Seminar titleEdible vaccines

Masume jahedM.Sc student of Agricultural

Biotechnology

Shahid beheshti university

Guided by Dr. hossein askari

Molecular farming

In putIsolation of gene of interest

Expression hosts for molecular farming

BacteriaYeast Mamalian cell cultureTransgenic Plants plant cells culture

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(Spök et al., 2008)

Out put

Vaccines antigens Therapeutics products Nutritional components Industrial products Bio plastics

Plant Molecular farming

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(Sharma ansd Sharma 2009)

What is 'molecular farming in plants'?

Advantages : Plant as Bioreactor

Significantly lower production costs than with transgenic animals, fermentation or bioreactors

Plants do not contain known human pathogens (such as virions, etc.) That could contaminate the final product.

(Sahu et al., 2014)4

low cost of production low cost of production

High safty High safty

ability to fold and assemble eucaryote complex proteins accurately ability to fold and assemble eucaryote complex proteins accurately

Rapid scalability Rapid scalability

Tyes of vaccines

Different types of currently available vaccines

Conventional vaccine Live attenuated vaccines Killed vaccines purified subunit vaccines (inactivated toxin)

Costly Longer time is needed for it to be produced Heat sensitive Prone to microbial contamination

recombinant subunit vaccineAdvances in molecular biology techniques during the 1980s, helped in the development of new strategies for the production of subunit vaccines.

Vaccines produced by biotechnological method are stable at room temperature, unlike traditional vaccine which needs cold chain storage which increases the yearly cost to preserve.

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History of Edible vaccines

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In 1990, Streptococcus mutans surface protein A was expressed in transgenic tobacco and given to mice. learning of a World Health Organization call for inexpensive oral vaccines that needed no refrigeration.

(Langridge, 2000)

In the early 1990s Charles J. Arntzen, then at Texas A&M University, conceived of a way to solve many of the problems that bar vaccines from reaching all too many children in developing nations.

History of Ecdible vaccines

Mason et al 1992 Haq et al 1995 McGarvey et al 1995 Mason et al 1996 Hein et al 1996 ….

Hepatitis

Hepatitis B surface antigen (HBsAg)

Tobacco/leaf

Norwalk virus (NV)

Gastroenteritis Norwalk virus capsid protein (NVCP)

Potato/tubertobacco/leaf

Rabies virus

Rabies

Rabies virus glycoprotein(RVG)

Tomato/leaf, fruit

V. Cholerae

Cholera

Cholera toxin B subunit (CTB)

Tobacco/leaf

E. Coli

Diarrhea

Heat labile toxin B subunit (LTB)

Potato/tuber, tobacco/leaf

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Production of Edible vaccine

1) stable nuclear transformation of a crop species that are grown in the field or a greenhouse

2) Stable plastid transformation of a crop species

3) transient transformation of a crop species

4) stable transformation of a plant species that is grown hydroponically or in in vitro systems

stable nuclear transformation of a crop species that are grown in the field or a greenhouse

Molecular analysis of T0 transgenic plants

expression cassette

Molecular analysis of T1,

T2, … generations of

transgenic plants

Putative transgenic plants

9transgenic plants

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Stable plastid transformation of a crop species

Advantages

Chloroplast system, represents the highest recombinant protein accumulation achieved so far in plants. plastids are inherited through maternal tissues in most species and the pollen does not contain chloroplasts, hence the transgene may not be transferable, thereby allaying public concerns.

Disadvantages

It is also envisaged that protein stability will change over time even with refrigeration.

(Meyers et al., 2010; Horn et al., 2004)

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transient transformation of a crop species

(Thomas et al., 2002)

Stable transformation of a plant species that is grown hydroponically or in in vitro systems

agroinfiltration (Agrobacterium-mediated delivery)

genetically modified plant viral vectors

Clinical trials

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Protein purification

Oral administration

Injected routes

(Daniell et al., 2009)

Application of Edible vaccines

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1) infectious diseaseAIDSMeaslesSmallpoxTransmissible gastroenteritis TuberculosisInfuenzaAnthraxTetanusSevere Acute Respiratory Syndrome (Sars)Human PapillomavirusCysticercosisPlagueFoot and mouth diseaseCentral nervous system diseaseNewcastle diseaseRabbit hemorrhagic syndromesCanine parvovirus diseaseBluetongue

Preventing :2) Autoimmune disease

Type-I Diabetes multiple sclerosis 

3) Cancer therapy Colon cancer Cervical cancer

(Tiwari et al., 2009; Hefferon, 2010; Sharma et al., 2004)

Features of different plant host systems

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(Saxena and Shweta Rawat, 2014)

Advantages Disadvantages

Tomato Relatively efficient transformation system fruit is edible raw

Relatively low fruit protein content; acidic fruit may be incompatible with some antigens or for delivery to 

infants

Tobaco Facile and efficient transformation system

Toxic alkaloids incompatible with oral delivery

Poatato Industrial tuber processing well established

unpalatablein raw form; cooking might cause denaturation

and poor immunogenicity of vaccine

Banana Cultivated widely in developing countries where vaccines are needed

Inefficient  transformation  system

Advantages of Edible vaccines

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  do not require administration by injection  do not require separation and purification of vaccines from plant materials  necessary syringe & needles not required                                                             Low cost  Eliminate trained medical personnel

  Enhance compliance in children  contamination risk is low as sterile conditions are not      and  Induction of both mucosul and systemic immunization

