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Production of sub unit vaccines in plant systems is increasing day by day due to many reasons. Traditional methods for production of vaccines are costly, time consuming and insufficient for rapidly spreading pathogenesis. Recombinant proteins expressed by transgenic plants are economical and convenient vehicles that produce protective antigens, abbreviated as PAs. They are preferred over traditional means of production of vaccines as they have little or no chance of contamination with animal pathogens, they are heat stable and plant engineering could lead to production of multiple vaccines using a single plant system. The production of vaccines using plant systems is cost effective, as it has low raw material cost and the capacity to scale up is rapid. Edible vaccines are produced that eliminate needle-associated risks and diminish pain and uneasiness caused thereby . Nuclear transformation is one way by which sub unit vaccines are being produced in plant systems. The gene construct is built and gene of interest is integrated into plant genome. This technique shows potential because many plants are compliant to transformations. Protein accumulation in tissues could be specified too, making the technique versatile. Expression of proteins is increased by targeting the protein to specific subcellular compartments or organalles, leading to increased expression and creating ease for post-translational modifications (such as glycosylation) (M. Manuela Rigano, 2009).
Chloroplast transformation is a technique wherein the target genes are incorporated into plastid genomes. Chloroplasts express a high copy number in each plants cell. This leads to expression of large amount of proteins in plants. The expression rate is uniform and it confers no gene silencing, making it preferable over nuclear transformation. Recent studies show that post-translational modifications as lipidation, protein oligomerization and disulphide bond formation can occur in chloroplasts too (M. Manuela Rigano, 2009).
Another approach to produce vaccine antigens in large quantities is transient expression of gene of interest in plants using hybrid vectors. Plant RNA viral vectors and Agrobacterium binary plasmids are developed that produce uniform high levels of target gene expression and rapid scale up (M. Manuela Rigano, 2009). Agrobacterium is a plant pathogen that infects plants. During the process of infection it transfers a segment of its DNA, T-DNA into plant genome. This process is being used to deliver desired gene(s) into plant genome. During tissue culture the transformed cells are selected followed by regeneration into transgenic plants (Amanda M Walmsley, 2000). Viral infections spread rapidly in a systemic manner thus the production of antigens in plants using virus-vector system yields products in large quantity (HAN Mei, April 2006).
The data gathered up till now shows that vaccine production using plant systems was initiated in 1990 with the expression of S. mutans surface protein antigen A (SpaA) in tobacco. Since then plants have been used as bioreactors for the production of sub unit vaccines. Rabies antigen has been expressed in tomato, cholera antigen expressed both in tobacco and potatoes, human cytomegalovirus antigen in tobacco and the expression of hepatitis antigen in tobacco and lettuce have been reported since then (Amanda M Walmsley, 2000). Vaccines production by plant systems effective against bioterrorists agents that cause anthrax, plague and small pox is also in limelight.
Plant-based anthrax vaccine
Anthrax is known as one of the most powerful bio-terror agents caused by Bacillus anthracis. Vaccination against anthrax cannot be neglected. The present licensed vaccine is prepared using attenuated B. anthracis strain in a complex and indefinite production process. New generation sub unit vaccines against anthrax have been produced in transgenic tobacco chloroplast in huge amounts that express PA. Statistical analysis demonstrates that 1 acre of chloroplast transgenic plants have the capacity to produce over 360 million doses of vaccine antigens of anthrax (M. Manuela Rigano, 2009).
Plant-based plague vaccine
Plague is caused by Y. pestis that spreads rapidly from individual to another. Anti-phagocytic capsular envelope glycoprotein (F1) and low calcium response virulent antigen (V) are the two Y. pestis antigens that are of particular interest in development of vaccines against plague. Tobacco mosaic virus-system is used to produce antigens F1 and V using transient expression system in N. benthamiana. The antigens produced using transient expression system stimulate strong immune response to provide protection against lethal challenge with Y. pestis. Similarly, sub unit vaccine was produced in transgenic tomato plants that is available for oral administration against plague. Chloroplast transformation in tobacco plants express fusion F1-V protein as high as 14.8% TSP (total soluble protein) and is effective against plague (M. Manuela Rigano, 2009).
