Curr Pharm Biotechnol. 2015;16(11):1002-11.
Induction of Oral Tolerance with Transgenic Plants Expressing Antigens for Prevention/Treatment of Autoimmune, Allergic and Inflammatory Diseases
Shengwu Ma1,2,*, Yu-Cai Liao3 and Anthony M. Jevnikar1
1Transplantation Immunology Group, Lawson Health Research Institute, London, Ontario, N6A 4G5, Canada;
2Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7, Canada;
3College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China
The prevalence and incidence of autoimmune and allergic diseases have increased dramatically over the last several decades, especially in the developed world. The treatment of autoimmune and allergic diseases is typically with the use of non-specific immunosuppressive agents that compromise the integrity of the host immune system and therefore, increase the risk of infections. Antigenspecific immunotherapy by reinstating immunological tolerance towards self antigens without compromising immune functions is a much desired goal for the treatment of autoimmune and allergic diseases. Mucosal administration of antigen is a long-recognized method of inducing antigen-specific immune tolerance known as oral tolerance, which is viewed as having promising potential in the treatment of autoimmune and allergic diseases. Plant-based expression and delivery of recombinant antigens provide a promising new platform to induce oral tolerance, having considerable advantages including reduced cost and increased safety. Indeed, in recent years the use of tolerogenic plants for oral tolerance induction has attracted increasing attention, and considerable progress has been made. This review summarizes recent advances in using plants to deliver tolerogens for induction of oral tolerance in the treatment of autoimmune, allergic and inflammatory diseases.
Key words: Oral tolerance induction, plant-based drug delivery system, bioencapsulation, autoantigen, allergen, autoimmune diseases, allergy.
Oral tolerance is a specific suppression of immune responses to an antigen following its oral ingestion. It represents a key feature of intestinal immunity, which will prevent the development of allergies and other immune-mediated diseases. Importantly, gut-induced tolerance is also involved in systemic immune regulation and the induction of oral tolerance offers attractive strategies to prevent or treat allergic inflammatory or autoimmune diseases resulting from the development of a deleterious immune response to self or exogenous (i.e., dietary) antigens. Such approaches would exploit effective yet selective natural immunosuppressive mechanisms, thereby avoiding unwanted side effects caused by long-term treatment with immunosuppressive drugs (1). The therapeutic efficacy of oral tolerance induction has been demonstrated in a range of animal models. The successful translation of promising preclinical research results into human clinical benefits may, however, critically depend on the development of an inexpensive antigen delivery platform, as oral tolerance induction requires repetitive administration of high doses of antigen that can make this therapy unaffordable. The use of transgenic plants for production and delivery of tolerogenic proteins provides a cheap, viable and practical platform for oral tolerance induction.
There is growing recognition of the benefits associated with the use of plants as platforms for production of high-value pharmaceutical proteins. The major benefits of plant-based production platforms include low-production cost, the ease of scaling-up and high-quality products. Additionally, another significant benefit is that pharmaceutical proteins expressed in edible transgenic plants can be delivered orally by ingestion of the protein-containing plant material, and this benefit makes transgenic plants particularly attractive for use in an oral tolerance induction strategy. Plant-made proteins delivered orally are naturally protected from degradation in the stomach because of the plant cell wall, which can provide protection to the protein that is bioencapsulated inside plant cells. Moreover, many plants contain chemicals such as lectins and saponins that could serve as natural adjuvant potentiating mucosal immune responses to mucosally delivered antigens (2)
Genetic transformation has now become a routine technique for many plant species (3). There are two main methods for transforming plant cells and tissues: the Agrobacterium-mediated method and the direct gene transfer (gene gun) method (illustrated in Figure 1). Agrobacterium tumefaciens is a soil phytopathogen with a natural ability to infect plant cells and insert a piece of its DNA (T-DNA) into the plant cell genome. Agrobacterium-mediated transformation has been widely used for production of transgenic dicot as well as some monocot plants (such as tobacco, tomato and wheat). The gene gun method, also called particle bombardment, is a direct physical method of introducing foreign DNA into plant cells. In this method, DNA that is to be delivered into plant cells is coated onto small tungsten or gold carrier particles, which is then fired into plant cells or tissues, usually using pressurised helium. The gene gun method has served as an excellent approaching for transforming cereal plants (rice and wheat) and recalcitrant agronomic crops (soybean).
