Vegetables as Edible Vaccines

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. Vegetables as Edible Vaccines Tamil Nadu Agricultural University Horticultural College and Research Institute, Periyakulam Doctoral Seminar 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 1 st 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 I n-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 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

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

14. Mechanism of Edible Vaccine

15. 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 & G othandam , 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

22. 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., 1 991 Heat Shock Method - - + Froger and Hall, 2007

23. 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 e t al ., 1989

24. 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

25. 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

26. 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 CASE STUDY 1

27. 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

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

29. 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 : I ran,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) CASE STUDY 2

30. 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

31. RESULT & CONCLUSION The highest expression (signal) was related to the conjugation of antibody to antigen @ 1/100 dilution M aximum expression of antigens - G reen tomato fruits (Not useful), tomato fruit is consumed at ripe and red state Since gene constructed using CaMV35S - T omato 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

32. 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 P RGSPRgpKDEL gene (64-66 kDa ) CASE STUDY 3 WESTERN BLOT ASSAY

33. 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 T ransgenic 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 ad juvant is effective in controlling Rabies in mice

34. 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 : C ysteine 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 CASE STUDY 4 Fasciola hepatica

35. 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 A mount of vaccine antigen - calculated using ELISA 6 Lambs and calves (3M & 3F) – administered with 500 μ g of freeze dried lettuce leaves orally 2 doses – orally – 4 week interval After 4 weeks – infected with parasite After 12 weeks Slaughtered and examined

36. Results & Conclusion Increased IgG levels - noted in vaccinated animals of both species – Peaked from 6 WPI to 10 WPI F emale animals had higher anti- CPFhW IgG levels when compared to male counterparts C ysteine protease family - C athepsin 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

37. 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

38. 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.

39. 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, O ral 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

40. 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

41. 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.

42. 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

43. 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

44. 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 .  Covishield : Rs.780 Covaxin : Rs.1410 Sputnik : Rs . 1145 MoFW , 2021

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