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ABSTRACT: The granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with many important applications and, due to its immunostimulatory properties, could also be used as a vaccine adjuvant. A simple strategy to produce recombinant mouse GM-CSF (mGM-CSF) in transgenic Nicotiana tabacum plants was used in this study. The mGM-CSF cDNA followed by the sequence encoding endoplasmic reticulum retention signal (KDEL) was cloned into the ImpactVector under the control of the strong promoter from the gene encoding a small subunit of Rubisco. In transgenic plants the accumulation level of recombinant mGM-CSF varied in the individual transformants from 8 to 19 microg/g of fresh leaf tissue, which makes up to 0.22% of total soluble protein. In most analyzed plants, the apparent molecular weight of the recombinant protein was larger than predicted due to its N-glycosylation, presumably in 2 sites. The recombinant plant-produced murine GM-CSF retained its biological activity as confirmed in vitro in proliferation assay using a mouse cell line, which is growth-dependent on GM-CSF.
Journal of interferon & cytokine research: the official journal of the International Society for Interferon and Cytokine Research 03/2010; 30(3):135-42. · 1.63 Impact Factor
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ABSTRACT: The hepatitis B core antigen (HBcAg) can generate a strong immune response and is recognized as an effective carrier for foreign epitopes. The domain-4 epitope of the anthrax protective antigen (PA-D4) plays an essential role in generating protective immunity against virulent Bacillus anthracis. Here we report the successful production of a recombinant protein comprised of the antigenic PA-D4 integrated into the c/e1 loop of HBcAg in transgenic low-alkaloid Nicotiana tabacum. Sera of mice injected with the plant-derived purified HB/PA-D4 protein exhibited significant anti-PA- and anti-HBcAg-specific IgG titers; however, formation of virus-like particles (VLP) was not observed. These data support the feasibility of producing complex protein chimeras in plants.
Hybridoma (2005) 09/2008; 27(4):241-7. · 0.42 Impact Factor
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ABSTRACT: Immunotherapy holds great promise for treatment of infectious and malignant diseases and might help to prevent the occurrence and recurrence of cancer. We produced a plant-derived tumor-associated colorectal cancer antigen EpCAM (pGA733) at high yields using two modern plant expression systems. The full antigenic domain of EpCAM was efficiently purified to confirm its antigenic and immunogenic properties as compared to those of the antigen expressed in the baculovirus system (bGA733). Recombinant plant-derived antigen induced a humoral immune response in BALB/c mice. Sera from those mice efficiently inhibited the growth of SW948 colorectal carcinoma cells xenografted in nude mice, as compared to the EpCAM-specific mAb CO17-1A. Our results support the feasibility of producing anti-cancer recombinant vaccines using plant expression systems.
Cancer Immunology and Immunotherapy 04/2008; 57(3):317-23. · 3.70 Impact Factor
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Maxim Golovkin,
Sergei Spitsin,
Vyacheslav Andrianov,
Yuriy Smirnov,
Yuhong Xiao,
Natalia Pogrebnyak,
Karen Markley, Robert Brodzik,
Yuri Gleba,
Stuart N Isaacs,
Hilary Koprowski
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ABSTRACT: We report here the in planta production of the recombinant vaccinia virus B5 antigenic domain (pB5), an attractive component of a subunit vaccine against smallpox. The antigenic domain was expressed by using efficient transient and constitutive plant expression systems and tested by various immunization routes in two animal models. Whereas oral administration in mice or the minipig with collard-derived insoluble pB5 did not generate an anti-B5 immune response, intranasal administration of soluble pB5 led to a rise of B5-specific immunoglobulins, and parenteral immunization led to a strong anti-B5 immune response in both mice and the minipig. Mice immunized i.m. with pB5 generated an antibody response that reduced virus spread in vitro and conferred protection from challenge with a lethal dose of vaccinia virus. These results indicate the feasibility of producing safe and inexpensive subunit vaccines by using plant production systems.
