Susan L. Woodard

Plant-based vaccines: unique advantages

Abstract Numerous studies have shown that viral epitopes and subunits of bacterial toxins can be expressed and correctly processed in transgenic plants. The recombinant proteins induce immune responses and have several benefits over current vaccine technologies, including increased safety, economy, stability, versatility and efficacy. Antigens expressed in corn are particularly advantageous since the seed can be produced in vast quantities and shipped over long distances at ambient temperature, potentially allowing global vaccination. We have expressed the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis virus at high levels in corn, and demonstrate that these antigens delivered in the seed elicit protective immune responses.

Maize (Zea mays)‐derived bovine trypsin: characterization of the first large‐scale, commercial protein product from transgenic plants

Bovine trypsin (EC 3.4.21.4) is an enzyme that is widely used for commercial purposes to digest or process other proteins, including some therapeutic proteins. The biopharmaceutical industry is trying to eliminate animal‐derived proteins from manufacturing processes due to the possible contamination of these products by human pathogens. Recombinant trypsin has been produced in a number of systems, including cell culture, bacteria and yeast. To date, these expression systems have not produced trypsin on a scale sufficient to fulfill the need of biopharmaceutical manufacturers where kilogram quantities are often required. The present paper describes commercial‐level production of trypsin in transgenic maize (Zea mays) and its physical and functional characterization. This protease, the first enzyme to be produced on a large‐scale using transgenic plant technology, is functionally equivalent to native bovine pancreatic trypsin. The availability of this reagent should allow for the replacement of animal‐derived trypsin in the processing of pharmaceutical proteins.

Monoclonal antibody manufacturing in transgenic plants: myths and realities

The number and types of antibodies expressed in plants has increased steadily since the first reports of this accomplishment in the 1980s, illustrating the versatility of plants as a production system for antibodies. Many recent reviews have detailed the antibody forms that have been derived from plant expression systems. This contribution focuses on the remaining challenges to develop plant-derived therapeutic antibodies into products, and some of the progress that is being made in addressing these challenges.

Delivery of subunit vaccines in maize seed

Abstract The use of recombinant gene technologies by the vaccine industry has revolutionized the way antigens are generated, and has provided safer, more effective means of protecting animals and humans against bacterial and viral pathogens. Viral and bacterial antigens for recombinant subunit vaccines have been produced in a variety of organisms. Transgenic plants are now recognized as legitimate sources for these proteins, especially in the developing area of oral vaccines, because antigens have been shown to be correctly processed in plants into forms that elicit immune responses when fed to animals or humans. Antigens expressed in maize (Zea mays) are particularly attractive since they can be deposited in the natural storage vessel, the corn seed, and can be conveniently delivered to any organism that consumes grain. We have previously demonstrated high level expression of the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis in corn, and have demonstrated that these antigens delivered in the seed elicit protective immune responses. Here we provide additional data to support the potency, efficacy, and stability of recombinant subunit vaccines delivered in maize seed.

Development of an edible subunit vaccine in corn against enterotoxigenic strains of escherichia coli

SummaryAdvances in the development of subunit vaccines and in the production of foreign proteins in plants together offer the prospect of stable and inexpensive vaccine delivery systems. Various bacterial and viral proteins stably produced in plants have been shown to elicit immune responses in feeding trials. We have extended this approach by using Zea mays as the plant production system. Corn has several advantages as a vaccine delivery vehicle, most notably established technologies to generate transgenic plants, to optimize traits through breeding and to process the seed into a palatable form. Here we report on the production in corn seed of the GM1 receptor binding (B) subunit of the heat-labile toxin (Lt) from enterotoxigenic strains of Escherichia coli. Versions of the Lt-B gene were synthesized to give optimum codon usage for corn and to target the protein to either the cell surface or the cytoplasm. These synthetic genes were fused to a strong promoter and transformed into corn. Lt-B was highly expressed in corn seed at up to 1.8% of the total soluble protein and this was further increased approximately five-fold through plant breeding. As in E. coli. Lt-B produced in corn forms a functional pentamer that can bind to the GM1 receptor. Furthermore, Lt-B pentamer stored in corn seed is much more resistant to heat than is the pure protein, allowing the transgenic corn to be readily processed into an edible form. This work demonstrates the potential of using products derived from transgenic corn seed as delivery vehicles for subunit vaccines.

Evaluation of monoclonal antibody and phenolic extraction from transgenic Lemna for purification process development.

