly spoil Acidic fruit may be unstable with some

iv Banana Charles Arntzen

Easily Consume in pure form No need to cook Grow well in tropical areas

Available in all seasons months to bear fruit

Hepatitis B Virus (HBV) Hepatitis B

v Maize Octavio Guerrero-Andrade

Cheaper crop Readily available in

develop-ing nations

Not eaten raw Cooking may

dena-ture the antigen

Newcastle disease virus (NDV)

Newcastle dis-ease

Edible Vaccines Recent Patents on Biotechnology, XXXX, Vol XX, No XX 5

disadvantages of edible vaccines However, a de-tailed comparison between the edible vaccine and the traditional vaccine is important to know how the edible vaccine is comparatively better than the conventional/traditional vaccines or how it is ben-eficial from the physical and economic point of

view [29] Table 2

3 METHOD OF PREPARATION OF EDIBLE VACCINES:

There are various types of methods are used for the preparation of edible vaccine, which are as fol-lows:

3.1 Plasmid /Vector Carrier System

As per Fig (3) (Plasmid /vector carrier system),

it is a simple and widely used method for

produc-ing edible vaccines through transgenic plants A

tumefaciens is a naturally occurring soil bacterium

used to transfer small DNA segments into the plant genome by the process of transformation We sterilized a small section of the plant in this process They suspended the fixed part of the plant into bacteria carrying antigen culture, allowing the bacteria to deliver the antigen genes into the plant

cells Then, expose the plant cells to an antibiotic solution to kill the cells lacking new genes and al-low the callus to form After some time, the callus sprouted, shoots, and roots After that, the whole plant is generated from individual plant cells The existing studies showed that genes are successfully expressed in experimental plants When it is given orally to animals, the transgenic plant extract con-taining antigen induces the production of serum antibodies in that animal [20, 21, 30-34] The method is explained below:

v Plasmid Selection and Modification

The plasmid is then modified by inserting the DNA of interest into the plasmid's DNA back-bone This DNA of interest can be a gene that needs to be expressed, a therapeutic gene for gene therapy, or an antigen for vaccine devel-opment

v Transformation or Transfection

The modified plasmid is introduced into the host cells This can be done through tech-niques like transformation (for bacteria) or transfection (for eukaryotic cells, such as hu-man cells)

Fig (2) Mechanism of action for edible vaccine (A higher resolution / colour version of this figure is available in the

elec-tronic copy of the article)

The cells take up the plasmid, and if the plas-mid contains a selectable marker, scientists can identify the cells that have successfully taken up the plasmid

v Gene Expression

Once inside the target cells, the plasmid can replicate along with the host cell's DNA If the plasmid contains a gene of interest under the control of a promoter, the host cell can ex-press that gene This leads to the production of the protein encoded by the gene In vaccine development, the plasmid can express an anti-gen, allowing the immune system to recognize and build immunity against it

v Harvesting the Product:

After gene expression or any other intended application, scientists can harvest the product

(e.g., protein, therapeutic effect, immune

re-sponse) for further analysis or use

3.2 Micro Projectile Bombardment (Biolistic) Method

As represented in Fig (4), the microprojectile

bombardment method is also called particle accel-eration or gene gun delivery It is the direct meth-od for transferring the foreign genes to the target cells using heavy metal particles coated with

ex-ogenous DNA It involves coating tiny particles (microprojectiles) with the desired genetic material and then propelling them into the cells using a high-pressure device, such as a gene gun Upon impact, the DNA-coated particles are taken up by the cells, facilitating genetic transformation In this physical method of gene transfer, a gene gun de-vice is used to bombard genes to target cells In this method, the plant cells are placed beneath the gene gun, and the antigen gen bombardment at high pressure penetrates the plant cells [32-35]

3.3 Electroporation Method

This method, as represented in Fig (5), is also

known as the electro-permeabilization method This is the microbiological technique in which an electrical field is applied to the cells to increase their permeability In this method, the plant mate-rial is incubated in a buffer solution containing DNA and subjected to a high voltage electric pulse by which the DNA migrates, and a pore is induced in the plasma membrane These pores carry anti-gen anti-genes to the plant cells This technique is used

for all cereal crops like rice, maize, wheat etc [34,

36]

3.4 Chimeric Virus Method

A chimeric virus is a virus that contains genetic material derived from two or more distinct viruses These plant viruses are genetically modified to

