The carotenoid biosynthetic pathway in higher plants was manipulated by using an RNA viral vector. A cDNA encoding phytoene synthase and a partial cDNA encoding phytoene desaturase (PDS) were placed under the transcriptional control of a tobamovirus subgenomic promoter. One to two weeks after inoculation, systemically infected Nicotiana benthamiana plants were analyzed for phytoene. Leaves from transfected plants expressing phytoene synthase developed a bright orange phenotype and accumulated high levels of phytoene. Cytoplasmic inhibition of plant gene expression by viral RNA was demonstrated with an antisense RNA transcript to a partial PDS cDNA derived from tomato. The leaves of the plants transfected with the antisense PDS sequence developed a white phenotype and also accumulated high levels of phytoene. A partial cDNA to the corresponding N. benthamiana PDS gene was isolated and found to share significant homology with the tomato antisense PDS transcript. This work demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a major, eukaryotic biosynthetic pathway. In addition, our results indicate that an antisense transcript generated in the cytoplasm of a plant cell can turn off endogenous gene expression.

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 (...

Plant-made vaccines have been the subject of intense interest because they can be produced economically in large scale without the use of animal-derived components. Plant-made therapeutic vaccines against challenging chronic diseases, such as cancer, have received little research attention, and no previous human clinical trials have been conducted in this vaccine category. We document the feasibility of using a plant viral expression system to produce personalized (patient-specific) recombinant idiotype vaccines against follicular B cell lymphoma and the results of administering these vaccines to lymphoma patients in a phase I safety and immunogenicity clinical trial. The system allowed rapid production and recovery of idiotypic single-chain antibodies (scFv) derived from each patient's tumor and immunization of patients with their own individual therapeutic antigen. Both low and high doses of vaccines, administered alone or co-administered with the adjuvant GM-CSF, were well tolerated with no serious adverse events. A majority (>70%) of the patients developed cellular or humoral immune responses, and 47% of the patients developed antigenspecific responses. Because 15 of 16 vaccines were glycosylated in plants, this study also shows that variation in patterns of antigen glycosylation do not impair the immunogenicity or affect the safety of the vaccines. Collectively, these findings support the conclusion that plant-produced idiotype vaccines are feasible to produce, safe to administer, and a viable option for idiotypespecific immune therapy in follicular lymphoma patients.phase I clinical trial ͉ plant-made pharmaceutical ͉ single-chain antibodies I n recent years, non-Hodgkin's lymphoma (NHL) has become the most common hematologic malignancy in the United States with an estimated 54,000 new cases each year. Approximately 30% of these cases are follicular B cell lymphoma (1), with a median survival of 8-10 years from diagnosis (2). Most patients subjected to standard treatments, such as chemotherapy, radiation, or antibodies (2-9), still relapse (10-12). Newer approaches have focused on active immunotherapy through vaccination. For B cell lymphoma, the cell-surface Ig (Ig), or idiotype, is the unique tumorspecific antigen. In contrast to passive therapy, an idiotype-induced immune response is (i) highly tumor specific, thus sparing normal cells; (ii) potentially more durable; and (iii) protective against tumor cell variants that might ''escape'' under selective pressure, because the response is polyclonal (13-15).There exists a 20-year history of idiotype vaccination for follicular lymphoma in animal models and clinical trials. The original vaccine manufacturing process, still used today in the majority of clinical research, used a patient's lymphoma B cells to derive a mouse/ human heteromyeloma cell line for production of the tumor's monoclonal idiotype (16). Once purified, the idiotype antibody was chemically coupled to a highly immunogenic carrier protein, keyhole limpet haemocyanin (KLH), and administered w...

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Lysosomal acid lipase (LAL) is an essential enzyme that hydrolyzes triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes.