A new approach for delivering vaccine antigens is the use of inexpensive, oral vaccines. Edible oral vaccines offer exciting possibilities for significantly reducing the burden of diseases like hepatitis and diarrhoea particularly in the developing world where storing and administering vaccines are often major problems. Even though they have some disadvantages like control of the “dosage” of the antigen that is present in the recombinant fruit or vegetable, they have many advantages as they trigger the immunity at the mucosal surfaces which is the body’s first line of defence. To overcome the disadvantage of adequate dosage, stable plant lines that produce fruits and vegetables with relatively constant amounts of the antigen need to be developed. The hope is that edible vaccines could be grown in many of the developing countries where their need is more. The traditional vaccines development requires more time and high cost and due to this, the disease outbreaks becomes more challenging. Now a days, plants have become more attractive platform for edible vaccine production than the other system. The development of an edible vaccine in a selected plant system has many significant advantages such as; easy and efficient oral delivery, low cost with higher scale production, avoidance of any trained medical personnel for delivery, lack of any pathogenic infection, multicomponent expression in a single plant. By using this plant-based platform, an edible vaccines can be produced in many crops like banana, cucumber, carrot, lettuce, and tomato against various diseases. Due to increasing cases glob¬ally with COVID-19, there is an urgent requirement to develop an ideal vaccine and antiviral therapy against this virus to control the disease worldwide.

1. Student
G. Sandeep
II PhD (Horticulture) Vegetable Science

2. Contents
• What is vaccine?
• Prophylactic Vaccine
• Therapeutic Vaccine
• Major outbreaks
• Types of Vaccine
• Molecular Farming
• Properties of ideal vaccine
• Edible Vaccine
• Types of Edible vaccine
• Mechanism of edible vaccine
• Candidate plant
• Vegetables as candidate plant
• Properties of candidate plant
• Production of edible vaccine
• Regulatory aspects
• Cases studies
• Advantages & Disadvantages
• Future thrust

3. What is vaccine?
• Vaccine is biological preparation that provide
active acquired immunity to a particular disease
• Vaccine contains particles resembles disease
causing microorganisms
• These are weakened or killed forms of
microbes (Toxins or surface protein)
• These particles stimulates body’s immune
system – Antigen
Edward Jenner (1796) used vaccine in
human beings – against smallpox

4. Vaccines Can be
• Prophylactic Vaccine
• Also known as Preventive Vaccines
• To prevent the effects of future infection by a pathogen
• Produces antibodies for those antigens
• Eg: Polio, Mumps etc.,
• Therapeutic Vaccine
• Vaccines are used for an individual who is already affected by a disease or
infection
• Therapeutic vaccine fights the existing infection in the body rather than
immunizing the body for future diseases and infections
• Vaccine used against viral infections (Human Papilloma virus) and cancer
thearpy
Shimasakii 2014 & fqsida.org

5. Major outbreaks in the history of human
civilizations

6. Types of Vaccines
• Live Attenuated
• Original and 1st Vaccine
• Weakened form of live infectious organism used as Vaccine
• Eg: Small pox, Measles, Mumps, TB, BCG, etc.
• Inactivated or Killed
• Debris of dead pathogens are used as vaccine
• In-activated using heat or chemicals - destroys replicating ability
• Eg: Rabies, Polio, Hepatitis A, etc. “COVAXIN”
• Toxoid / Inactivated toxin
• Toxins by organism is used as vaccine
• Eg: Tetanus (TT)- Clostridium tetani – Neurotoxin (Tetanospasmin)- inactivated
• Subunit / Conjugate
• A part of target pathogen or gene code for target protein
• Eg: NOVAVAX - COVID, Hepatitis B, Human papilloma-virus
Plotkin et al., 2013

7. • RNA Vaccine
• A piece of Messenger RNA (mRNA) that produce antigenic proteins used as Vaccine
• Eg: COVID 19 mRNA Vaccine
• Biosynthetic Vaccine
• Man made vaccines with similar shape and properties of pathogens
• Eg: Hepatitis B Vaccine
• DNA vaccines
• Plasmid DNA with sequence encoding antigen
• Directly injected into muscle or tissue to get expressed
• Eg: Malaria, Herpes Virus
• Recombinant Vaccine
• Gene encoding antigen is expressed in bacteria or Yeast cells
• Protein is then purified and used as vaccine – “COVISHIELD”
• Eg: 6-in-1 vaccine - UK - 6 serious diseases: diphtheria, tetanus, whooping cough
(pertussis), polio, Hib disease (Haemophilus influenzae type b) and hepatitis B
• Edible Vaccine
• Edible part of plant is genetically modified to express antigens to stimulate immunity
Plotkin et al., 2013

