A chain, B chain, desoxyinsulin, and pro-insulin, as well as human GAD. In this case, only GAD was effective in providing protection.
Subcutaneous Immunization Experiments. Researchers immunized animals at 4, 8, and 12 weeks. Controls received incomplete Freund’s adjuvant (IFA) with a diluent, which had been shown to give no protection. (Mycobacterium antigen in a complete Freund’s does give marked but nonspecific protection.) Experimentals received A chain, B chain, or whole insulin with IFA. The A chain provided insignificant protection, but animals immunized with B chain developed marked protection that was transferrable to irradiated animals. The protective epitope turns out to be amino acids 9 through 23 on the B chain of the insulin molecule. Similar experiments are now under way with GAD peptides and various whole GAD molecules.
Since oily adjuvants such as IFA cannot be used in humans, researchers have investigated other adjuvants that might be approvable. Alum plus B chain proved to be effective, although alum by itself may have some effect as well. Diphtheria, tetanus toxoids, and pertussis vaccine (DTP), a common childhood vaccine that contains alum, also proved to be a good adjuvant, at least in mice. Clearly, however, DTP itself causes a nonspecific stimulation that biases the animal to a Th-2-type peripheral phenotype. Analysis of cytokines produced in DTP-treated animals shows an increase in IL-4 and a big increase in IL-10, but not the same decrease in IFN-gamma that was seen following oral administration.
This process is not anergy but instead an immune response. Responses to GAD are not decreased but actually enhanced. Researchers believe that this is an IL-4-driven response. Regardless of regime, including DTP by itself, the treated animals had increased responses to GAD. Consequently, this is an active process.
Conclusions. Oral administration of autoantigens may be leading to tolerance. Intravenous administration of insulin is relatively ineffective in delaying diabetes. Subcutaneous administration in IFA proved to be the easiest route and to have the longest-lasting effect. Alum and DTP are effective substitutes for IFA as an adjuvant. The process seems to involve, at least in part, switching from a destructive Th-1 to a protective Th-2 response. These findings could now be subjected to human trials, if the B chain epitope is similarly reproduced in humans.
Other groups have reported that the T-cells of NOD mice have a high density of receptors for insulin, and that transfer of these cells to irradiated mice uniformly transferred diabetes as well. It is possible that immunization against the B chain of insulin deviates these cells from homing on the pancreatic islets that express insulin. Researchers have already constructed a viral vaccine designed to immunize against the B chain of insulin experiments are ongoing, and they look forward to subjecting it to provisional human feasibility studies.
In response to questions from the audience, Dr. McLaren added the following: