The drastic intestinal secretion of fluid and electrolytes that is characteristic of cholera is the result of reasonably well understood cellular and biochemical actions of the toxin secreted by Vibrio cholerae. Based on this understanding it is possible to devise new techniques for the treatment and prophylaxis of cholera to complement those based on fluid replacement therapy and sanitation.

Ganglioside GM, was isolated from the small intestinal mucosa of man, pig, and beef and amounted to 0.1, 2.0, and 43 nmol per g fresh weight, respectively.

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...

This study addressed the question of whether the mucosal adjuvant property of cholera toxin (CT) and the structurally closely related Escherichia coli heat-labile toxin (LT) requires the enterotoxic and adenylate cyclase/cAMP activating property of these molecules. Therefore, we investigated the cytotoxic and adjuvant abilities of the enterotoxins and compared the results with those obtained with the non-toxic CT and LT derivatives; recombinant CTB (rCTB) and a mutated LT (mLT), which had a single amino acid substitution in position 112 (Glu----Lys) of the A subunit. Detailed functional studies revealed that, in contrast to the enterotoxins, both rCTB and mLT lacked ADP-ribosylating and cAMP-stimulating abilities. However, similar membrane ganglioside GM1-receptor binding ability of all the putative adjuvants was demonstrated. When the probe antigen, keyhole limpet hemocyanin (KLH), was given perorally together with CT or LT strong gut mucosal anti-KLH immune responses were stimulated, whereas no or very low anti-KLH responses were seen in the groups which received antigen admixed with rCTB or the mLT. Moreover, the specific serum antibody responses to the various immunization protocols closely paralleled the local anti-KLH response in the gut. From these results it appears that the adjuvant mechanism of LT, and probably also of CT, is linked to the ability to ADP-ribosylate and to stimulate cAMP formation. However, this study does not unequivocally rule out other possibilities such as interactions by the A1 fragment of CT or LT with other G-proteins than Gs alpha or events that parallel or precede the effects on the adenylate cyclase/cAMP system. Thus, the levels of ADP-ribosylation and cAMP-induction that are required and the key event or target cell that is responsible for the adjuvant effect of CT and LT remain to be elucidated. Studies are underway to address these issues.

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