8. Molecular Farming
• Molecular Farming –
experimental
application of
“Biotechnology”
• Genetic modification
of crops (GMO) – to
produce proteins
and Phytochemicals
– Biopharmaceutical
systems - for
commercial purpose
MOLECULAR
FARMING
Nutraceuticals
Therapeutical
Products
Vaccines
Antibodies
Molecular biology, 2016

9. Edible Vaccine
• Edible vaccines involves introduction of desired genes
into plant system to manufacture altered protein
(antigen proteins)
• Antigen proteins are genetically engineered into the crops
that are consumed
• Genes encoding bacterial and viral antigens can be
made to express in plants
• In 1989, Hiatt and co-workers formulated plant vaccine
protocol
• In 1990s Concept of edible vaccine was developed by
Charles Arntzen
Concha et al., 2017
Charles Arntzen

10. History of Edible Vaccines
• Tobacco leaf
• Surface
Antigen of
Hepatitis B
Hepatitis
B (1992)
• Tomato leaf
& fruit
• Rabies Virus
Glycoprotein
Rabies
Virus
(1995)
• Potato tuber
• Norwalk
Virus Capsid
Protein
Norwalk
Virus
(1996)

11. Properties of Ideal Vaccine

12. Why Edible vaccine?
• Needle Free
• Oral vaccine provide “Mucosal Immunity”
• Don’t require sterilization and low risk of
infection
• Cheap
• Production cost can be reduced
• Storage
• Don’t required cold chain maintenance
• Safe
• Activates both mucosal and systemic
immunity
Mason et al., 1992 & Mishra et al., 2008

13. Types of Edible Vaccines
• Plant Based Edible Vaccine
• Eg: Vegetables, fruits, etc.
• Algae Based Edible Vaccine
• Eg: Single Cell Micro-Aglae
• Chlamdomonas reinhardtii
• Dunaliella salina
• Phaeodactylum tricornutum
• Insect Cell based Vaccine
• NOVAVAX – Expressed in Moth Cells of Fall Armyworm by Baculovirus + Adjuvant
soapbark tree extract
• “CERVARIX”- Expressed in Cabbage Looper – VLP - Human Papillomavirus L1 protein
• Whole Cell Yeast Based Edible Vaccine
• Eg: Saccharomyces cerevisiae – HPV, Hepatitis C virus Vaccine
• Lactic Acid Bacteria Based Edible Vaccine
• Eg: Lactobacillus spp & Bacillus subtilis - expressing Helicobacter pylori urease B -ulcer
JAYARAM, 2018

15. Main Goal : To stimulate Mucosal and systemic immunity
Oral intake Edible vaccine – Mastication and degradation occur in
intestine by digestive enzymes
Mucosal Associated Lymphoid Tissue (MALT) – Peyer’s Patch (PP)
component of GALT- enriched source of IgA producing plasma cells
Edible Vaccine breaks at Peyer’s Patch – Follicle site allows antigen
penetration in intestine epithelium – M CELLS ARE PRESENT
Jayaraman et al., 2018 & William, 2000

16. Jayaraman et al ., 2018 & William, 2000

17. Candidate Plant and its properties
Candidate plant : Plant suitable for edible vaccine production
Properties of candidate plant
• Long shelf life : long storage Without degradation
• Faster growth : Plants are preferred than trees
• More biomass
• More protein content
• Easy Transformation
Gunasekaran & Gothandam, 2020

18. Candidate Plants Suitable for Edible vaccine
• Lettuce
• Spinach
• Tomato
• Cucumber
• Muskmelon
• Carrot
• Sweet Corn
• Potato
• Legumes
• Quinoa (Chenopodium)
• Amaranthus
• Banana
• Tobacco
• Rice
Kurup and Thomas, 2021

19. Why Vegetables as Candidate Plant?
• Vegetable has lesser growth cycle
• Transgenic vegetables development is
easy
• Vegetables are hardy and palatable plant -
high nutritive value and protein content
• Most of them can be consumed raw as
salads
• More possibility of developing plants
expressing more than one antigenic
protein

