Plant cells cultures have a quick development cycle, have contained production and suitable for transient expression for rapid production of high protein yields of vaccines and prophylactic antibodies required during medical emergencies or outbreaks such as the Ebola virus disease in West Africa, but are the least scalable . Also, large number of bacteria introduced into the leaves increases the endotoxin load . Cell suspension cultures generated de novo by transformation of wild-type cells are always polyclonal because transformation is not 100% efficient and the transgenic cell lines can also undergo somaclonal variation, to generate cell populations with heterogeneous expression levels, necessitating screening and selection of the productive cell lines, optimizing the expression construct and culture conditions. Plantglycans also can affect the stability and functionality of recombinant proteins.
Plant-based systems still face one major bottleneck that needs to be overcome—their lower yields compared to mammalian cell cultures . The first licensed recombinant pharmaceutical protein derived from plants, Taliglucerasealfa was produced in plant cell suspension cultures. Vaccine against Newcastle disease virus produced in tobacco suspension cultures by DowAgrosciences, LLC, Indianapolis, USA was the first tobacco cell-based vaccine approved by the FDA against Newcastle disease virus in poultry. Physcomitrella patens suspension cultures were used to produce α-galactosidase for Fabry disease and βα for Gaucher disease by Greenovation Biotech BMBH company in Germany . Cells from tobacco, rice, medicago, carrot, tomato, sweet potato, soybean, Siberian and Korean ginseng are already used for production of Hepatitis B vaccine, Hepatitis antibody, Anti-HIV antibody, Norwalk virus capsidprotein, immunomodulators, growth hormone, lactoferrin, interferons etc.
Screening of high producing genotypes, selection of adequate medium, and optimization of the culture environment for plant cell culture may increase production. The plant virus-based expression systems can be used as transient expression systems, which use whole plants and avoid tedious regeneration processes. This is desired when rapid protein yield is needed and overcomes the difficulties of stable transformation. The plant virus-based expression systems can be either epitope presentation systems or polypeptide expression systems. Short antigenicpeptides are fused with the coat protein and are displayed on the surface of assemble dviral particles in epitope presentation systems. In polypeptide expression systems,whole unfused recombinant proteins are expressed and accumulated in plants. But insert size and limited host range are major constraints in this system. Recombinant plant virus-based nanoparticles made from genetically engineered isometric or helical viruses, with antigenic epitopes from pathogens elicit effective immune responses. Self-assembling coat proteins from viruses form the protein nanostructures free of genetic material and are referred to as virus-like particles , which are non-infectious and lack replication potential.
Non-enveloped helical plant viral capsids which are flexible and stable in terms of expressing foreign genes, are ideal platforms for epitope presentation system. Helical plant viruses, such as, bamboo mosaic virus , cardamommosaic virus , johnsongrass mosaic virus , papaya mosaic virus, papaya ring spot virus , plum pox potyvirus , potato virusX , potato virus Y , tobacco etch virus , tobacco mosaicvirus , and zucchini yellow mosaic virus have been genetically engineered to display immunogenic epitopes on their surfaces for vaccination against several diseases .Tobacco mosaic virus -based expression vectors are the most widely used vectors to produce foreign proteins in plants. First-generation viral vector retain infectivity in the plant, but have raised safety concerns. Second-generation viral vectors called viral “deconstructed” vectors have minimum of viral elements required for replication of the vector, and most DNA delivery to the target plant is via non-viral elements. A launch vector is developed with characteristics of Agrobacterium binary plasmid and plant virus expression vector. Tobacco Mosaic Virus is the viral vector widely used for the expression off oreign proteins in plants. Single-stranded positive-sense RNA genome of TMVen codes viral replicase genes for virus replication, while cell-to-cell movement protein , and coat protein genes are needed for recombination, effective spread and survival of the virus in the environment.
The target antigen gene that replaces CP gene which is inserted into a unique cloning site, under the transcriptional control of the coat protein sub-genomic mRNA promoter replicase,MP and viral replicase gene required for the replication of TMV gene was inserted between left and right border sequences of the Agrobacteriumbinary plasmid to form a launch vector, which is transferred into plant cells by agroinfiltration. Multiple single-stranded DNA copies of sequence between LB and RB are generated and released. Thus, viral vector is launched into plant tissue .These types of vectors have been used as an expression system for monoclonal antibodies due to their high and stable levels of protein expression.