DNA immunization to prevent autoimmune diabetes

Abstract

Mice expressing lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) as a transgene in their beta cells develop insulin-dependent diabetes mellitus (IDDM) only after LCMV infection. Inoculation of plasmid DNA encoding the insulin B chain reduced the incidence of IDDM by 50% in this model. The insulin B-chain DNA vaccination was effective through induction of regulatory CD4 lymphocytes that react with the insulin B chain, secrete IL-4, and locally reduce activity of LCMV-NP-autoreactive cytotoxic T lymphocytes in the pancreatic draining lymph node. In contrast, similar vaccination with plasmids expressing the LCMV viral ("self") protein did not prevent IDDM, because no such regulatory cells were induced. Thus, DNA immunization with plasmids expressing self-antigens might constitute a novel and attractive therapeutic approach to prevent autoimmune diseases, if the antigens are carefully preelected for an ability to induce regulatory lymphocytes in vivo.

Figure 1

Immunization with the insulin B chains, but not the insulin A chain, is effective in preventing IDDM in RIP-NP mice. RIP-NP transgenic mice were immunized with 100 μg of porcine insulin A or B chains in IFA (1:1) once 5 days after LCMV infection, or were fed oral whole-porcine insulin as a positive control (see Methods for feeding protocol). Diabetes was measured weekly by Accucheck (see Methods); the total observation period was 3 months. Group sizes were as follows: 5 mice received insulin A; 6 mice received insulin B; 10 mice received oral porcine insulin (5); 10 mice were untreated controls. Definition of diabetes is blood glucose consistently >350 mg/dL.

Figure 2

DNA immunization protocols. Constructs were generated as described in Methods (see also reference 25), and plasmid injections were given intramuscularly into the quadriceps femoris muscle on each side (50 μg in 50 μL saline per injection), by 1 of the 3 protocols shown. For each protocol, the paired DNA injections are indicated by arrows; time between each set of injections was 7 days (protocol 1) or 3 days (protocols 2 and 3). The development of IDDM was followed for >3 months after LCMV infection. *Time at which LCMV-specific CTL responses were evaluated.

Figure 3

Immunization with DNA plasmids expressing the insulin B chain, but not the LCMV-NP, is effective in preventing IDDM in RIP-NP mice. RIP-NP transgenic mice were treated with pCMV-NP (protocols 1, 2, or 3; Figure 2), with pCMV-insB (protocol 1; Figure 2), or were fed oral porcine insulin (see Methods). Diabetes was measured weekly by Accucheck (see Methods); the total observation period was 3 months. Group sizes were as follows: 10 mice received pCMV-insB; 10 mice received pCMV-NP; 10 mice received oral porcine insulin (5); 10 mice were untreated controls; 4 mice received pCMV. Definition of diabetes is blood glucose consistently >350 mg/dL. Note that injection of pCMV (protocol 3) did not lower diabetes incidence.

Figure 4

Affinities of NP-specific CTL following DNA immunization. LCMV-NP CTLs were determined by using serial log dilutions of LCMV-NP H-2^d peptide on syngeneic BALB/c targets in a 5-hour ⁵¹Cr-release assay. The overall plateau release was decreased in pCMV-NP–treated groups compared with pCMV-treated controls; however, the fall-off of the curve was not shifted, indicating that there were no significant affinity differences. Decrease in CTL numbers was confirmed by pCTL analysis (Table 1). Three mice per group were injected with pCMV or pCMV-NP, according to protocol 2 in Figure 2.