Proceedings of the National Academy of Sciences 05/2007; 104(16):6864-9. · 9.68 Impact Factor
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Robert Brodzik,
Magdalena Glogowska,
Katarzyna Bandurska,
Monika Okulicz,
Deepali Deka,
Kisung Ko,
Joke van der Linden,
Jeanette H W Leusen,
Natalia Pogrebnyak,
Maxim Golovkin,
Zenon Steplewski,
Hilary Koprowski
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ABSTRACT: Although current demands for therapeutic mAbs are growing quickly, production methods to date, including in vitro mammalian tissue culture and transgenic animals, provide only limited quantities at high cost. Several tumor-associated antigens in tumor cells have been identified as targets for therapeutic mAbs. Here we describe the production of mAb BR55-2 (IgG2a) in transgenic plants that recognizes the nonprotein tumor-associated antigen Lewis Y oligosaccharide overexpressed in human carcinomas, particularly breast and colorectal cancers. Heavy and light chains of mAb BR55-2 were expressed separately and assembled in plant cells of low-alkaloid tobacco transgenic plants (Nicotiana tabacum cv. LAMD609). Expression levels of plant-derived mAb (mAbP) were high (30 mg/kg of fresh leaves) in T1 generation plants. Like the mammalian-derived mAbM, the plant mAbP bound specifically to both SK-BR3 breast cancer cells and SW948 colorectal cancer cells. The Fc domain of both mAbP and mAbM showed the similar binding to FcgammaRI receptor (CD64). Comparable levels of cytotoxicity against SK-BR3 cells were also shown for both mAbs in antibody-dependent cell-mediated cytotoxicity assay. Furthermore, plant-derived BR55-2 efficiently inhibited SW948 tumor growth xenografted in nude mice. Altogether, these findings suggest that mAbP originating from low-alkaloid tobacco exhibit biological activities suitable for efficient immunotherapy.
Proceedings of the National Academy of Sciences 07/2006; 103(23):8804-9. · 9.68 Impact Factor
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Phytopathology 01/2003; 92(12):1260-1. · 2.80 Impact Factor
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ABSTRACT: Plant expression cassettes containing the Escherichia coli cysE gene alleles (encoding SAT) were constructed. After the Agrobacterium-mediated transformation of tobacco, we identified stable transformed plants containing several-fold higher SAT activity in comparison to the control plant. Determination of non-protein thiol contents indicated two- to threefold higher cysteine and glutathione levels in some of these transgenic plants. The maximal elevation of the cysteine level was about fourfold while that of GSH was about twofold higher than in the controls. The most striking physiological consequence of the modification of sulfur metabolite levels in the transgenic plants, however, was their several-fold increased resistance to oxidative stress generated by exogenous hydrogen peroxide.
The Plant Journal 09/1999; 20(2):237 - 243. · 6.16 Impact Factor
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ABSTRACT: Plants have emerged as a modern production system to produce recombinant proteins—antigens that can be used as subunit vaccines. The ideal plant candidate for this purpose should be capable to sustain high levels of expression of foreign proteins without adverse effects on its growth and development. It is also essential that it has large biomass, is edible and suitable for long-term storage and delivery.This work is a part of an effort to develop Cruciferae-based production system using transgenic vegetable plants collard and cauliflower. Several parameters were tested and optimized to achieve an efficient stable transformation of these recalcitrant species with constructs containing expression cassettes for the known viral antigens. Using the original procedure we obtained transgenic collard cv Morris Heading that express high levels of smallpox vaccine candidate (B5) in leaves and retain its normal phenotype. Transgenic cauliflower plants cv Early Snowball were obtained in similar procedure and have shown detectable amounts of SARS coronavirus spike-protein (SARS-CoV S1) in floret tissue of mature curd.To our knowledge, this is the first report on generation of transgenic collard plants ever and the first successful attempt to use these vegetables for production of pharmaceutical proteins.
Plant Science.
