Oral Delivery of Glutamic Acid Decarboxylase (GAD)-65 and IL10 byLactococcus lactisReverses Diabetes in Recent-Onset NOD Mice

Abstract

Growing insight into the pathogenesis of type 1 diabetes (T1D) and numerous studies in preclinical models highlight the potential of antigen-specific approaches to restore tolerance efficiently and safely. Oral administration of protein antigens is a preferred method for tolerance induction, but degradation during gastrointestinal passage can impede such protein-based therapies, reducing their efficacy and making them cost-ineffective. To overcome these limitations, we generated a tolerogenic bacterial delivery technology based on live Lactococcus lactis (LL) bacteria for controlled secretion of the T1D autoantigen GAD65 and the anti-inflammatory cytokine interleukin-10 in the gut. In combination with short-course low-dose anti-CD3, this treatment stabilized insulitis, preserved functional b-cell mass, and restored normoglycemia in recent-onset NOD mice, even when hyperglycemia was severe at diagnosis. Combination therapy did not eliminate pathogenic effector T cells, but increased the presence of functional CD4+ regulatory T cells. These preclinical data indicate a great therapeutic potential of orally administered autoantigen-secreting LL for tolerance induction in T1D.Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of insulin-producing b-cells in the islets of Langerhans in the pancreas (1). Past and current attempts to cure this immune-mediated disease included antigen-specific therapies and systemic immunomodulatory and immunosuppressive agents (1-4), but many of these trials suffered from a lack of efficacy, as reflected by only transient b-cell preservation, or adverse effects due to immune suppression. Autoantigenspecific therapies remain appealing to restore tolerance in T1D patients because they hold promise in targeting only b-cell-reactive T cells without impairing normal immune responses to diabetes-unrelated antigens. Studies in man and NOD mice, the most widely used preclinical model of T1D, have identified glutamic acid decarboxylase (GAD65) as one of the major autoantigens in this disease (5,6). GAD65 is expressed not only in the central nervous system but also in the b-cell (7), and peptide sequences within GAD65 behave as autoantigens in T1D both in man and in NOD mice (5,8). The majority of prediabetic and newly diagnosed T1D patients test positive for anti-GAD65 autoantibodies (6,9) or GAD65-reactive T cells (10). In NOD mice, GAD65-specific T-cell responses can be detected as early as 4 weeks of age (5). Although its role in initiating human T1D remains controversial, preclinical studies in NOD mice have shown that vaccines based on GAD65

Engineered bacteria as therapeutic agents

“…Responding mice showed normal glucose levels in blood, and remained responsive to other antigens, suggesting an antigen-specific immunosuppression. A similar combination therapy based on anti-CD3 Abs and the oral delivery of engineered L. lactis secreting IL-10 and the diabetes-related auto-antigen glutamic acid decarboxylase (GAD)-65 has been reported [17]. This combination was effective in the treatment of advanced stages of T1D with severe hyperglycemia.…”

Sustainable therapies by engineered bacteria

“…LAB have been also engineered to induce mucosal immune tolerance of causative antigens in food allergies, such coeliac disease (Huibregtse et al ., 2009). Induction of tolerance has also been applied to downregulate autoimmune diseases like type 1 diabetes (T1D), in which self‐antigens from pancreatic β‐cells are recognized by the immune system (Takiishi et al ., 2012; Robert et al ., 2014). In addition, engineered bacteria can modulate levels of cytokines outside the GIT, preventing cardiovascular pathologies (Jing et al ., 2011) and encephalomyelitis (Rezende et al ., 2013).…”

Emerging technologies to achieve oral delivery of GLP-1 and GLP-1 analogs for treatment of type 2 diabetes mellitus (T2DM)

“…L. lactis (AG013) has also been modified to secrete trefoil factor 1 for the treatment of oral mucositis, and is currently in Phase Ib clinical trials [50]. Mucosal delivery of recombinant L. lactis MG1363 secreting the diabetic autoantigen proinsulin or glutamic acid decarboxylase (GAD 65) along with IL-10 reversed T1D in NOD (non obese diabetic) mice [51,52], while oral vaccination with recombinant L. plantarum NCL21 expressing a common Japanese cedar pollen allergen, Cry j 1(Cry j 1-LAB), ameliorated the symptoms of cedar pollinosis and decreased allergen-specific IgE production in a murine model of cedar pollinosis [53]. A recent progress made in the application of LAB as live mucosal vectors is the production and delivery of GLP-1 and GLP-1analogs for the treatment of T2DM.…”