Cholera toxin B subunit: an efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance.

Abstract

Oral administration of antigens, including allergens and autoantigens, may be an efficient way to prevent disease associated with untoward immune responses to self. and non-self-antigens. However, this approach has met with limitations because it usually requires repeated administrations of large doses of antigen and is less efficient in an already immune host, and the effect is ofshort duration. We report that a single oral adinistration ofminute Oral administration of antigens is a long-recognized method for inducing peripheral immunological tolerance (1, 2) and has been proposed as a means to prevent or treat allergic reactions (3,4), Rh alloimmunization (5), and experimental autoimmune diseases (6-13). Efforts to develop optimal tolerogenic formulations based on this strategy have been stimulated by recent studies reporting beneficial effects of oral administration of antigens in patients with autoimmune diseases (14)(15)(16).Although oral administration of antigens offers a convenient way to induce systemic tolerance, its therapeutic potential has been seriously limited. Indeed, unless tolerogens are administered repeatedly and in large doses, tolerance is usually modest and of short duration (17, 18), being rather difficult to induce in an already-immune host (19)(20)(21)(22).We now report that a single oral administration of a small dose of a soluble or particulate antigen conjugated to the B subunit of cholera toxin (CTB), rather than abrogating systemic tolerance to conjugated antigens, as generally assumed (23-25), can profoundly enhance it in naive as well as in immune animals.MATERIALS AND METHODS Animals. BALB/c and C57BL/6J female mice, 6-8 weeks old at the start of experiments, were used.Antigens. Purified human -y-globulin (HGG) was purchased from Pharmacia. Sheep red blood cells (SRBCs) and horse red blood cells (HRBCs) were obtained from the National Institute of Veterinary Medicine (HAtunaholm, Sweden).Cholera toxin (CT) was obtained from List Biological Laboratories (Campbell, CA).Preparation of CTB-Conjugated Antigens. CTB was purified from the culture supernatant of a mutant strain of Vibrio cholerae deleted of the CT genes and transfected with a plasmid encoding CTB (26, 27).To facilitate coupling to CTB, SRBCs and HRBCs were first derivatized with monosialoganglioside (GM1). A solution of GM1 (Sigma) [300 nmol/ml in phosphate-buffered saline (PBS)] was added to packed red cells at a ratio of 1:2 (vol/vol) and the mixture was incubated for 2 hr at 370C with shaking. After three washes with PBS, GM1-coated red cells were resuspended in PBS. CTB was added to the cell suspension in molar excess to the cell-bound GM1 (50 ,g of CTB per 5 x 109 GM1-SRBCs per ml). After 2 hr at 370C, the erythrocytes were washed twice with PBS to remove unbound CTB. A solid-phase hemadsorption assay using GM1 immobilized on plastic wells was employed to ascertain that the CTB molecules (pentamers) had bound to GM1-coupled SRBCs or HRBCs and were still able to bind additional GM1 molecules.HGG was covale...

Oral Tolerance Induction with Antigen Conjugated to Cholera Toxin B Subunit Generates Both Foxp3+CD25+ and Foxp3−CD25− CD4+ Regulatory T Cells

“…administration of OVA/CTB conjugate effectively induces OVA-specific peripheral immunological tolerance, whether tested in normal BALB/c mice, OVA TCR Tg (DO11.10) mice, or in either BALB/c mice or Rag1 Ϫ/Ϫ mice transferred with OVA TCR Tg T cells. Consistent with our previous findings in other systems (17)(18)(19)(20)(21)(22), oral tolerance induction with Ag/CTB conjugate was superior to that induced with free Ag both in markedly increasing the efficacy of tolerization and in dramatically decreasing the effective amounts of Ag. The oral tolerance by i.g.…”

“…OVA protein was chemically coupled to CTB using N-succininmidyl (3-(2-pyridyl)-dithio)propionate (Pierce) as a bifunctional coupling reagent as described (17). Coupled OVA/CTB was purified and quantified by fast protein liquid chromatography (FPLC) gel filtration (Superdex 200 16/60 column; Pharmacia Biotech) using the Biologic Workstation FPLC system (Bio-Rad)).…”

“…In earlier studies, we and others have shown (17) that even microgram amounts of relevant Ag conjugated to the nontoxic protein of cholera toxin (CT) B subunit (CTB) can induce oral tolerance with unique efficacy and in an Ag-specific manner, in experimental models, suppress development of autoimmune diseases (18 -21). Oral tolerance induction by CTB-conjugated Ag is associated with enhanced IL-10 and TGF-␤1 mRNA expression and cytokine production by T reg (19,22).…”

Strategies of mucosal immunotherapy for allergic diseases

“…Despite being a transmucosal carrier-delivery system for induction of peripheral immunological tolerance, CTB has also been used as a nontoxic mucosal immunomodulatory adjuvant through its binding ability to the asialo-GM-1 receptor on B cells, T cells and DCs. 51 Smits et al found that intratracheal administration of CTB can suppress allergic inflammation through the induction of airway luminal IgA secretions in a TGF-b-dependent manner, which is necessary for its preventive and curative effect. 52 CpG oligodeoxynucleotides (CpG ODNs).…”

“…Sun et al 51 found that using N-suc-cinimidyl [3-(2-pyridyl)dithio]-propionate as a conjugator to link different antigens can achieve immunotolerance through a single oral administration of low-dose antigen. In a study of the HDM allergen, Lee and Mo found that immune tolerance could be induced through intranasal application of a HDM and CTB conjugate in the murine allergic rhinitis model and that the effect can last for 4 weeks.…”