A cholera toxoid-insulin conjugate as an oral vaccine against spontaneous autoimmune diabetes

Abstract

Mucosally induced immunological tolerance is an attractive strategy for preventing or treating illnesses resulting from untoward inflammatory immune reactions against self- or non-self-antigens. Oral administration of relevant autoantigens and allergens has been reported to delay or suppress onset of clinical disease in a number of experimental autoimmune and allergic disorders. However, the approach often requires repeated feeding of large amounts of tolerogens over long periods and is only partly effective in animals already systemically sensitized to the ingested antigen such as in animals already harboring autoreactive T cells, and thus presumably also in humans with an autoimmune disease. We have recently shown that oral administration of microgram amounts of antigen coupled to cholera toxin B subunit (CTB), can effectively suppress systemic T cell reactivity in naive as well as in immune animals. We now report that feeding small amounts (2-20 microg) of human insulin conjugated to CTB can effectively suppress beta cell destruction and clinical diabetes in adult nonobese diabetic (NOD) mice. The protective effect could be transferred by T cells from CTB-insulin-treated animals and was associated with reduced lesions of insulitis. Furthermore, adoptive co-transfer experiments involving injection of Thy-1,2 recipients with diabetogenic T cells from syngeneic mice and T cells from congenic Thy-1,1 mice fed with CTB-insulin demonstrated a selective recruitment of Thy-1,1 donor cells in the peripancreatic lymph nodes concomitant with reduced islet cell infiltration. These results suggest that protection against autoimmune diabetes can be achieved by feeding minute amounts of a pancreas islet cell autoantigen linked to CTB and appears to involve the selective migration and retention of protective T cells into lymphoid tissues draining the site of organ injury.

Figure 1

Suppression of spontaneous autoimmune diabetes after oral treatment with CTB-insulin conjugate. NOD females were treated at 8 weeks of age with a single oral administration of 20 μg of insulin conjugated to CTB (○), insulin alone (▴), or CTB alone (•). Data are combined from two independent experiments.

Figure 2

T cells from NOD mice that were fed CTB-insulin conjugate abrogate adoptive transfer of autoimmune diabetes. A depicts the incidence curves of diabetes in mice co-injected with 5 × 10⁶ T cells from the spleens of diabetic females and 5 × 10⁶ T cells from the spleens of 8-week-old females fed a single dose of 2 μg of CTB-conjugated insulin (○) or 2 μg of CTB-conjugated ovalbumin (•). The degree of insulitis was determined histologically on pancreas specimens obtained 34 days after co-transfer and is expressed as mean percentages of infiltrated islets ± SD (B).

Figure 3

T cells from CTB-insulin-fed NOD mice suppress beta cell infiltration by diabetogenic T cells. Immunofluorescence staining of pancreatic islets from irradiated NOD males 30 days after co-injection of syngeneic Thy-1,2+ T cells from 20-week-old diabetic female mice with Thy-1,1+ T cells from congenic NOD mice that were fed CTB-insulin (A, C) or CTB-ovalbumin (B, D). Immunostainings were performed with monoclonal antibodies to Thy-1,2+ (A, B) and Thy-1,1+ (C, D). Original magnifications ×250.