Table 2 A comparison between traditional and edible vaccines is given as follows

i The edible vaccines are the transgenic plants that provide oral immunization against a particular disease The traditional vaccines contain the killed or attenuated form of virus antigen, providing systemic immunization ii It provides both systemic as well as mucosal immunity It provides only systemic immunity

iii It is administered orally and directly stimulates the immune system It cannot directly stimulate the immune system iv No syringe is required for the vaccine administration A syringe is essential for vaccination

v It is easily affordable for the poor people of developing nations It is too expensive, so poor people cannot afford the vaccines vi It doesn't spread any environmental pollution The process of traditional vaccine manufacturing involves environmental

pollution vii Its production of edible vaccines does not produce any toxic

chemical substances Traditional vaccines produce toxic chemicals in plants viii No professional help is required for the vaccine administration Without an experienced doctor, it is impossible to administer the vaccine

ix No sterilization, purification, or processing is required The sterilization, packaging, and storage of traditional vaccines are very expensive

x It is easily stored at room temperature Marinating a cold temperature is required for the storage of vaccines

Edible Vaccines Recent Patents on Biotechnology, XXXX, Vol XX, No XX 7

Fig (3) Plasmid /vector carrier system (A higher resolution / colour version of this figure is available in the electronic copy of

the article)

Fig (4) Micro Projectile bombardment (Biolistic method) (A higher resolution / colour version of this figure is available in

the electronic copy of the article)

Fig (5) Electroporation method (A higher resolution / colour version of this figure is available in the electronic copy of the

article)

carry particular genes and used to infect their natu-ral hosts In these edible plants, the cloned genes expressed themselves to varying degrees in vari-ous edible parts of the host plant Particular viruses can be redesigned to represent fragments of anti-genic proteins on their surfaces, like cauliflower mosaic virus (CaMV), alfalfa mosaic virus, tobac-co mosaic virus, tobac-cowpea mosaic virus, tomato bushy stunt virus, and potato virus [33-34,37] These chimeric viruses can serve as the basis for vaccines or help researchers study virus interac-tions and mechanisms For instance, in the devel-opment of vaccines, the chimeric virus can stimu-late an immune response against a particular path-ogen while lacking the harmful effects of the orig-inal virus

4 EDIBLE VACCINES: CHALLENGES OR CONSTRAINTS

Although the plant expression system has sev-eral applications for human and veterinary vaccine production, only a few vaccine candidates are in clinical trials [6] Commercial human vaccines are unavailable due to low levels of expression, low

efficacy, and a lack of knowledge about the prop-erties of plant-made antigens and production sys-tems The following are some of the challenges or constraints with plant-based vaccines [36, 37]:

• Transgenics Generation Time

• Environmental and Human Health Risks • Acceptance by peoples

Edible vaccines, a groundbreaking concept in immunization, hold immense promise in revolu-tionizing how we prevent infectious diseases In-stead of traditional injections, they are adminis-tered through the consumption of genetically mod-ified plants or fruits, offering the potential for

eas-Edible Vaccines Recent Patents on Biotechnology, XXXX, Vol XX, No XX 9

ier and more accessible vaccination However, while the concept is tantalizing, it is not without its significant challenges and constraints One of the primary challenges faced by edible vaccines is the complex regulatory landscape Developing genet-ically modified organisms (GMOs) for vaccine production requires stringent safety testing to en-sure their suitability for human consumption and minimal environmental impact Regulatory agen-cies worldwide must establish comprehensive guidelines for developing, producing, distributing, and labelling edible vaccines Navigating this reg-ulatory maze is time-consuming and costly, often acting as a significant barrier to progress in this field [38] Stability and dosage control are funda-mental concerns when it comes to edible vaccines The stability of vaccine antigens within the plant or fruit matrix is critical for their efficacy Factors such as environmental conditions, pests, and plant age can influence antigen stability Moreover, en-suring consistent dosage control in every fruit or plant is challenging, potentially leading to variabil-ity in vaccine effectiveness among individuals Allergenicity is another pressing issue associated with edible vaccines Genetically modifying plants to produce vaccine antigens may introduce new proteins that could provoke allergic reactions in some individuals Extensive allergenicity testing is essential to identify and mitigate potential risks, but achieving complete certainty is difficult [39]

Storage and distribution of edible vaccines are more challenging than their traditional counter-parts Traditional vaccines are stored under con-trolled conditions, ensuring their stability and effi-cacy In contrast, edible vaccines are exposed to a range of environmental conditions during storage and transportation Safeguarding the integrity of the vaccine in different climates and logistics sys-tems is a formidable task that needs effective solu-tions Public perception and acceptance represent a substantial constraint for edible vaccines Some individuals harbor concerns about consuming ge-netically modified organisms, and there may be a lack of understanding or trust in the technology Successful adoption of edible vaccines hinges on effective public education and communication to build trust and address misconceptions Cost and scalability are practical constraints that cannot be ignored Developing and producing edible vac-cines can be expensive, particularly during the ear-ly stages Scaling up production to meet global

demand necessitates substantial infrastructure and investment Cross-contamination between genet-ically modified crops and non-GMO crops poses ecological risks that demand robust containment strategies Unintended consequences for ecosys-tems and agriculture must be averted Assessing the long-term safety of consuming edible vaccines is paramount [38-40]

