Protection of NOD Mice From Type 1 Diabetes After Oral Inoculation with Vaccinia Viruses Expressing Adjuvanted Islet Autoantigens

Abstract

Oral administration of autoantigens and allergens can delay or suppress clinical disease in experimental autoimmune and allergic disorders. However, repeated feeding of large amounts of the tolerogens is required over long periods and is only partially effective in animals systemically sensitized to the ingested antigen. Enhanced suppression of type 1 autoimmune diabetes insulitis and hyperglycemia was demonstrated in both naive and immune animals following oral inoculation with plant-based antigens coupled to the cholera toxin B subunit (CTB). Thus, plant-synthesized antigens linked to the CTB adjuvant, can enhance suppression of inflammatory TH1 lymphocyte-mediated autoreactivity in both naive and immune animals. To stimulate adjuvant-autoantigen fusion protein biosynthesis in the gut mucosae, the authors evaluated oral inoculation of juvenile non-obese diabetic (NOD) mice with recombinant vaccinia virus (rVV) expressing fusion genes encoding CTB linked to the pancreatic islet autoantigens proinsulin (INS) and a 55-kDa C-terminal peptide from glutamate decarboxylase (GAD55). Hyperglycemia in both rVV-CTB:: INS and rVV-CTB:: GAD inoculated mice was substantially reduced in comparison with the uninoculated mouse control. Oral inoculation with rVV carrying the CTB::INS fusion gene generated a significant reduction in insulitis. An increase in IgG1 in comparison with IgG2c antibody isotype titers in rVV-CTB::INS infected mice suggested possible activation of autoantigen specific Th2 lymphocytes. The experimental results demonstrate feasibility of using vaccinia virus oral delivery of adjuvanted autoantigens to the mucosae of prediabetic mice for suppression and therapy of type 1 autoimmune diabetes.

Autoantigens Plus Interleukin-10 Suppress Diabetes Autoimmunity

“…Construction, propagation, and purification of the recombinant viruses expressing CTB::GAD and IL-10 (recombinant VV [rVV]-CTB::GAD and rVV-IL10) and of control virus rVV-L15 were previously described. [48][49][50] Virus titers were determined by plaque assay on CV-1 cells. The virus constructs used in this study are presented in Figure 1.…”

“…48 The Lister vaccine (LIVP) strain of VV was used as the parental virus. Construction, propagation, and purification of the recombinant viruses expressing CTB::GAD and IL-10 (recombinant VV [rVV]-CTB::GAD and rVV-IL10) and of control virus rVV-L15 were previously described.…”

“…A score of 4 indicated massive intra-islet T-cell infiltration involving 70-100% of the islet. 48 Analyses of cytokine-secreting splenocytes After more than 1 year (64 weeks), the surviving mice were euthanized by CO 2 asphyxiation. The spleens were immediately excised from each animal (n ¼ 4-9 mice per group) and teased apart with scissors into cold RPMI 1640 medium without fetal bovine serum.…”

“…42 Low-level diabetes suppression was observed following vaccinia virus (VV)-mediated mucosal or intraperitoneal inoculation of NOD mice with CTB::GAD fusion or IL-10. 48,49 Here we show that a systemically delivered combination of VVs expressing autoantigen CTB::GAD and the cytokine IL-10 is highly effective in long-term prevention of the onset of diabetes in NOD mice.…”

“…Insulitis scores of 1-2 indicated progressively increasing levels of periislet insulitis, and scores of 3-4 indicated progressive levels of intra-islet insulitis, with a score of 4 equivalent to total invasion of the islet by autoreactive lymphocytes. 48 Computer-assisted morphometry measurement of pancreatic islets…”