Plant-based smallpox vaccine
Variola virus is the causative agent of smallpox whose vaccine is produced from vaccinia virus. Low reserves of this vaccine and adverse affects on cardiac system along with other contradictions has led to the production of new generation vaccines in plants systems. A27L, L1R, A33R and B5R are protein candidates for development of vaccines against smallpox. Transient expression of these proteins in tobacco protoplasts verified protein synthesis and constancy in pulse chain reactions. Nuclear genome transformation by Agrobacterium expressed high levels of A27L and A33R. A27L was integrated into chloroplast genome. Chloroplast culture gave high yields of A27L protein (M. Manuela Rigano, 2009).
Plant-based ETEC vaccine
Enteropathogenic E. coli (ETEC) strains produce heat-labile toxin (LT) whose B subunit is similar to cholera toxin. It causes diarrhea in children. LT-B was expressed in corn and its immunogenicity tested in mice. For the construction of plasmid LT-B gene variant was synthesized whose codon was optimized for high expression of maize genes and fused with barley a-amylase signal sequence. Then these plant expression constructs were amplified using overlapping oligonucleotide sequences in polymerase chain reaction followed by their introduction in maize using A. tumefacians. After pollination, ears of maize line were harvested so as to extract immature embryos under sterile conditions. A suspension of A. tumefacians was directly added to the embryos and embryos grew along with calli in selection medium. The callus obtained was finally transferred onto regeneration medium. After the germination of mature somatic embryos, plants were transplanted to soil to generate T1 seed. Soluble extract of proteins from dried corn seeds was prepared by homogenization and centrifugation. Quantification of recombinant LT-B in corn was done by performing ELISA. Production of transgenic seed was increased by backcrossing T1 seed to commercial seed lines. This seed based production of vaccine was found effective in generating immune response when seeds were fed to mice (Stephan J. Streatfield J. M., 2001). 1mg of required dosage could be delivered in as little as only 2g plant material (processed), making it cost effective (Streatfield, 2006). Expression of vaccines in seeds has advantages as ease of purification, high level of accumulation and stability of recombinant protein (Rurick K Salyaev, 2010).
LT-B has also been expressed in potato tubers using nuclear expression system and a total of 0.2% of total soluble proteins obtained (Stephan J. Streatfield J. A., 2003).
Plant-based Dengue vaccine
Plant viral vector transient expression system has been used to produce vaccines against DENV-2 E (dengue 2 envelope protein) in mature plant hosts has been employed. Although little success has been achieved but it may be promising in future with further advancement to eradicate dengue fever. A modified TMV-based vector system was used and N. benthamiana infected manually to obtain expression of the protein D2EIII. The subunit vaccine when administered to mice represented immunogenicity. Cucumis melo was also used for the production of truncated EBV that is protein binding region of DENV-2. 1:1 ratio was observed of CGMMV CP (cucumber green mottle mosaic virus coat protein) to wild-type CGMMV CP, that suggested the potential of this strategy to yield production of VLPs that express dengue virus epitope. More recently a binary vector system introduced in plants, containing Agrobacterium with plant RNA virus that has high antigen expression showed some potential results. C-terminal truncated DENV-2 E protein, truncated fragment of CprME (capsid-premembrane-envelope), and fusion of hepatitis B core antigen with DENV-2 E domain III were expressed using this plant system. Approximately 0.4-0.6 mg/g of fresh weight after 7-10 days inoculation of these antigens were produced (Yun-Kiam Yap, 2010).
Plant-based HIV vaccines
Tomato was used for the expression of HIV vaccines. HIV is responsible for the weakening of human immune system and is accompanied by many other diseases that eventually lead to the death of the patient. Synthetic gene construct of TBI-HBS was made by incorporating epitopes of TBI, neutralizing epitope of Env protein (IQRGPGRAF) and HBsAg. The expression of synthetic gene was studied in tomato from where fresh and incapsulated vaccines were obtained. Synthesis of antigenic proteins was induced by the gene TBI-HBS. Animal trials are being carried out using tomato as the candidate vaccine against HIV infections (Rurick K Salyaev, 2010).