20. Developing Edible Vaccine
• There are 2 ways
• First Case : Entire structural gene is
inserted into plant transformation
vector between 5’ and 3’
• Second Case: Epitope within the
antigen are identified - DNA
fragment encoding these protein is
used to construct

21. Production of Edible Vaccines
• Direct Gene Delivery Method
• Biolistic Method
• Electroporation Method
• Indirect Gene Delivery system
• Agrobacterium Mediated Gene
transfer
• Genetically Engineered Plant
Virus
Eg: CaMV, TMV, etc

23. Other Transformation Techniques
Transformation Method Plant Microagale Bacteria References
Agrobacterium Mediated gene
transfer + + -
Silin et al., 2002
Gene Gun Method + + -
Muynck et al ., 2010
Electroporation + + +
Doshi et al., 2013
Glass Beads method + -
Green et al., 1993
Electrospray - - +
Mozo et al., 1991
Heat Shock Method - - +
Froger and Hall, 2007

24. Current status of plant-based vaccines and therapeutic proteins
Disease Product Plant References
Hepatitis B HBsAg Lettuce, Cherry Tomato Ritrcher et al., 2000
Hepatitis E HEV-E2 Potato Ma et al., 2003
Rabies Rabies Virus GP/NP Spinach Modelska et al., 1998
Gaucher Disease Glucocerebrosidase Carrot sell suspension Sayed et al.,2017
Cholera Cholera Toxin B (CTB) Tomato, Potato Arakawa et al.,1997
Gastroenteritis Tetraspanin proteins Potato tuber & Tomato Lamphang 2005
Norwalk Virus Capsid Protein Potato Zhang et al., 2006
Measles Loop B cell epitope Carrot Yu & Langridge, 2003
HIV HIV1 TAT protein Spinach Karasev et al., 2005
HIV CP24 Protein Carrot Lindh et al., 2009
Human Cytomegalovirus glycoprotein B Beans Yan et al., 2010
Systemic lupus
erythematosus
INF alpha D Turnip Zoeten et al., 1989

25. Regulatory Aspects of Edible Vaccine
• Care Taken – from contamination in food,
medicine or agricultural products
• Ensure to grown in greenhouse or other
structures – to avoid release of
antigenic proteins into environment
• Transgenes may spread by sucking
insects, pollens, soil microbes – pollute
surface and ground water
• Labelling of edible pharmaceutical
plants to preserve their identity, and
avoid the contamination of the food
supply.
Taccket, 2009 & Butelli et al., 2008

26. Gene Transfer into the environment
• Different approaches suggested to stop the flow of gene from
Edible vaccine crops (GM crops) to environment
• Physical Isolation
• Tough and expensive – frequently done
• Crops are grown in isolated areas
• Grown in contained greenhouse conditions
• Genetic Containment
• Achieved through different technological means
• Infertility & incompatibility systems to limit – transfer of pollens
• Genetic Use Restriction Technologies (GURT) – hinder seed formation
• Chloroplast transformation – chloroplast genome inherited maternally –
not in pollen
Chow et al., 2016

27. AIM
• EpCAM (Epithelial Cell Adhesion Molecule) is a cell-surface glycoprotein – expressed high in Colorectal
Carcinoma
• Agrobacterium Mediated Transformation in 2 Plants
• One with genes encoding EpCAM recombinant protein
• Second with J chain with KDEL Endoplasmic Reticulum Retention Motif
• Materials and Methods
• Plant : Chinese Cabbage (B.rapa)
• Proteins : EpCAM (837 bp) with Fragment crystallizable region of IgM (1053 bp) & J chain K (543 bp)
• Place : Korea, 2020

28. • EpCAM – Cancer antigenic Protein – Prevents & Inhibit Cancer
• Fused with Fc (Fragment Crystallizable) region of IgG – enhance protein stability – Induce
“humoral Immunity”
• J Chain - protein component of the antibodies IgM and IgA
• KDEL sequence prevents a protein from being secreted from the endoplasmic reticulum (ER)
• Promotor : CaMV
• Vector : pRCV2 & pCAMBIA 1301

