Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigens (1)
Immunization by the gastrointestinal or respiratory mucosal route is usually more effective at stimulating a SIgA response than parenteral delivery of antigen. Cholera toxin has induced significant intestinal SIgA responses when administered by oral, intraduodenal, and even parenteral routes.
Ten days separated the first two and the final two immunizations, while 6 days separated all other doses given. Immunizations with KLH alone gave rise to 255+/-22 SFC/10^7 cells. An enhancing effect of CT on this response was first seen with a dose of 1 ug,which evoked 1200+/-200 anti-KLH SFC/10^7 cells. Higher doses of CT increased the response further until a plateau was reached with 10 ug of of CT, which promoted 3200 +/- 225 specific SFC/10^7cells.
Multiple oral immunizations with KLH together with CT resulted in an interesting dichotomy of the immune response to the specific antigens in the spleen. Whereas anti-CT SFC numbers were high after two immunizations (250 SFC/10^7 cells), third and fourth immunizations resulted in much lower anti-CT SFC numbers (<25 SFC/10^7 cells). At variance with the spleen responses, the lamina propria lymphocytes demonstrated increased numbers of anti-KLH as well as anti-CT SFCs with each of the four oral immunizations. The specific SFC in the spleen comprised 48% IgA, 48% IgG and 4% IgM, and those in the lamina propria 90% IgA, 8% IgM and 2% IgG.
Despite a strong anti-CT SFC response in the spleen and mesenteric lymph nodes after the i.v. administration of CT, no anti-KLH SFCs were detected in lamina propria, the spleen or elsewhere. The p.o. administered CT, in contrast, gave poor anti-toxic response in lamina propria and no detectable response in the spleen, while significant numbers of anti-KLH SFC were observed in all lymphoid tissues examined.
Mice were fed KLH twice with 10 days between the immunizations and CT added as adjuvant on one, both or neither occasion. The strongest response, however, was clearly seen when CT was added to both the priming and booster immunizations (5320 SFC/10^7 lamna propria cells).
The adjuvant effect of oral CT was found exclusively in immunizations that involved the simultaneous administration of KLH and CT.
No adjuvant activity was demonstrated in the B subunit. The B subunit was much less immunogenic than the holotoxin. No dose of B-subunit up to 500 ug given in three p.o. immunizations gave rise to more than 250 anti-toxin SFC/10^7 lamina propria cells as compared with 5600 SFC/10^7 cells evoked by 10 ug of CT.
Higher doses of CT together with 10 pg of B subunit were more effective in increasing the anti-toxin SFC numbers; with 500 ng of CT, over 10,000 anti-toxin SFC/10^7 cells were seen, which was more than a 50-fold increase as compared with the response evoked by B subunit given alone.
A CT booster evoked 8574 anti-toxin SFC/10^7 cells, corresponding to a five-fold increase in SFC numbers in 6 days following the CT booster as compared to anti-toxin SFC/10^7 cells after the B subunit booster.
CT is known to be a potent activator of adenylate cyclase in most cell types, including both epithelial and lymphoid cells. CT increased the uptake of antigen from the intestine and/or stimulated the function of antigen-presenting cells in the gut. The mucosal antibody response, which is mainly in the IgA class, to most orally presented antigens depends on and is regulated by interactions between B cells and different T cells with inducer/helper/ differentiator versus suppressor functions. cAMP is an essential signal in the induction of antibody production by B cells. A need for a close temporal relation- ship, less than 6 hr, between the administration of CT and KLH in order to see enhanced anti-KLH responses. In mice, 100-500 ng of CT enhanced the gut mucosal immune response to cholera B subunit 50-fold, and also generated long-term memory that could be boosted efficiently into a vigorous mucosal SFC response 10 weeks later. In contrast to these low adjuvant-active doses, it takes 5-10 ug of p.o. administered CT to evoke detectable fluid secretion in the mouse small intestine.