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The recombinant vaccinal proteins can provide a solution for prevention of the contagious diseases, especially in deprived countries. While using eukaryotic platforms such as E. coli lack the post-translational modifications for expression of the proteins and using mammalian cells and plant cells encounter a labor-intensive and continuous process of cell culture and the final product requires special transportation logistics, the plant seeds can provide a good solution for expression of the vaccinal proteins as an oral vaccine. Seeds contain a low amount of water content and can show a considerable bioencapsulation to protect the recombinant products from environmental degradation. However, for the expression of a recombinant protein, there are steps and considerations that should be considered. These considerations include the choice of an appropriate seed to be used as an expression platform (bioreactor), the choice of the promoter, adjustment of the codon preference, targeting the protein for in the cell and finally purification of the vaccinal protein in case of need. This article aims to describe the details of the recombinant vaccine production in the seed platform with the focus on oral delivery of the vaccine at a glance.

Recombinant vaccine, oral delivery system, transgenic seeds.

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Arntzen C. Plant-derived vaccines and antibodies: Potential and limitation. Vaccine. 2005;23:1753–6.

Streatfield SJ, Howard JA. Plant production systems for vaccines; 2003.

Streatfield SJ, Howard JA. Plant-based vaccines. International Journal for Parasitology. 2003;33(5):479-493.

Chen M, Liu X, Wang Z, Song J, Qi Q, Wang PG. Modification of plant N‐glycans processing: The future of producing therapeutic protein by transgenic plants. Medicinal Research Reviews. 2005;25(3): 343-360.

Tiwari S, Verma PC, Singh PK, Tuli R. Plants as bioreactors for the production of vaccine antigens. Biotechnology Advances. 2009;27(4):449-67.
DOI: 10.1016/j.biotechadv.2009.03.006

Merlin M, Pezzotti M, Avesani L. Edible plants for oral delivery of biophar-maceuticals. British Journal of Clinical Pharmacology. 2017;83(1):71-81.

Lau OS, Sun SS. Plant seeds as bioreactors for recombinant protein production. Biotechnol Adv. 2009;27(6):1015-22.
DOI: 10.1016/j.biotechadv.2009.05.005

Hegedus DD, et al. A strategy for targeting recombinant proteins to protein storage vacuoles by fusion to Brassica napus napin in napin-depleted seeds. Protein Expression and Purification. 2014;95:162-168.

Sun SS. Application of agricultural biotechnology to improve food nutrition and healthcare products. Asia Pac J Clin Nutr. 2008;17(S1):87-90.

Basaran P, Rodríguez-Cerezo E. Plant molecular farming: Opportunities and challenges. Critical Reviews in Biotechno-logy. 2008;28(3):153-172.

Fukuda K, et al. Efficacy of oral immunotherapy with a rice-based edible vaccine containing hypoallergenic Japanese cedar pollen allergens for treatment of established allergic conjunctivitis in mice. Allergology International. 2018;67(1):119-123.

Nochi T, et al. A rice-based oral cholera vaccine induces macaque-specific systemic neutralizing antibodies but does not influence pre-existing intestinal immunity. The Journal of Immunology. 2009;183(10): 6538-6544.

Iizuka M, et al. Prophylactic effect of the oral administration of transgenic rice seeds containing altered peptide ligands of type II collagen on rheumatoid arthritis. Bioscience, Biotechnology and Biochemistry. 2014;78(10):1662-8.
DOI: 10.1080/09168451.2014.936349

Yuki Y, et al. Differential analyses of major allergen proteins in wild-type rice and rice producing a fragment of anti-rotavirus antibody. Regulatory Toxicology and Pharmacology. 2016;76:128-136.

Shojaei Jeshvaghani F, et al. Oral immunization with a plant-derived chimeric protein in mice: Toward the development of a multipotent edible vaccine against E. coli O157: H7 and ETEC. Immunobiology. 2019;224(2):262-269.
DOI: 10.1016/j.imbio.2018.12.001

Hayden CA, et al. Oral delivery of wafers made from HBsAg-expressing maize germ induces long-term immunological systemic and mucosal responses. Vaccine. 2015; 33(25):2881-2886.

