Coupling of palmitate to ovalbumin inhibits the induction of oral tolerance

Abstract

Oral tolerance is a phenomenon that may occur in animals exposed to protein antigens for the first time by the oral route. They become unable to produce immune responses at the levels normally observed when they are immunized parenterally with antigen in the presence of adjuvants. Lipids have been used as adjuvants for both parenteral and oral immunization. In the present study we coupled ovalbumin with palmitate residues by incubating the protein with the N-hydroxysuccinimide palmitate ester and tested the preparation for its ability to induce oral tolerance. This was performed by giving 20 mg of antigen to mice by the oral route 7 days prior to parenteral immunization in the presence of Al(OH) 3 . Mice were bled one week after receiving a booster that was given 2 weeks after primary immunization. Specific antibodies were detected by ELISA. Despite the fact that the conjugates are as immunogenic as the unmodified protein when parenterally injected in mice, they failed to induce oral tolerance. This discrepancy could be explained by differences in the intestinal absorption of the two forms of the antigen. In fact, when compared to the non-conjugated ovalbumin, a fast and high absorption of the lipid-conjugated form of ovalbumin was observed by "sandwich" ELISA.

Expression of costimulatory molecules in antigen-activated peritoneal macrophages treated with either ovalbumin or palmitoyl-ova conjugates

“…It has been previously shown that the coupling of palmitoyl residues to Ova abolished the induction of oral tolerance in B6D2F1 mice (Oliveira et al 1998;Oliveira et al 2002). To determine whether this coupling might influence the activation of APCs, cells from PerC were cultured with Ova or palmitoyl-Ova at two different concentrations and evaluated for the expression of activation markers.…”

“…In an earlier studies, the nature of a soluble protein antigen (ovalbumin; Ova) was changed by coupling it to palmitoyl residues, which showed that this procedure did not interfere with the induction of immune responses in the animals that were injected with one, or another form of the antigen (Oliveira et al 1998;Oliveira et al 2002). However, when both the forms of the antigen were orally administered to mice, opposite effects were obtained, induction of oral toleranc by the native protein and development of humoral immune response by the lipid-conjugated protein (Oliveira et al 1998).…”

“…However, when both the forms of the antigen were orally administered to mice, opposite effects were obtained, induction of oral toleranc by the native protein and development of humoral immune response by the lipid-conjugated protein (Oliveira et al 1998). The present study investigated the role of the accessory cells in the immune responses triggered by the oral route and focused on the pattern of expression of co-stimulatory molecules on Ova or palmitoyl-Ova-activated peritoneal macrophages and the induction of cellular immune responses.…”

Ethanol affects the absorption and tissue distribution of orally administered antigens in mice

“…Lipid-ova conjugates (i.e., palmitoyl-ova) were prepared as described previously (Oliveira et al 1998). In brief, 1.0 g of the N-hydroxysuccinamide ester of palmitate (Sigma) was dissolved in 100 mL of absolute ethanol at 55°C.…”

Biodistribution of free 99mTc-ovalbumin and 99mTc-ovalbumin encapsulated in liposomes

“…For this reason, the oral route is being considered as an appropriate choice for immunization (Boyaka et al, 1999;Cripps et al, 2001;William and Gibbons, 1972;Majumdar and Ghose, 1982;Killian et al, 1988). Different and contradictory results have been observed when animals are orally treated with the antigen in distinct manners, such as in its free or soluble form; conjugated with lipid residues or encapsulated in liposomes (Oliveira et al, 1998;Fujii et al, 1993;Clark et al, 2002). These results suggest that the form of the antigen may, in some cases, protect it from the attack of digestive fluids and/or modify its absorption and biodistribution, leading either to the induction or the suppression of an immune response.…”