Induction of mucosul immunization

16(Mishra et al., 2008)

Plant cell protect the antigent against enzymatic attack

o Plant cell degradation by digestive/ bacterial enzyme in intestineo Antigens released near the Intestinal lining over Peyer’s patcheso Stimulate follicles develop germinal centreo Taken up by M cellso Penetrates the intestinal epitheliumo Accumulates within organized lymphoid structureo M cells express class II MHCo transported across the mucous membrane by M cellso Activate B cells within lymphoid follicleso Migration to MALTo Differentiate into Plasma cells Ig A production in plasma cells and memory cells.oTransporte to Lumeno Interacte with antigeno Neutralize the infectious agent 

limitation, Concerns and Safety Aspects of Edible Vaccine

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 Selection of best plant is difficult           Some food cannot be eaten raw (e.g. potato) and needs cooking which will denature or weaken the protein present in it. Consistency of dosage form fruit to fruit, plant-to-plant, and generation-to-generation is not similar             Dosage of vaccines would be variable           Possibility of development of immunotolerance  to  the  vaccine  protein  or  peptide.  Standardization  of  dosage  in  case  of edible vaccine, is very important. Possible allergic reaction to plant componant is yet another remote concern.

From Seeds, freeze-dried fruits and leaves:  powder form vaccines are produced. Recent Development : “Edible Vaccines” now more popular as “Plant Vaccines”

  Not convenient for infants.

  The  potential  gene  flow  to  weeds  or  related  crops  through  pollination  or  seed contamination              there are  issues about PDMs accidentally entering  the  food chain and being consumed by non-target organisms.

Conclusions

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www.who.int/ Global Immunization Data

  an  estimated  21.8  million  infants  worldwide  are  still  not  being  reached  by  routine immunization services. Inadequate supply of vaccines, lack of access to health workers, and insufficient political and  financial  support  account  for  a  large  proportion  of  people  who  start  but  don’t  finish national immunization schedules.

future prospects

There are several technical and logistic problems which need to be addressed before plant derived edible vaccine becomes a reality )Tiwari et al., 2009).

The optimal dosage required to produce immunity and duration of the immune response Distribution and marketing issue Examining issues related to commercialization Issues relating to the ethical, social, biosafety and environmental impact.

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References

1- Floss, D. M., Falkenburg, D., & Conrad, U. (2007). Production of vaccines and therapeutic antibodies for veterinary applications in transgenic plants: an overview. Transgenic Research, 16(3), 315-332. 2- Sahu, P. K., Patel, T. S., Sahu, P., Singh, S., Tirkey, P., & Sharma, D. (2014). Molecular Farming : A biotechnological approach in agriculture for production of useful metabolites. International Journal of Research in Biotechnology and Biochemistry, 4(2): 23-30.3- Daniell, H., Singh, N. D., Mason, H., & Streatfield, S. J. (2009). Plant-made vaccine antigens and biopharmaceuticals. Trends in plant science, 14(12), 669-679.4- Lal, P., Ramachandran, V. G., Goyal, R., & Sharma, R. (2007). Edible vaccines: current status and future. Indian journal of medical microbiology, 25(2), 93.5- Tiwari, S., Verma, P. C., Singh, P. K., & Tuli, R. (2009). Plants as bioreactors for the production of vaccine antigens. Biotechnology advances, 27(4), 449-467.6- Mason, H. S., Lam, D. M., & Arntzen, C. J. (1992). Expression of hepatitis B surface antigen in transgenic plants. Proceedings of the National Academy of Sciences, 89(24), 11745-11749.7- Streatfield, S. J., & Howard, J. A. (2003). Plant-based vaccines. International journal for parasitology, 33(5), 479-493.

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References

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8- Saxena, J., & Rawat, S. (2014). Edible Vaccines. In Advances in Biotechnology (pp. 207-226). Springer India.9- Shah, C. P., Trivedi, M. N., Vachhani, U. D., & Joshi, V. J. (1990). Edible Vaccine: A Better Way for Immunization. pharmaceuticals, 4, 5.10- Sabalza, M., Christou, P., & Capell, T. (2014). Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks. Biotechnology letters, 36(12), 2367-2379.11- Obembe, O. O., Popoola, J. O., Leelavathi, S., & Reddy, S. V. (2011). Advances in plant molecular farming. Biotechnology advances, 29(2), 210-222.12- Naithani, M., Viswanath, D., & Urs, P. (2014). Edible vaccines-a review. International Journal of Pharmacotherapy, 4(1), 58-61.13- Fahad, S., Khan, F. A., Pandupuspitasari, N. S., Ahmed, M. M., Liao, Y. C., Waheed, M. T., ... & Huang, J. (2014). Recent developments in therapeutic protein expression technologies in plants. Biotechnology letters, 1-15.14- Spök, A., Karner, S., Stein, A. J., & Rodríguez-Cerezo, E. (2008). Plant molecular farming. Opportunities and challenges. JRC Scientific and Technical Reports.

References

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15- Hefferon, K. (2010). Clinical trials fuel the promise of plant-derived vaccines. Am J Clin Med, 7, 30-7.16- Langridge, William HR. (2000). "Edible vaccines.“ Scientific american-american edition- 283.3 : 48-53.17- Sharma, A. K., & Sharma, M. K. (2009). Plants as bioreactors: Recent developments and emerging opportunities. Biotechnology advances, 27(6), 811-832.