Joseph M. Jilka

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.

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.

Corn as a production system for human and animal vaccines

The synthesis of selected antigens in plants and their oral delivery has great potential for reducing the costs of vaccine production and administration. The application of this technology requires antigen concentrations in final plant material to be uniform to ensure consistent dosing. In addition, antigen levels should be such as to allow the volume of each dose, containing a set amount of antigen, to be practical for oral delivery. Here, we demonstrate that the Lt-B protein of enterotoxigenic E. coli is evenly distributed in defatted corn germ prepared from transgenic grain. Furthermore, the choice of sub-cellular location for Lt-B affects accumulation of the protein in excess of four orders of magnitude.

PLANT-BASED PRODUCTION OF XENOGENIC PROTEINS

Foreign protein production in transgenic plants has been successful, from the generation of transgenic plant lines to the marketing of purified proteins. Antigenic proteins from disease organisms, monoclonal antibodies raised against antigens of disease organisms, and proteins with industrial process applications have been produced and tested. For vaccines, clinical trials in humans and feeding trials in animals are in progress to demonstrate their efficacy. For industrial proteins, high expression and downstream processing efficiency are key concerns, with application and test market trials in progress.

A corn-based delivery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immunity in swine.

Abstract Recombinant plant expression systems offer a means to produce large quantities of selected antigens for subunit vaccines. Cereals are particularly well-suited expression vehicles since the expressed proteins can be stored at relatively high concentrations for extended periods of time without degradation and dry seed can be formulated into oral vaccines suitable for commercial applications. A subunit vaccine candidate directed against porcine transmissible gastroenteritis virus and expressed in corn seed has been developed for oral delivery to swine. Here, we show that this vaccine, when administered to previously sensitized gilts, can boost neutralizing antibody levels in the animals’ serum, colostrum and milk. Thus, this vaccine candidate is effective at boosting lactogenic immunity and is appropriate to pursue through large-scale field trials preceding commercialization.

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.

Analysis of the maize polyubiquitin-1 promoter heat shock elements and generation of promoter variants with modified expression characteristics.

The maize polyubiquitin-1(Ubi-1) promoter is one of a few select promoters used to express foreign genes in monocots, such that recombinant proteins can be produced at commercially viable levels. Modifying the activity, specificity and responsiveness of such promoters provides a means to achieve desired levels and patterns of expression of genes encoding target products. Ubi-1 is constitutively expressed but is further induced by heat shock. The promoter contains two overlapping sequences with similarity to defined heat shock elements and we show that these sequences are also present upstream of the Ubi-1 homologue isolated from teosinte. Both the maize and teosinte promoters can mediate a heat shock response in transgenic maize. We have dissected the overlapping maize Ubi-1 promoter heat shock elements and demonstrate that the 3′ element is required to mediate a heat shock response. The Ubi-1 promoter is particularly active in tissues consisting of rapidly dividing cells, and within the seed it is strongly biased towards driving expression in the embryo. However, replacement of the heat shock elements with a trimer of a basic domain/leucine zipper factor binding site of a pea lectin promoter shifts the balance in seed expression towards the endosperm. The Ubi-1 variants described here differ in their overall activity in the seed, but they all show potential for driving high levels of heterologous gene expression in maize.

Advantageous Features of Plant-based Systems for the Development of HIV Vaccines

Plants have recently become an attractive option for the production of recombinant proteins. Plant-based systems can be used to produce many classes of foreign proteins including candidate vaccine antigens. The selected antigen can be purified from plant material prior to delivery by the preferred route, or alternatively delivered orally in edible plant material that has been processed to give a homogeneous and stable product. Several plant species have been used to express a wide range of vaccine candidates with tobacco, potato and corn being particularly favored. Corn seed is especially well suited to various food processing technologies that generate dry homogeneous material suitable for extended storage and refrigeration-free transport and distribution. Many antigens have been expressed in corn and assessed for efficacy in trials with generally positive results. Candidate HIV vaccines are particularly good targets for plant-based oral delivery since there is a great need for an easily distributed affordable vaccine that could be administered without injection and induce strong mucosal immune responses. As a first step in evaluating plant expression technology with a relevant antigen that might easily be tested in an animal system, we expressed the SIV major surface glycoprotein gp130 (analogous to HIV gp120) in corn seed. Expression levels were achieved that are compatible with conducting oral delivery trials in animals.

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.

Interactions between plants, pathogens and insects: Possibilities for engineering resistance

Abstract Genetically engineered agricultural crops that resist viral and insect pathogens have been produced and tested under both environmentally controlled and field test conditions. Recent advances in creating plants that resist viruses, and plants that contain a bacterial protein, which confers selected insect resistance are reviewed. Recent studies on plant defense mechanisms are also discussed. In the future, plant defense mechanisms will be genetically modified to produce plants that resist insect and fungal pathogens.