Feng H, et al. Oral administration of a seed-based bivalent rotavirus vaccine containing VP6 and NSP4 induces specific immune responses in mice. (in eng), Front Plant Sci. 2017;8:910-910.
DOI: 10.3389/fpls.2017.00910

Lau OS, NG DWK, Chan WWL, Chang SP, Sun SSM. Production of the 42-kDa fragment of Plasmodium falciparum merozoite surface protein 1, a leading malaria vaccine antigen, in Arabidopsis thaliana seeds. Plant Biotechnology Journal. 2010;8(9):994-1004.
DOI: 10.1111/j.1467-7652.2010.00526.x

Moravec T, Schmidt MA, Herman EM, Woodford-Thomas T. Production of Escherichia coli heat labile toxin (LT) B subunit in soybean seed and analysis of its immunogenicity as an oral vaccine. Vaccine. 2007;25(9):1647-1657.

Jonsdottir S, Svansson V, Stefansdottir SB, Mäntylä E, Marti E, Torsteinsdottir S. Oral administration of transgenic barley expressing a Culicoides allergen induces specific antibody response. Equine Veterinary Journal. 2017;49(4):512-518.
DOI: 10.1111/evj.12655

Joensuu JJ, et al. Glycosylated F4 (K88) fimbrial adhesin FaeG expressed in barley endosperm induces ETEC-neutralizing antibodies in mice. Transgenic Research. 2006;15(3):359.

Zimmermann J, et al. Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens. (in eng), BMC Biotechnol. 2009;9:79-79.

DOI: 10.1186/1472-6750-9-79

Ramessar K, Capell T, Christou P. Molecular pharming in cereal crops. Phytochemistry Reviews. 2008;7(3):579-592.

Ma JK, Drake PM, Christou P. The production of recombinant pharmaceutical proteins in plants. Nature Reviews Genetics. 2003;4(10):794-805.

Hood EE, et al. Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification. Molecular Breeding. 1997;3(4):291-306.

Hayden CA, et al. Bioencapsulation of the hepatitis B surface antigen and its use as an effective oral immunogen. Vaccine. 2012; 30(19):2937-2942.

Hayden CA, et al. Supercritical fluid extraction provides an enhancement to the immune response for orally-delivered hepatitis B surface antigen. Vaccine. 2014;32(11):1240-1246.

Spök A. Molecular farming on the rise–GMO regulators still walking a tightrope. Trends in Biotechnology. 2007;25(2):74-82.

Yang L, Wakasa Y, Takaiwa F. Biopharming to increase bioactive peptides in rice seed. AOAC Int. 2008;91:957-964.

Takagi H, et al. A rice-based edible vaccine expressing multiple T cell epitopes induces oral tolerance for inhibition of Th2-mediated IgE responses. Proceedings of the National Academy of Sciences of the United States of America. 2005;102:48: 17525-17530.
DOI: 10.1073/pnas.0503428102

Takaiwa F. A rice-based edible vaccine expressing multiple T-cell epitopes to induce oral tolerance and inhibit allergy. Immunology and Allergy Clinics of North America. 2007;27(1):129-139.

Yang L, Tada Y, Yamamoto MP, Zhao H, Yoshikawa M, Takaiwa F. A transgenic rice seed accumulating an anti-hypertensive peptide reduces the blood pressure of spontaneously hypertensive rats. FEBS Letters. 2006;580(13):3315-3320.

Xie T, et al. A biologically active rhIGF-1 fusion accumulated in transgenic rice seeds can reduce blood glucose in diabetic mice via oral delivery. Peptides. 2008;29(11): 1862-1870.

Takeyama N, et al. Oral rice-based vaccine induces passive and active immunity against enterotoxigenic E. coli-mediated diarrhea in pigs. Vaccine. 2015;33(39): 5204-5211.