30. • The expected quaternary structure of EpCAM–IgM
Fc X J-chain K in transgenic plant F1 is pentameric
• Conclusion
• Cross-fertilization results revealed that both
transgenes were stably inserted EpCAM–IgM Fc
and J-chain K T1 transgenic plants.
• Transgenic Chinese cabbage expressing EpCAM–
IgM Fc express anti-colorectal cancer IgM Fc
fusion recombinant vaccine candidate proteins

31. AIM
• To Produce Oral Vaccine against Shigellosis, Anthrax and Cholera antigens in tomato tissue
• To Fuse PA20, ipaD and CTxB as gene cassette
Materials and Method
• Place : Iran,2018
• Plant : Tomato
• Gene : PA20, ipaD and CTxB
• Method of Transformation: Agroinfilteration of A.tumifaciens
• Vectors:
• pBI121 (containing extension single peptide and CTxB)
• pET28 (ipaD and PA20)

32. • Agrobacterium Strain GV 3101 was
transformed by Heat shock
• Kanamycin and Rifampin – uses to
select colonies
• Agro - Infiltration was carried out in
2 month old tomato leaves & Red
State Fruit in green house
• Inoculated samples – growth
chamber – 16/8h Dark & Light
condition 5-7 days @ 26 C

33. • RESULT & CONCLUSION
• The highest expression (signal) was related to the conjugation of antibody to
antigen @ 1/100 dilution
• Maximum expression of antigens - Green tomato fruits (Not useful), tomato
fruit is consumed at ripe and red state
• Since gene constructed using CaMV35S - Tomato fruit specific promoter
involved in ethylene biosynthesis can be used to get expressed at ripening
stage
ipaD
PA20
PA20 Control
Data diagram obtained from ELISA reader

34. • Aim
• To produce cost effective Plant based vaccine for Rabies Virus
• Expression of Rabies Virus (PRGSpRgp) glycoprotein in Melon
• Materials & Methods
• Plant: Cantaloupe melon – Cotyledon as explant
• Transformation: Agrobacterium Mediated
• Vector: Agrobacterium tumefaciens pBin19 strain EHA105 with
PRGSPRgpKDEL gene (64-66 kDa)
WESTERN BLOT ASSAY

35. 66 kDA
• Study conducted with 48 Swiss albino rats
• Con A (Concanavalin A) protein extract - Transgenic melon plant
• Intramuscular (0.2ml)
• Intramuscular + Freund’s Adjuvant (0.2ml + 0.1ml)
• Intraperitoneal (0.5 ml)
Result & Conclusion
• Transgenic cantaloupe fruit expressed sufficient levels of rabies
glycoprotein - Neutralizing antibodies in mice.
• No adverse effects were observed in the inoculated mice
• Intramuscular injection with Freund’s adjuvant is effective in
controlling Rabies in mice

36. • Aim
• Fasciolosis - chronic disease – Affecting cattle and sheep - Loss of
approximately 3 billion dollars annually
• Oral vaccination for Fasciolosis against sheep and cow
• Materials and Methods
• Candidate Plant : Lettuce
• Gene : Cysteine proteinase of the trematode Fasciola hepatica
(CPFhW)
• Transformation : Agrobacterium Mediated using Strain LBA 4404
• Parasite : Weybridge Strain of Fasciola hepatica
• Host : Fluke Free- 12 Corriedale lambs and 12 Holstein-Friesian
calves – 5 Month old Fasciola hepatica

37. Vaccine Construct & Transgenic lettuce plant
• cDNA encoding CPFhW cloned into the
pcDNA3.1 to amplify sequence encoding
CPFhW
• HBV 321 plasmid of hepatitis B virus used to
amplify the encoding core protein
• Fused protein HBcAg(T) with CPFhW by
GlyRich Linkers - placed in a pROK2 plant
expression vector of A. tumefaciens LBA 4404
strain
• Lettuce leaves are transformed with
A.tumefaciens
• Amount of vaccine antigen - calculated using
ELISA
• 6 Lambs and calves (3M & 3F) – administered
with 500 μg of freeze dried lettuce leaves
orally
• 2 doses – orally – 4 week interval
• After 4 weeks – infected with parasite
• After 12 weeks Slaughtered and examined

