Reversal of Hyperglycemia and Suppression of Type 1 Diabetes in the NOD Mouse with Apoptotic DNA Immunotherapy™ (ADi™), ADi-100.

The antigen-specific apoptotic DNA immunotherapeutic, ADi-100, is designed to suppress type 1 diabetes and consists of two DNA plasmids encoding genetic sequences of the apoptosis-inducing molecule, BAX, and the secreted form of the autoantigen, glutamic acid decarboxylase 65, that is CpG hyper-methylated to avoid inflammatory signaling (msGAD55). Upon a four-day treatment with ADi-100 of young female non-obese diabetic (NOD) mice, the frequency of various tolerogenic dendritic cell populations increased in draining lymph nodes; these cells lost the capacity to stimulate glutamic acid decarboxylase (GAD)-specific CD4+ T lymphocytes and were associated with the previously demonstrated enhancement of GAD-specific regulatory T cells. The efficacy of two ADi-100 formulations containing different proportions of BAX and msGAD55, 1:4 (10/40 µg) and 1:2 (17/33 µg), was evaluated in mildly hyperglycemic pre-diabetic NOD female mice. Both formulations suppressed the incidence of diabetes by 80% in an antigen-specific manner, while all untreated mice developed diabetes. However, treatment of pre-diabetic mice with significantly higher hyperglycemia, denoting progressive disease, showed that ADi-100 1:2 strongly suppressed diabetes incidence by 80% whereas the ADi-100 1:4 was less effective (50%). As an antigen-specific monotherapy, ADi-100 is highly efficacious in reversing elevated hyperglycemia to prevent diabetes, in which increasing apoptosis-inducing BAX content is a promising immune tolerance feature.

Recent patents on immunoregulatory DNA vaccines for autoimmune diseases and allograft rejection.

The goal of immunoregulatory DNA vaccination is the antigen- and tissue-specific suppression of pathological inflammation that underlies immune-mediated inflammatory disorders like autoimmune diseases and allograft rejection. Recent patents and patent applications have applied immunoregulatory DNA vaccines in rodent model systems and human clinical trials using plasmid DNA coding for autoantigens such as insulin and glutamic acid decarboxylase for type 1 diabetes, myelin-associated proteins for multiple sclerosis, and heat-sock protein 60 for rheumatoid arthritis. In these cases, the objective is to induce a homeostatic-like regulatory immune response to suppress pathological inflammation. In addition, patent applications have disclosed the use of DNA vaccines encoding the pro-inflammatory MIF cytokine and the CD25 IL-2 receptor subunit to interfere with the inflammatory process. Approaches have also been taken to improve DNA vaccination efficacy, including covalent modification of plasmid DNA, engineering secretion of vaccine-encoded antigen, and co-delivery of DNA coding for anti-inflammatory cytokines, a mutant co-stimulatory molecule, a growth factor, or a pro-apoptotic protein. Furthermore, a patent application has disclosed the use of a DNA vaccine previously shown to treat successfully an autoimmune disease to prolong allograft survival. Taken together, these patents and patent applications indicate a promising bench-to-bedside potential for immunoregulatory DNA vaccination applied to autoimmune diseases and allograft rejection.

DNA vaccines for transplantation.

IMPORTANCE OF THE FIELD: DNA vaccination for transplantation has been less investigated compared with DNA vaccines for infectious disease, cancer and pathological autoimmunity. However, the emerging role of transplant-induced autoimmunity in allograft rejection may lead to the development of new DNA vaccination approaches where peripheral delivery of an antigen shared by recipient and allograft could be used to prevent rejection of a given organ for large numbers of individuals. In addition, apoptosis-inducing DNA vaccines could further minimize the need to identify specific donor antigens for induction of tolerance by generating apoptotic cells carrying donor or recipient antigens depending on site of vaccine delivery. AREA COVERED IN THIS REVIEW: The review covers DNA vaccination approaches that have been taken to prolong allograft survival in different animal model systems and their possible immune mechanisms and speculates on the future role of DNA vaccines for transplantation. WHAT THE READER WILL GAIN: The review gives insight into how new DNA vaccination strategies might be developed to take into account recent development in allotransplantation. TAKE HOME MESSAGE: DNA vaccination could reduce the need for systemic immunosuppressants and be applied to prevention of chronic rejection, which remains a major barrier to successful allotransplantation.