Figure 1. Schematic diagram showing various steps of stable genetic transformation of plants using Agrobacterium-mediated and direct gene transfer (gene gun) methods.
Since many plant species can now be genetically transformed and regenerated, this allows flexibility in the choice of plant hosts that can be used to deliver oral tolerogens for the induction of oral tolerance, increasing palatability for plant-delivered tolerogenic products. Vegetable plants or fruit plants offer the most convenient way to deliver bioencapsulated tolerogen, as they are palatable and can be eaten raw without cooking. Thus, proteins expressed in them do not risk to be denatured by heat treatments. Alternatively, leaf materials can be harvested, freeze-dried to remove water, ground into powder and then put into capsules or tablets for oral administration. Compared to fresh leaf material, lyophilized leaf powder offers advantages in terms of long-term storage, increase of therapeutic protein content, protein stability and uniform dose delivery. Similarly, fresh fruits can also be processed into dry powder, then incorporated into drinks or made into cookies for consumption (illustrated in Figure 2). Recently, Hansson et al. (4) showed that oral feeding of mice with fresh transgenic Arabidopsis thaliana leaves expressing a tolerogenic CTA1(R7K)-COL-DD fusion protein protected against arthritis through oral tolerance induction. Su et al. (5) demonstrated that feeding of Hemophilia B mice with lyophilized lettuce powder containing a fusion protein composed of factor IX and CTB (CTB-FIX ) by oral gavage induced oral tolerance characterized by induction of LAP+ regulatory T cells, suppression of inhibitor/IgE formation and anaphylaxis against FIX. Plant seeds offer another high-performance oral delivery platform for tolerogens for oral tolerance induction. Compared to vegetable crops and fruits, plant seeds enable long-term storage of the foreign protein produced as dry seeds have very low water contents. Additionally, plant seeds are high in protein content, facilitating foreign protein accumulation to high levels. Seeds also contain protease inhibitors in high concentrations, which is able to protect target proteins from proteolytic degradation by gastrointestinal enzymes during oral delivery via the stomach. Recently, Wakasa et al. (6) reported a new method for using transgenic rice seed to deliver tolerogens for allergen-specific immunotherapy via oral tolerance induction. They produced transgenic rice plants expressing the T cell epitope peptide of Cryj I and Cry j II, the major Japanese cedar pollen allergens, deposited into endoplasmic reticulum (ER)-derived type I protein bodies (PB-I) in rice seeds. Allergen-containing PBs were then concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The Cry j 1 and Cry j 2 antigens in this concentrated PB product were found to be more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. Importantly, the concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered.
Figure 2. Schematic diagram showing the use of transgenic vegetable and fruit plants to deliver edible tolerogens for oral tolerance induction.
- Bégin P, Winterroth LC, Dominguez T, Wilson SP, Bacal L, Mehrotra A, Kausch B, Trela A, Hoyte E, O’Riordan G, Seki S, Blakemore A, Woch M, Hamilton RG, Nadeau KC Safety and feasibility of oral immunotherapy to multiple allergens for food allergy. Allergy Asthma Clin Immunol. 2014; 15;10:1. doi: 10.1186/1710-1492-10-1.
- Granell, A.; Fernandez-del-Carmen, A.; Orzaez, D. In planta production of plant-derived and non-plant-derived adjuvants. Expert Rev Vaccines 2010; 9:843–858.
- Barampuram S, Zhang ZJ. Recent advances in plant transformation. Methods Mol Biol. 2011;701:1-35.
- Hansson C, Schön K, Kalbina I, Strid Å, Andersson S, Bokarewa MI, Lycke NY. Feeding transgenic plants that express a tolerogenic fusion protein effectively protects against arthritis. Plant Biotechnol J. 2015; doi: 10.1111/pbi.12479.
- Su J, Zhu L, Sherman A, Wang X, Lin S, Kamesh A, Norikane JH, Streatfield SJ, Herzog RW, Daniell H. Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B. Biomaterials 2015; 70:84-93.
- Wakasa Y, Takagi H, Watanabe N, Kitamura N, Fujiwara Y, Ogo Y, Hayashi S, Yang L, Ohta M, Thet Tin WW, Sekikawa K, Takano M, Ozawa K, Hiroi T, Takaiwa F. Concentrated protein body product derived from rice endosperm as an oral tolerogen for allergen-specific immunotherapy–a new mucosal vaccine formulation against Japanese cedar pollen allergy. PLoS One 2015; 10(3):e0120209.