Oral tolerance, induced by the feeding of autoantigens, has been applied successfully as a therapeutic tool in experimental models of autoimmune diseases (Strobel and Mowat, 1998
). The basic mechanism of oral tolerance in humans is currently a work in progress, and oral antigen administration regimens have resulted in limited success when applied to patients (Garside et al., 1999
; Chaillous et al., 2000
; Pozzilli et al., 2000
). A possible explanation for this limited success may be that the doses of antigens administered orally to humans were low compared with those delivered to mice, considering the surface area of the intestinal absorptive epithelium (Pozzilli and Cavallo, 2000
). In this case, CTB may serve as the necessary cofactor required to overcome the inefficient presentation of insulin to mucosal T-cells, resulting from the limited transport of native insulin across the epithelial layer. In order for oral tolerance to become a realistic therapy for human autoimmune diseases, adjuvants that possess the ability to enhance the tolerogenic potential of orally delivered antigens need to be identified. The coupling of autoantigens – in this case, proinsulin – to non-toxic CTB dramatically increases their tolerogenic potential (Sun et al., 1994
; Bergerot et al., 1997
; Arakawa et al., 1998
). This effect is mediated by the ability of CTB to act as a transmucosal carrier, although CTB may have a direct affect on the immune system (Burkart et al., 1999
; Li and Fox, 1999
). The current primary limitation in advancing this concept in clinical trials is the low levels of expression in transgenic plants (Bergerot et al., 1997
). This limitation can be overcome by the hyper-expression of the CTB-Pins fusion protein in transgenic chloroplasts.
Previous studies expressing the CTB-Pins fusion protein in plants have been performed in potato (Arakawa et al., 1998
). The expression level in nuclear transgenic potato tubers was 0.1% of TSP. This low expression level required the feeding of NOD mice with large amounts of fresh potatoes. In the present study, the CTB-Pins fusion protein accumulated in transplastomic tobacco to up to ~16% of TSP, 160-fold greater than that achieved in nuclear transgenic potatoes. NOD mice were given 8 mg of CTB-Pins tobacco leaf tissue (containing ~14 µg of the fusion protein) per week by gavage; this represents a 375-fold decrease in the amount of plant tissue administered compared with the 3 g per week used previously (Arakawa et al., 1998
). The use of these small concentrated doses reduces the possibility of the potential confounding effects of leaf tissue, and eliminates the need to process or purify large quantities of plant material. Hyper-expression of CTB-Pins in plant plastids should make this fusion protein abundantly available for animal studies or human clinical trials.
Although lower levels of CTB-Pins accumulation were observed in transplastomic lettuce when compared with tobacco (approximately sixfold less in lettuce), the average value determined for lettuce (~1.8% of TSP) represents a level of protein sufficient to proceed with animal or preclinical studies. For example, in this study, using tobacco, we delivered approximately 14 µg of CTB-Pins to NOD mice; a comparable dose could be derived from 100 mg of fresh lettuce leaf, a feasible quantity for weekly oral delivery. The difference observed between tobacco and lettuce may be attributed, in part, to the 5′ regulatory elements used in our study. Tobacco expression of CTB-Pins is driven by the endogenous psbA
5′ UTR, whereas lettuce expression is regulated by the inclusion of the translational control region of bacteriophage T7 gene 10. Previous studies have demonstrated that the level of foreign protein accumulation is lower when these translation elements are used to drive expression of the same gene, with psbA
5′ UTR being more efficient (Dhingra et al., 2004
). In addition, intrinsic variation in the nature of the leaves from tobacco and lettuce may influence the accumulation of foreign protein expressed in chloroplasts. We are developing new transformation constructs for lettuce for CTB-Pins expression which will employ lettuce endogenous translation elements, such as psbA
5′ UTR, to further increase the level of expression.
The oral administration of self-antigens, such as insulin, leads to their uptake by gut-associated lymphoid tissue (GALT), including intestinal mucosal M-cells, which pass the antigen to underlying antigen-presenting cells (Limaye et al., 2006
). This leads to the activation of T-cells and the induction of a Th2 cell response, which is characterized by the up-regulation of immunosuppressive cytokines (such as IL-10 and IL-4) and serum antibodies (such as IgG1, but not IgG2a) (Salmond et al., 2002
; Faria and Weiner, 2005
). No significant increase in mucosal IgA was seen in our study in CTB-Pins-treated mice vs. the control groups. CTB-Pins-treated animals showed very high levels of IgG1, but not IgG2a, whereas the control groups showed no variation (). The serum IgG1 values of CTB-Pins-treated animals confirm the activated Th2 response, which is also supported by the histology, with less lymphocytic infiltration, the up-regulation of immunosuppressive cytokine levels in the tissues, and the trend in the blood and urine glucose levels (which were higher in the control groups than in the CTB-Pins-treated group). The presence of CTB in the intestine ensures effective receptor-mediated oral delivery of intact plant-derived fusion protein across the intestinal mucosa via the binding of CTB to the GM1
ganglioside receptor and uptake by intestinal M-cells and enterocytes.
Taken together, the data presented here suggest that the suppression of insulitis is mediated by regulatory Th2 cells. As T-cell regulation plays a major role in mucosal immunity, the oral administration of an autoantigen can be used to treat autoimmune diseases in animal models by generating active T-cell suppression. Several autoimmune diseases and their antigens are known: multiple sclerosis (myelin basic protein and proteolipid protein), arthritis (type II collagen), uveitis (S-antigen and interphotoreceptor retinoid binding protein), myasthenia gravis (acetylcholine receptor) and thyroiditis (thyroglobin) (Hafler and Weiner, 1997
). In the USA, 5.5 million people suffer from psoriasis, 3 million from Graves' disease, 2.5 million from rheumatoid arthritis, 2–5 million from vitiligo, 3.5 million from thyroiditis, 1–4 million from Sjogren's syndrome, 0.5 million from Crohn's disease and multiple sclerosis, 370 000 from type 1 diabetes, etc. This study opens up the possibility for new investigations on autoimmune diseases.
Five-week-old mice were used in this study to demonstrate the alleviation of symptomatic pancreatic insulitis and the preservation of insulin-producing β-cells, a condition that mimics human type 1 diabetes. Based on the success of the concept in older mice (Harrison et al., 1996
), this strategy is likely to work not only prior to the onset of diabetes, but also at later stages of this autoimmune disease, and this will be explored in future experiments. One previous human clinical study on the oral delivery of insulin was unsuccessful (Skyler et al., 2005
), because insulin was not protected from digestive enzymes and acid hydrolysis. In our study, however, insulin was protected by bioencapsulation within plant cells. On the basis of the results obtained in this study, human clinical trials have been initiated.