T. U. O. T. Institute of Medical Science. MucoRice CTB - Astellas/The Institute of Medical Science/The University of Tokyo [Online].

Nochi T, et al. Rice-based mucosal vaccine as a global strategy for cold-chain-and needle-free vaccination. Proceedings of the National Academy of Sciences. 2007; 104(26):10986-10991.

Bruchmüller A, et al. Expression of influenza A (H5N1) vaccine in barley grains for oral bird immunization. Journal Für Verbraucherschutz und Lebensmittelsicherheit. 2007;2:118-118.

Okay S, Sezgin M. Transgenic plants for the production of immunogenic proteins. AIMS Bioengineering. 2018;5(3).

Stöger E, et al. Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Molecular Biology. 2000;42(4):583-590.

Rasheed A, Xia X, Yan Y, Appels R, Mahmood T, He Z. Wheat seed storage proteins: Advances in molecular genetics, diversity and breeding applications. Journal of Cereal Science. 2014;60(1):11-24.

Stöger E, Parker M, Christou P, Casey R. Pea legumin overexpressed in wheat endosperm assembles into an ordered paracrystalline matrix. Plant Physiology. 2001;125(4):1732-1742.

Liew PS, Hair-Bejo M. Farming of plant-based veterinary vaccines and their applications for disease prevention in animals. Advances in Virology; 2015.

Yonesi M, Kamrani A. Oral vaccines and bioreactors: Physical and chemical determination of Balagu seeds for bioreactor and medical usage; 2017.

Yonesi M, Kamrani A. An efficient isolation plan for extraction of the mucilage collected from the seeds within the lallemantia for pharmaceutical purpose. Plant Cell Biotechnology and Molecular Biology. 2019;294-304.

Dean GH, et al. Identification of a seed coat-specific promoter fragment from the Arabidopsis Mucilage-MODIFIED4 gene. Plant Molecular Biology. 2017;95(1-2):33-50.

Loos A, et al. Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis. Plant Physiol. 2011;155(4):2036-2048.

Rosales-Mendoza S, Salazar-Gonzalez JA. Immunological aspects of using plant cells as delivery vehicles for oral vaccines. Expert Review of Vaccines. 2014;13(6): 737-749.

Juarez P, Virdi V, Depicker A, Orzaez D. Biomanufacturing of protective antibodies and other therapeutics in edible plant tissues for oral applications. Plant Biotechnology Journal. 2016;14(9):1791-1799.

Appaiahgari MB, Kiran U, Ali A, Vrati S, Abdin MZ. Plant-based edible vaccines: Issues and advantages. In Plant Biotechno-logy: Principles and Applications: Springer. 2017;329-366.

Zavallo D, Bilbao ML, Hopp HE, Heinz R. Isolation and functional characterization of two novel seed-specific promoters from sunflower (Helianthus annuus L.). Plant Cell Reports. 2010;29(3):239-248.

Koia J, Moyle R, Hendry C, Lim L, Botella JR. Pineapple translation factor SUI1 and ribosomal protein L36 promoters drive constitutive transgene expression patterns in Arabidopsis thaliana. Plant Molecular Biology. 2013;81(4-5):327-336.

Hudson LC, Garg R, Bost KL, Piller KJ. Soybean seeds: A practical host for the production of functional subunit vaccines. BioMed Research International; 2014.

El-Mezawy A, Wu L, Shah S. A seed coat-specific promoter for canola. Biotechnology Letters. 2009;31(12):1961-1965.

Wu L, El-Mezawy A, Shah S. A seed coat outer integument-specific promoter for Brassica napus. Plant Cell Reports. 2011;30(1):75-80.

Bang SW, et al. Characterization of the stress-inducible OsNCED3 promoter in different transgenic rice organs and over three homozygous generations. Planta. 2013;237(1):211-224.