38. Results & Conclusion
• Increased IgG levels - noted in vaccinated animals of both species – Peaked from 6 WPI to 10 WPI
• Female animals had higher anti-CPFhW IgG levels when compared to male counterparts
• Cysteine protease family - Cathepsin L1 - the protease is known to play pivotal roles in liver migration, tissue
feeding and blood digestion
• CPFhW fused with HBcAg is responsible for the enhanced immunogenicity
• Enzyme in vaccine study (CPFhW) - showed reduction in F. hepatica fecundity
• Oral immunization with a plant-made vaccine expressing CPFhW fused to an HBcAg carrier is highly efficient
in controlling fasicolosis
Total IgG levels in sera collected from experimental animals

39. Advantages
• Easy administration.
• Easy transportation.
• Extensive storage facilities like cold storage are not required.
• Heat stable and no need of refrigeration.
• Antigen is protected through bio encapsulation
• Stimulation of both systemic and mucosal immunity.
• Multiple antigens can be delivered – Gene cassette technique
• Cheap

40. Disadvantages
• Stability of fruit vaccine in fruit is not known.
• Evaluating dosage requirement is tedious.
• Chances of food allergic reactions due to the presence of antigens
• Selection of specific plant for specific gene is difficult.
• Certain foods like potato are not eaten raw and cooking the food might
weaken the medicine present in it.

41. Edible Vaccine Dosage
• Right Dosage – Person weight & Age ; Plants size & Protein content is considered
• Foreign proteins in plants - accumulate at low amounts (0.01–2 %) - less immunogenic
• So, Oral dose far exceeds the intranasal/parenteral dose
• Low doses fail to induce immunity
For example
• Oral hepatitis B dose require 10 – 100 times more than parenteral dose
• 100 g potato expressing B subunit of labile toxin of ETEC (LT-B) requires in 3 different
doses to be immunogenic.
• Attempts to boost amount of antigens cause stunted growth of plants and reduced tuber/fruit
formation
• Result: More mRNA from the transgene causes gene silencing in plant genome.
Plant biotechnology, Umesha, 2019

42. Steps to Over Come Edible Vaccine Dosage Problem
• Optimization of coding sequence of bacterial/ viral gene for
expression
• Plant virus expressing foreign gene
• Expression in plastids
• Coat protein fusion
• Promoter elements with reporter genes
Plant biotechnology, Umesha, 2019

43. ELELYSO™ (TALIGLUCERASE ALFA)
Elelyso (Taliglucerase alfa)
The US Food and Drug
Administration (USFDA) Approved
therapeutic enzyme based vaccine
produced from genetically engineered
carrot Cells - Treat type 1 Gaucher’s
disease in 2012
Disease cause fatty substances to
build up in the bone marrow, liver and
spleen.
A rare genetic disorder in which
individuals fail to produce the enzyme
glucocerebrosidase
A recombinant form of human
Glucocerebrosidase made to express in
transgenic carrot cells.

44. Edible Cholera vaccine made of powdered
rice proves safe in phase 1 human trials
University of Tokyo and Chiba University
DR. HIROSHI KIYONO
For the study, 30 volunteers received a
placebo and groups of 10 volunteers received a total of
four doses spaced every two weeks of either 3
milligrams (mg), 6 mg or 18 mg each of the vaccine.
NEWS RELEASE 25-JUN-2021

45. • US Researchers Are Engineering Lettuce and Spinach to Carry mRNA
COVID Jabs
• Spinach and lettuce are being genetically engineered with COVID-
19 mRNA vaccines
• mRNA, a molecule contained in the Pfizer-BioNTech and
Moderna COVID-19 vaccines that is normally used by our cells
to make protein
• The mRNA in the vaccine teaches your cells how to make
copies of the spike protein.
• Genetic material contained in mRNA vaccines will be inserted
into small, disk-like structures within plant cells
called chloroplasts
• Ideally, a single plant would produce enough mRNA to
vaccinate a single person

46. Future Aspects
• Farmers have widely adopted GM technology –
increased from 1.7 million hectares in 1996 to
191.7 million hectares in 2018, 113 fold increase
• Future edible vaccine against smallpox, anthrax, plague, etc. can be produced
on a large scale within a short span of time
• Edible vaccines can be produced at large quantity with low cost
• New vaccine production systems using rDNA or mRNA technologies -
emerging diseases - COVID-19, MERS-CoV, Avian influenza, Ebola, Zika and
possible future infections.
MoFW, 2021