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...

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