Xu R, et al. Isolation of four rice seed-specific promoters and evaluation of endosperm activity. Plant Cell, Tissue and Organ Culture (PCTOC). 2017;128(1):125-132.

Yi N, et al. Functional analysis of six drought-inducible promoters in transgenic rice plants throughout all stages of plant growth. Planta. 2010;232(3):743-754.

Joshi JB, et al. A maize α-zein promoter drives an endosperm-specific expression of transgene in rice. (in eng). Physiol Mol Biol Plants. 2015;21(1):35-42.
DOI: 10.1007/s12298-014-0268-9

Coussens G, et al. Brachypodium distachyon promoters as efficient building blocks for transgenic research in maize. Journal of Experimental Botany. 2012; 63(11):4263-4273.

Streatfield SJ, et al. Identification of maize embryo-preferred promoters suitable for high-level heterologous protein production. GM Crops. 2010;1(3):162-172.

Li C, Yue Y, Chen H, Qi W, Song R.The ZmbZIP22 transcription factor regulates 27-kD γ-Zein gene transcription during maize endosperm development. (in eng), The Plant Cell. 2018;30(10):2402-2424.
DOI: 10.1105/tpc.18.00422

Kawakatsu T, Takaiwa F. 4 - Rice proteins and essential amino acids. in Rice (Fourth Edition). J. Bao Ed.: AACC International Press. 2019;109-130.

Takaiwa F. Update on the use of transgenic rice seeds in oral immunotherapy. Immuno-therapy. 2013;5(3):301-312.

Sriraman R, et al. Recombinant anti‐hCG antibodies retained in the endoplasmic reticulum of transformed plants lack core‐xylose and core‐α (1, 3)‐fucose residues. Plant Biotechnology Journal. 2004;2(4):279-287.

Boothe J, et al. Seed‐based expression systems for plant molecular farming. Plant Biotechnology Journal. 2010;8(5):588-606.

De Jaeger G, et al. Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences. Nature Biotechnology. 2002;20(12):1265.

Guerche P, De Almeida ER, Schwarztein MA, Gander E, Krebbers E, Pelletier G. Expression of the 2S albumin from Bertholletia excelsa in Brassica napus. Molecular and General Genetics MGG. 1990;221(3):306-314.

BäUmlein H, et al. A novel seed protein gene from Vicia faba is developmentally regulated in transgenic tobacco and Arabidopsis plants. Molecular and General Genetics MGG. 1991;225(3):459-467.

Bäumlein H, Wobus U, Pustell J, Kafatos FC. The legumin gene family: Structure of a B type gene of Vicia faba and a possible legumin gene specific regulatory element. Nucleic Acids Research. 1986;14(6):2707-2720.

Hoffman LM, Donaldson DD, Bookland R, Rashka K, Herman EM. Synthesis and protein body deposition of maize 15‐kd zein in transgenic tobacco seeds. The EMBO Journal. 1987;6(11):3213-3221.

Al-Babili S, Hoa TTC, Schaub P. Exploring the potential of the bacterial carotene desaturase CrtI to increase the β-carotene content in golden rice. Journal of Experimental Botany. 2006;57(4):1007-1014.

Horvath H, et al. The production of recombinant proteins in transgenic barley grains. Proceedings of the National Academy of Sciences. 2000;97(4):1914-1919.

Marin Viegas VS, Ocampo CG, Petruccelli S. Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields. (in eng), Bioengineered. 2017;8(3):203-211.
DOI: 10.1080/21655979.2016.1222994

Chikwamba RK, Scott MP, Mejía LB, Mason HS, Wang K. Localization of a bacterial protein in starch granules of transgenic maize kernels. Proceedings of the National Academy of Sciences. 2003; 100(19):11127.
DOI: 10.1073/pnas.1836901100

Drakakaki G, et al. The intracellular fate of a recombinant protein is tissue dependent. (in eng), Plant Physiol. 2006;141(2):578-586.
DOI: 10.1104/pp.106.076661

Niu C, et al. N-glycosylation improves the pepsin resistance of histidine acid phosphatase phytases by enhancing their stability at acidic pHs and reducing pepsin's accessibility to its cleavage sites. Appl. Environ. Microbiol. 2016;82(4):1004-1014.