Several pharmaceutical protein products made in transgenic plant hosts are advancing through clinical trials. Plant hosts present a different set of impurities from which the proteins must be purified compared to other expression hosts such as mammalian cells. In this work, phenolic compounds present in extracts of monoclonal antibody (mAb)‐expressing Lemna minor were examined. Two different extraction pHs were evaluated to assess the effect of extraction condition on the concentration of mAb and phenolics in the extracts. The extract prepared at pH 4.5 had an enriched level of mAb relative to native protein when compared to a pH 7.5 extract although similar overall mAb was extracted at either pH. Slightly more mAb was recovered from the pH 3 elution of the pH 4.5 extract run on a MabSelect column than was recovered from the pH 7.5 extract. Phenolic levels in extracts were assessed by spectrophotometry, Folin–Ciocalteu assay and by profiling on RP‐HPLC. The Folin–Ciocalteu assay results did not agree with those obtained by the other two methods. Therefore phenolic levels were quantified by RP‐HPLC comparing the total area of phenolic peaks to those of reference phenolic compounds. The pH 7.5 extract had 22% less phenolics than the pH 4.5 extract. Acidic precipitation of the pH 7.5 extract resulted in further reduction of phenolics originally present in the pH 7.5 extract. The total phenolics present in the extracts were effectively removed by incubation of extracts with a commercially available anion exchange resin, Amberlite IRA‐402. We anticipate that early removal of phenolic compounds will prolong the life of more expensive affinity columns used for the purification of potential pharmaceutical proteins and should therefore be considered in process development involving proteins extracted from transgenic plant hosts. Biotechnol. Bioeng. 2009; 104: 562–571

Industrial Proteins Produced from Transgenic Plants

Industrial proteins include those used for industrial applications such as purification, diagnostics and enzymes for industrial processes. Enzymes as natural products are obtained from animal, plant and/or microbial tissues. When the industrial enzyme business began, enzymes were primarily obtained from plant and animal sources. However, today the bulk of commercial enzymes are derived from microbial sources either natural or recombinant. As a result, the extraction of commercial enzymes from plant and animal tissue has declined in recent years and continues to decline.

Phenolics removal from transgenic Lemna minor extracts expressing mAb and impact on mAb production cost

Transgenic Lemna minor has been used successfully to produce several biotherapeutic proteins. For plant‐produced mAbs specifically, the cost of protein A capture step is critical as the economic benefits of plant production systems could be erased if the downstream processing ends up being expensive. To avoid potential modification of mAb or fouling of expensive protein A resins, a rapid and efficient removal of phenolics from plant extracts is desirable. We identified major phenolics in Lemna extracts and evaluated their removal by adsorption to PVPP, XAD‐4, IRA‐402, and Q‐Sepharose. Forms of apigenin, ferulic acid, and vitexin comprised ∼75% of the total phenolics. Screening of the resins with pure ferulic acid and vitexin indicated that PVPP would not be efficient for phenolics removal. Analysis of the breakthrough fractions of phenolics adsorption to XAD‐4, IRA‐402, and Q‐Sepharose showed differences in adsorption with pH and in the type of phenolics adsorbed. Superior dynamic binding capacities (DBC) were observed at pH 4.5 than at 7.5. To evaluate the cost impact of a phenolics removal step before protein A chromatography, a mAb purification process was simulated using SuperPro Designer 7.0. The economic analysis indicated that addition of a phenolics adsorption step would increase mAb production cost only 20% by using IRA‐402 compared to 35% for XAD‐4 resin. The cost of the adsorption step is offset by increasing the lifespan of protein A resin and a reduction of overall mAb production cost could be achieved by using a phenolics removal step.

Production of Aprotinin in Transgenic Maize Seeds for the Pharmaceutical and Cell Culture Markets

Aprotinin is a serine protease inhibitor found in several bovine organs that has a number of applications in both the pharmaceutical and cell culture markets. In cell culture it is used as a component of serum-free media to preserve the integrity of recombinant proteins during fermentation and downstream purification. As a pharmaceutical, it is used to reduce blood loss during major surgeries, such as cardiopulmonary bypass surgery, and as a component of fibrin sealant kits for sutureless wound closure. Current commercial production of aprotinin is from bovine lungs with the inherent concern that contamination with pathogens such as BSE may occur. Using transgenic maize as a production vehicle for aprotinin would provide an inexpensive, easily scalable source of the protein that would be free of human pathogens. Transgenic maize plants expressing aprotinin were generated using both particle bombardment and Agrobacterium-mediated transformation. In addition, transformations were performed using three different maize genotypes and seven different constructs incorporating three promoters, two targeting sequences and multiple plant transformation units.

Enhanced Expression Levels of Cellulase Enzymes Using Multiple Transcription Units

Transgenic cereals are an attractive option for the accumulation of foreign proteins when large volumes and low cost are required. Previous work has shown maize germ to be a particularly good location for accumulating enzymes that target cellulose for degradation. In this study, recently identified embryo-preferred promoters were used to investigate their ability to increase the accumulation of the enzymes endoglucanase E1 and cellobiohydrolase CBHI. The effect of increasing copy numbers of identical transcription units, as well as multiple copies of the enzyme driven by different promoters, was explored. Results show that accumulation of the E1 or CBHI enzymes can be significantly increased, particularly when using constructs with multiple copies of the transcription units. These findings demonstrate the highest levels of these enzymes obtained in a commercially relevant plant species observed thus far. The methodology described here may provide a low-cost plant-based source of enzymes enabling an economically viable solution for the conversion of cellulose to ethanol.