SummaryWhen grown for energy production instead for smoking, tobacco can generate a large amount of inexpensive biomass more efficiently than almost any other agricultural crop. Tobacco possesses potent oil biosynthesis machinery and can accumulate up to 40% of seed weight in oil. In this work, we explored two metabolic engineering approaches to enhance the oil content in tobacco green tissues for potential biofuel production. First, an Arabidopsis thaliana gene diacylglycerol acyltransferase

In view of a recent spread of severe acute respiratory syndrome (SARS), there is a high demand for production of a vaccine to prevent this disease. Recent studies indicate that SARS-coronavirus (CoV) spike protein (S protein) and its truncated fragments are considered the best candidates for generation of the recombinant vaccine. Toward the development of a safe, effective, and inexpensive vaccine candidate, we have expressed the N-terminal fragment of SARS-CoV S protein (S1) in tomato and low-nicotine tobacco plants. Incorporation of the S1 fragment into plant genomes as well as its transcription was confirmed by PCR and RT-PCR analyses. High levels of expression of recombinant S1 protein were observed in several transgenic lines by Western blot analysis using specific antibodies. Plant-derived antigen was evaluated to induce the systemic and mucosal immune responses in mice. Mice showed significantly increased levels of SARS-CoV-specific IgA after oral ingestion of tomato fruits expressing S1 protein. Sera of mice parenterally primed with tobacco-derived S1 protein revealed the presence of SARS-CoV-specific IgG as detected by Western blot and ELISA analysis.immune response ͉ plant biotechnology ͉ severe acute respiratory syndrome-coronavirus ͉ recombinant subunit vaccine T he recent spread of severe acute respiratory syndrome (SARS) has heightened demand for a SARS vaccine that is safe, effective, economical, and easily administered. The production of recombinant subunit vaccines has become a valuable modern strategy for prevention of infectious diseases (1, 2). Development of such vaccines relies on the identification of the best antigen and the choice of expression system. The causative agent of SARS has been identified as SARScoronavirus (CoV) (3-5), which is similar to other CoVs in both virion structure and genomic organization (5, 6). Efforts to develop a vaccine against SARS are necessarily based on only the limited knowledge gained from studies of SARS-CoV, as well as on anti-CoV strategies that have been devised over the years. Several groups have shown that the spike glycoproteins are major inducers of neutralizing antibodies, providing protective immunity against many CoVs (7-11). Currently, the SARS-CoV S glycoprotein and its truncated versions are considered the best candidates for generation of a recombinant vaccine against this disease (12)(13)(14)(15)(16)(17)(18)(19)(20). The full-length SARS-CoV spike protein (S protein) is large (139 kDa) and probably not cleaved into S1 and S2 subunits (21,22). The N-terminal part of the SARS-CoV S protein (S1) contains a putative receptor-binding domain (RBD) that is responsible for cell attachment (15,23). Antibodies against this region were found to be highly effective in blocking RBD-receptor interaction in other CoVs (24). Moreover, vaccinated animals developed an antibody response against the S1 fragment of SARS S protein (25), and SARS patient sera showed significant immunoreactivity with peptide located within the S1 region (19).Recent studies have s...

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.plant biotechnology ͉ transgenic plants ͉ B5 glycoprotein ͉ recombinant antigen P lants have emerged as an excellent alternative to other expression systems for the production of complex pharmaceutical proteins, including recombinant subunit vaccines (1-6). It was shown that some plant-derived antigens can induce systemic and mucosal immune responses and, in some cases, confer protection against challenge (1-3). Plants provide the additional advantage of direct delivery through oral or other mucosal routes (1, 6). Despite some difficulties with the expression of certain recombinant proteins, especially those of viral origin, plant biotechnology holds the promise of producing medicinal proteins to be used in vaccine formulations.Interest in a safe smallpox vaccine has been reawakened by the threat of bioterrorism (7, 8) and continuous outbreaks of orthopoxvirus diseases (9, 10). A live vaccinia virus (VV)-based vaccine has been used to eventually eradicate smallpox disease (11, 12), but does display side effects (13). Although one approach for developing a safer vaccine is to use a highly attenuated live virus, recombinant protein-based vaccines are likely to be safer. For orthopoxviruses, there are several candidate antigens that can protect mice and nonhuman primates from lethal challenge (14-20). The extracellular virus (EV)-specific membrane glycoprotein encoded by the B5R gene (21-24) is the main target of EV-neutralizing antibodies present in human-derived vaccinia gamma globulin used to treat complications arising from smallpox vaccination (25).In this study, we demonstrate that the VV B5 protein can be produced in two plant expression systems. The use of the magnifection transient expression system (26-28) enabled rapid high-yield production of soluble B5 as well as selection of optimal subcellular targeting signals to use in stable plant transformation. Preparations of purified plant-derived B5 antigen (pB5) induced a strong immune response when administered parenterally and intranasally, and mice vaccinated i.m. with pB5 were protected from VV challenge. ResultsExpression Ca...

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 (mAb P ) were high (30 mg͞kg of fresh leaves) in T 1 generation plants. Like the mammalian-derived mAb M , the plant mAb P bound specifically to both SK-BR3 breast cancer cells and SW948 colorectal cancer cells. The Fc domain of both mAb P and mAb M showed the similar binding to Fc␥RI 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 mAb P originating from low-alkaloid tobacco exhibit biological activities suitable for efficient immunotherapy.breast and colorectal cancer ͉ plant biotechnology ͉ transgenic low-alkaloid tobacco ͉ tumor growth inhibition

Objective The aims of the following experiments were to characterize anti-diabetic in vitro and in vivo activity of the polyphenol-rich aqueous extract of Rutgers Scarlet Lettuce. Materials / Methods Rutgers Scarlet Lettuce (RSL) extract (RSLE) and isolated compounds were evaluated for inhibitory effects on glucose production as well as tumor necrosis factor alpha (TNFα)-dependent inhibition of insulin activity in H4IIE rat hepatoma cells. Additionally, high fat diet-induced obese mice were treated with RSLE (100 or 300 mg/kg), Metformin (250 mg/kg) or vehicle (water) for 28 days by oral administration and insulin and oral glucose tolerance tests were conducted. Tissues were harvested at the end of the study and evaluated for biochemical and physiological improvements in metabolic syndrome conditions. Results A polyphenol-rich RSLE, containing chlorogenic acid, cyanidin malonyl-glucoside and quercetin malonyl-glucoside, was produced by simple boiling water extraction at pH 2. In vitro, RSLE and chlorogenic acid demonstrated dose-dependent inhibition of glucose production. In vivo, RSLE treatment improved glucose metabolism measured by oral glucose tolerance tests, but not insulin tolerance tests. RSLE treated groups had a lower ratio of liver weight to body weight as well as decreased total liver lipids compared to control group after 28 days of treatment. No significant differences in plasma glucose, insulin, cholesterol, and triglycerides were observed with RSLE treated groups compared to vehicle control. Conclusion RSLE demonstrated anti-diabetic effects in vitro and in vivo and may improve metabolic syndrome conditions of fatty liver and glucose metabolism.