Khan I, Twyman RM, Arcalis E, Stoger E. Using storage organelles for the accumulation and encapsulation of recombinant proteins. Biotechnology Journal. 2012;7(9):1099-1108.
DOI: 10.1002/biot.201100089

Hills MJ, Watson MD, Murphy DJ. Targeting of oleosins to the oil bodies of oilseed rape (Brassica napus L.). Planta. 1993;189(1):24-29.

Müntz K. Deposition of storage proteins. In Protein Trafficking in Plant Cells: Springer. 1998;77-99.

Jauh GY, Fischer AM, Grimes HD, Ryan CA, Rogers JC. δ-tonoplast intrinsic protein defines unique plant vacuole functions. Proceedings of the National Academy of Sciences. 1998;95(22):12995-12999.

Jiang L, et al. The protein storage vacuole: A unique compound organelle. J Cell Biol. 2001;155(6):991-1002.

Frigerio L, de Virgilio M, Prada A, Faoro F, Vitale A. Sorting of phaseolin to the vacuole is saturable and requires a short C-terminal peptide. The Plant Cell. 1998; 10(6):1031-1042.

Nishizawa K, Maruyama N, Satoh R, Fuchikami Y, Higasa T, Utsumi S. AC‐terminal sequence of soybean β‐conglycinin α′ subunit acts as a vacuolar sorting determinant in seed cells. The Plant Journal. 2003;34(5):647-659.

Bednarek SY, Raikhel NV. The barley lectin carboxyl-terminal propeptide is a vacuolar protein sorting determinant in plants. The Plant Cell. 1991;3(11):1195-1206.

Lahm T, Yakubov B, Mangu V, Park J, Daniell H. Oral delivery of angiotensin converting enzyme 2 and angiotensin-(1-7) bioencapsulated in lettuce cells attenuates experimental pulmonary hypertension (PH). Circulation. 2018;138(Suppl-1):A16984-A16984.

Takaiwa F, Wakasa Y, Hayashi S, Kawakatsu T. An overview on the strategies to exploit rice endosperm as production platform for biopharmaceuticals. Plant Science. 2017;263:201-209.

Sun YX. Immunological adjuvant effect of a water-soluble polysaccharide, CPP, from the roots of Codonopsis pilosula on the immune responses to ovalbumin in mice. Chemistry & Biodiversity. 2009;6(6):890-896.
DOI: 10.1002/cbdv.200800154

Moeller L, Taylor‐Vokes R, Fox S, Gan Q, Johnson L, Wang K. Wet‐milling transgenic maize seed for fraction enrichment of recombinant subunit vaccine. Biotechnology Progress. 2010;26(2):458-465.

Paraman I, Moeller L, Scott MP, Wang K, Glatz CE, Johnson LA. Utilizing protein-lean coproducts from corn containing recombinant pharmaceutical proteins for ethanol production. Journal of Agricultural and Food Chemistry. 2010;58(19):10419-10425.

Paraman I, Fox SR, Aspelund MT, Glatz CE, Johnson LA. Recovering corn germ enriched in recombinant protein by wet-fractionation. Bioresource Technology. 2010;101(1):239-244.

Zhang C, Glatz CE, Fox SR, Johnson LA. Fractionation of transgenic corn seed by dry and wet milling to recover recombinant collagen-related proteins. Biotechnology Progress. 2009;25(5):1396-1401.
DOI: 10.1002/btpr.220

Zhang D, et al. Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.). Protein Expression and Purification. 2010; 74(1):69-79.

Wilken LR, Nikolov ZL. Recovery and purification of plant-made recombinant proteins. Biotechnology Advances. 2012; 30(2):419-433.