Plant-based TB vaccine
TB is caused by the infection of bacteria Mycobacterium tuberculosis thus making it inevitable to induce immunity in individuals. Ag85B and ESAT-6 are the two antigens that are primarily important in inducing immunity in animal models studied so far. Tobacco leaves (Nicotiana tabacum) have been used as the plant system for expression of ESAT-6 protein using PVX vector system. The yield obtained from tobacco leaves ranged from 0.5% to 1% of TSP. Agroinjection of binary vector into Nicotiana benthamiana led to a high expression of TB antigens in plants cell. The accumulation of Ag85B ranged up to 800mg/kg of fresh leaves. Transgenic Arabidopsis thaliana was used to produce fusion proteins of heat-labile enterotoxin of E. coli and ESAT-6 for tagetted TB vaccine. The yield of LTB-ESAT-6 ranged from 11 to 24.5 ug/g fresh weight. Lactuca sativa, commonly known as lettuce leaves was transformed genetically to produce TB antigens- Ag58B and ESAT-6. Lettuce leaves were chosen because of its ability to grow rapidly and it does not require any thermal pretreatment before consumption. Transgenic carrots have also been reported to produce TB antigens (Rurick K Salyaev, 2010).
Plant-based cholera vaccine
Potato tubers and tobacco leaves have been used to express cholera vaccine in plants. The B subunit of cholera toxin when expressed in potato tubers produced 0.3% of the total soluble protein that was primarily targeted for retention in ER (endoplasmic reticulum.) Chloroplast expression system used in tobacco leaves produced about 4% soluble proteins in its leaves (Stephan J. Streatfield J. A., 2003).
Plant-based vaccine for human hydatidosis
Echinococcus granulosus is the causative agent of hydatid diseaase both in humans and animals. Nuclear transformation of transgenic alfafa plants has been used to produce vaccine for human hydatosis. Transgenic alfafa was produced by the combination of antigens EgA31 and Eg95. Mouse models were used to detect immunogenicity which proved successful as administration of oral and intranasal transgenic alfafa resulted in reduction in size of larva and evoked a pro-inflammatory response.
Multiple vaccines against hydatidosis and cysticercosis have been produced in papaya. Oral administration of transgenic papaya could lead to possible prevention of both diseases simultaneously (Sergio Rosales-Mendoza D. O.-A.-E., 2012).
Plant-based vaccine for helminthiasis
Necator americanus, Trichuris trichiura, Ancylostoma duodenale and Ascaris lumbricoides are the pathogens that cause helminthiasis mostly in poor populations. Seed culture was used to produce the antigen As16 for heminthiasis. Rice seeds were used for the production. 50ug/g of the desired product was expressed in each seed. When fed to mouse models, As16 induced immunity in them that shows potential for use in humans too (Sergio Rosales-Mendoza D. O.-A.-E., 2012).
Plant-based rabies vaccine
Rabies caused by lyssavirus is the major cause of fatality among people worldwide. Tomato has been engineered by nuclear transformation to produce G-protein that induces immunity in mouse models. Spinach leaves infected with virus have also been reported to express Drg24 peptide of rabies virus. Nictoniana tabacum was used as binary virus system. G-protein and nucleoprotein were fused to alfafa mosaic virus and expressed antigens against rabies that elicited humoral response when fed to mice. Transgenic maize has also been used to express glycoprotein of rabies virus. The expression levels obtained using transgenic maize raised up to 1% of the total soluble proteins (Sergio Rosales-Mendoza D. O.-A.-E., 2012).
Plant-based FH vaccine
Physcomitrella patens, a bryophyte is used as a bioreactor for production of an important component of human innate response. It is regulated by complement FH. P. patens have the potential to express FH in its system. The gene expression could be carried out using protoplast culture, plastid transformation, and transient nuclear expression. A recombinant FH (rFH) vector was formed by co-transfection of desired gene and marker gene. rFH was expressed in P. patens and secreted into culture supernatant. 25.8 ug/g yield was obtained when cultivated after 1 week (Sergio Rosales-Mendoza L. O.-E.-M., 2014).
Plant-based vaccine for atherosclerosis
Atherosclerosis is caused by the accumulation of lipoproteins that is triggered by low-density lipoprotein (LDL) particles. It is marked as a chronic inflammatory disease. Plants are used to produce chimeric proteins that comprise either LTB or CTB along with MDA epitopes (p210, p45). Plants based expression systems are used to express these candidate genes by transient expression by viral vectors and/or stable transformation by nuclear and plastid transformation. The oral administration of vaccines produced using plant systems induces systemic immune response and leads to immunity against atherosclerosis (Jorge Alberto Salazar-Gonzalez, 2013).
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