Plant viral vectors delivered by Agrobacterium are the basis of several manufacturing processes that are currently in use for producing a wide range of proteins for multiple applications, including vaccine antigens, antibodies, protein nanoparticles such as virus-like particles (VLPs), and other protein and protein-RNA scaffolds. Viral vectors delivered by agrobacterial T-DNA transfer (magnifection) have also become important tools in research. In recent years, essential advances have been made both in the development of second-generation vectors designed using the 'deconstructed virus' approach, as well as in the development of upstream manufacturing processes that are robust and fully scalable. The strategy relies on Agrobacterium as a vector to deliver DNA copies of one or more viral RNA/DNA replicons; the bacteria are delivered into leaves by vacuum infiltration, and the viral machinery takes over from the point of T-DNA transfer to the plant cell nucleus, driving massive RNA and protein production and, if required, cell-to-cell spread of the replicons. Among the most often used viral backbones are those of the RNA viruses Tobacco mosaic virus (TMV), Potato virus X (PVX) and Cowpea mosaic virus (CPMV), and the DNA geminivirus Bean yellow dwarf virus. Prototypes of industrial processes that provide for high yield, rapid scale up and fast manufacturing cycles have been designed, and several GMP-compliant and GMP-certified manufacturing facilities are in place. These efforts have been successful as evidenced by the fact that several antibodies and vaccine antigens produced by magnifection are currently in clinical development.
This manufacturing process is reliable and robust, the manufacturing time from biopsy to vaccine is <12 weeks and the expression and purification of antigens require only 2 weeks. The process is also broadly applicable for manufacturing monoclonal antibodies in plants, providing 50- to 1000-fold higher yields than alternative plant expression methods.
Production of recombinant biologics in plants has received considerable attention as an alternative platform to traditional microbial and animal cell culture. Industrially relevant features of plant systems include proper eukaryotic protein processing, inherent safety due to lack of adventitious agents, more facile scalability, faster production (transient systems), and potentially lower costs. Lower manufacturing cost has been widely claimed as an intuitive feature of the platform by the plant-made biologics community, even though cost information resides within a few private companies and studies accurately documenting such an advantage have been lacking. We present two technoeconomic case studies representing plant-made enzymes for diverse applications: human butyrylcholinesterase produced indoors for use as a medical countermeasure and cellulases produced in the field for the conversion of cellulosic biomass into ethanol as a fuel extender. Production economics were modeled based on results reported with the latest-generation expression technologies on Nicotiana host plants. We evaluated process unit operations and calculated bulk active and per-dose or per-unit costs using SuperPro Designer modeling software. Our analyses indicate that substantial cost advantages over alternative platforms can be achieved with plant systems, but these advantages are molecule/product-specific and depend on the relative cost-efficiencies of alternative sources of the same product.
Rapid production of protein-based tumorspecific vaccines for the treatment of malignancies is possible with the plant-based transient expression system described here. We created a modified tobamoviral vector that encodes the idiotype-specific single-chain Fv fragment (scFv) of the immunoglobulin from the 38C13 mouse B cell lymphoma. Infected Nicotiana benthamiana plants contain high levels of secreted scFv protein in the extracellular compartment. This material reacts with an anti-idiotype antibody by Western blotting, ELISA, and affinity chromatography, suggesting that the plant-produced 38C13 scFv protein is properly folded in solution. Mice vaccinated with the affinity-purified 38C13 scFv generate >10 g͞ml anti-idiotype immunoglobulins. These mice were protected from challenge by a lethal dose of the syngeneic 38C13 tumor, similar to mice immunized with the native 38C13 IgM-keyhole limpet hemocyanin conjugate vaccine. This rapid production system for generating tumorspecific protein vaccines may provide a viable strategy for the treatment of non-Hodgkin's lymphoma.Most B cell malignancies are incurable and are increasing in frequency in the populations of industrial nations (1, 2). Although B cell tumors are characterized by extreme variability in treatment and prognosis (3), they share a common feature that makes them ideal for the development of patientspecific cancer vaccines. Each clone of malignant B cells expresses a unique cell surface immunoglobulin (Ig)-a tumorspecific marker. The tumor's surface Ig, when made immunogenic by conjugation to keyhole limpet hemocyanin (KLH) and administered with an adjuvant, has been used to vaccinate patients in chemotherapy-induced remission (4, 5). When an immune response is triggered by such vaccination, patients have a superior clinical outcome (6, 7).Unfortunately, Igs are difficult proteins to produce. Currently, Igs for patient therapies are created by fusion of tumor cells to a transformed human͞mouse heteromyeloma cell (8, 9). Hybridomas are screened for secreted patient tumorspecific Ig and expanded for large-scale production of the protein. Besides the labor and expense involved, a drawback of hybridoma production systems is the unpredictable loss of chromosomes and of tumor-specific Ig protein expression over time. This phenomenon currently limits the application of the therapy, in terms of both the quantity of vaccine per patient and the total number of patients that can be treated. To expand the scope of individualized non-Hodgkin's lymphoma (NHL) therapies, a source of abundant, safe, easily purified vaccine needs to be generated in a time frame of weeks rather than months or years.An appealing alternative to multichain whole Ig vaccines is singe-chain variable region (scFv) vaccines. Consisting of just the hypervariable domains from the tumor-specific Ig, these proteins recreate the antigen-binding site of the native Ig (10-12), are a fraction of the size, and can be expressed in several expression systems (13-17), including transgenic plants (...
Enterohemorrhagic Escherichia coli (EHEC) is one of the leading causes of bacterial enteric infections worldwide, causing ∼100,000 illnesses, 3,000 hospitalizations, and 90 deaths annually in the United States alone. These illnesses have been linked to consumption of contaminated animal products and vegetables. Currently, other than thermal inactivation, there are no effective methods to eliminate pathogenic bacteria in food. Colicins are nonantibiotic antimicrobial proteins, produced by E. coli strains that kill or inhibit the growth of other E. coli strains. Several colicins are highly effective against key EHEC strains. Here we demonstrate very high levels of colicin expression (up to 3 g/kg of fresh biomass) in tobacco and edible plants (spinach and leafy beets) at costs that will allow commercialization. Among the colicins examined, plant-expressed colicin M had the broadest antimicrobial activity against EHEC and complemented the potency of other colicins. A mixture of colicin M and colicin E7 showed very high activity against all major EHEC strains, as defined by the US Department of Agriculture/Food and Drug Administration. Treatments with low (less than 10 mg colicins per L) concentrations reduced the pathogenic bacterial load in broth culture by 2 to over 6 logs depending on the strain. In experiments using meats spiked with E. coli O157:H7, colicins efficiently reduced the population of the pathogen by at least 2 logs. Plant-produced colicins could be effectively used for the broad control of pathogenic E. coli in both plant-and animal-based food products and, in the United States, colicins could be approved using the generally recognized as safe (GRAS) regulatory approval pathway.antimicrobials | colicin | EHEC | food safety | plant-made recombinant proteins
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