5 PATENTS ON EDIBLE VACCINES

Vaccines have long been essential in preventing the spread of infectious diseases and protecting global health Traditional vaccines are typically administered through injections, requiring trained personnel, cold storage, and proper disposal of needles However, recent advancements in bio-technology have paved the way for a groundbreak-ing concept: edible vaccines This innovative ap-proach involves producing vaccines in genetically modified plants, where the plant tissues become the delivery system This paper explores the con-cept of the production process of edible vaccines and their potential benefits, challenges, and future prospects The production of edible vaccines in-volves isolating the DNA sequence encoding a specific surface antigen from a pathogen This gene is then fused with a plant-specific promoter, ensuring the antigen's expression in transgenic plants Using various techniques like Agrobacte-rium-mediated transformation or biolistic particle bombardment, the modified gene is introduced in-to plant cells, integrating inin-to their genetic materi-al As the plant grows, it synthesizes the desired antigen in its edible parts, such as fruits, leaves, or seeds Once consumed, the plant-based vaccine stimulates the immune system, producing protec-tive antibodies against the targeted pathogen De-spite the promise of edible vaccines, several chal-lenges need to be addressed Regulatory concerns, public acceptance, and the potential for unintended environmental effects are critical issues that re-quire careful consideration Additionally, develop-ing edible vaccines for complex diseases requirdevelop-ing multiple antigen components remains a significant

challenge [41-69] Table 3

6 FUTURE OF EDIBLE VACCINES

The future of edible vaccines depends upon the various types of physical and social factors, some of which are listed below:

Table 3 List of edible vaccine Patent

Year Patent Number

1 Toshiyuki Sasagwa, Hideki Tohda, Yuko Hama Edible vaccine 2009 US 2009/0017063 A1 2 Toshiyuki Sasagwa, Hideki Tohda, Yuko

3

Sayed Sartaj Sohrab, Esam Ibraheem Ahmed Ashar, Sherif Aly Abdelkhalek Elkafrawy, Ayman Talaat Abbas Abdelhad, Edward P Rybicki, Emmanuel Aubrey Margolin,

Development of an edible vaccine 2022 US 2022/0054628 A1

4 Kenneth John Piller, Kenneth Lee Bost Edible vaccines expressed in soybeans 2018 US 10,030,250 B2 5 Dominic Man-Kit LAM, Yuhong XU

Oral vaccines are produced and administered using edible microorganisms, including lactic acid bacterial

strains

2013 US 2013/0004547 A1 6 Heli Salmela, Dalial Freitak Edible vaccination against microbial pathogens 2021 US 2021/0275658 A1

7 Henry Daniell Expression of protective antigens in transgenic

chlo-roplasts and the production of improved vaccines 2014 US 2014/0294895 A1

8 Moshe Flaishman, Avi Pearl, Sara Gol-obowicz Transgenic ficus, a method for producing the same and use thereof 2012 US 8,148,603 B2 9 Takeshi Arakawa, William H.R Langridge Methods and substances for preventing and treating autoimmune disease 2002 AU 750623 B2

10 Dominic Man - Kit Lam, Olivia Yee - Yee Lam, Han Lei

Edible vaccines expressed in yeast for preventing and treating infectious diseases, including hepatitis b, in

humans

2020 US 10,793,866 B2

11 Guy Cardineau, Hugh Mason, Joyce VanEck, Dwayne Kirk, Amanda Walmsley

Vectors and cells for preparing immunoprotective

compositions derived from transgenic plants 2005 US 2005/0048074 A1

12

Yoshikazu Yuki, Hiroshi Kiyono, Takachika Hiroi, Tomonori Nochi, Fumio Takaiwa, Hidenori Takagi, Lijyun Yang, Kazuya

Su-zuki, Hiroyasu Ebinuma, Koichi Sugita, Saori Kasahara

Rice plants having vaccine genes transferred

13 Yoseph Shaaltiel, Einat Almon Mucosal or enteral administration of biologically

active macromolecules 2017 EP2 441 840B1 14 Dionisius Elisabeth Antonius Florack, Hen-drik Jan Bosch Chimeric carrier molecules for the production of mucosal vaccines 2008 US 2008/0286297 A1

15 Fumio Takaiwa, Hidenori Takagi

Method of accumulating allergen-specific T-cell antigen determinant in plant and plant having the

antigen determinant accumulated therein

2007 US 2007/0136896 A1

16 Dominic Man-Kit Lam, Yuhong Xu Immunoprotection by oral administration of

recombi-nant lactococcus lactis mini-capsules 2012 US 2012/0276167 A1

17 Henry Deniell Chloroplasts engineered to express pharmaceutical

proteins in edible plants 2016 EP2 141 981B1 18 Mee Chye, Hong Li, Sathiskumar Ramalin-gam, Leo Poon, Joseph Peiris

Genetically modified plants comprising SARS-CoV viral nucleotide sequences and methods of use thereof

for immunization against SARS

2006 US 2006/0053516A1

(Table 3) Contd…