A therapeutic DNA vaccination strategy for autoimmunity and transplantation.

De novo autoimmunity induced by an allograft may play a significant role in chronic organ rejection, which remains a major barrier to successful transplantation. Accordingly, immunization with non-polymorphic antigens found in both donor allograft and recipient would be an attractive means to prevent long-term graft rejection, because it would rely on recipient mechanisms of immune homeostasis and could minimize the need to identify appropriate donor polymorphic antigens for induction of graft tolerance. Here we show that intradermal injection of plasmid DNA encoding glutamic acid decarboxylase (GAD) polypeptide, which is synthesized in both pancreatic islet and skin tissue, ameliorated new-onset type 1 diabetes in NOD mice and increased skin allograft survival in a BALB/c-C57BL/6 model system in a donor-specific manner. Successful therapy of autoimmune diabetes required CpG-methylation of plasmid DNA and co-delivery of a cDNA coding for the pro-apoptotic BAX protein, which was shown previously to induce Foxp3(+) regulatory T cells in NOD mice. In contrast, significantly increased skin allograft survival after immunization of recipient only required CpG-methylation of plasmid DNA coding for GAD alone. Injection of unmethylated plasmid DNA coding for BAX alone near the allograft also promoted graft survival, but induced a pro-inflammatory response to self-antigens. Our results reveal a promising potential for autoimmunity-targeting DNA vaccination to be applied to transplantation.

The effect of hyperglycemia on pulpal healing in rats.

Diabetes mellitus (DM) may impede healing of dental pulps. In this study, the effect of hyperglycemia on pulpal healing was determined in exposed rat pulps capped with mineral trioxide aggregate. Two groups of 11 rats received injections of saline (control group) or streptozotocin to induce hyperglycemia (DM group). The pulps of the maxillary first molars of all rats were exposed and capped. Intact teeth and teeth with exposed pulps without restorations served as positive and negative controls, respectively. Histologic samples were prepared and evaluated for dentin bridge formation and pulpal inflammation. Data were analyzed by using Fisher exact, Mann-Whitney U, and Spearman correlation tests. Dentin bridge formation was inhibited in diabetic rats (p = 0.029) along with more inflammation in these pulps (p = 0.005). There was an inverse association between dentin bridge formation and inflammatory cell infiltration (p = 0.001). Based on these results, it appears that hyperglycemia adversely affects pulpal healing in rats.

Bioluminescent mammalian cells grown in sponge matrices to monitor immune rejection.

The growth and bioluminescence of cells seeded in collagen and gelatin sponge matrices were compared in vitro under different conditions, and immune rejection was quantified and visualized directly in situ based on loss of bioluminescence activity. Mammalian cells expressing a Renilla luciferase complementary deoxyribonucleic acid (cDNA) were used to seed collagen and gelatin sponge matrices soaked in either polylysine or gelatin to determine optimal growth conditions in vitro. The sponges were incubated in tissue culture plates for 3 weeks and received 2, 9, or 15 injections of coelenterazine. Measurements of bioluminescence activity indicated that gelatin sponges soaked in gelatin emitted the highest levels of light emission, multiple injections of coelenterazine did not affect light emission significantly, and light emission from live cells grown in sponges could be measured qualitatively but not quantitatively. Histologic analysis of sponge matrices cultured in vitro showed that cells grew best in gelatin matrices. Visualization of subcutaneously implanted sponges in mice showed accelerated loss of light emission in immunocompetent BALB/c mice compared with immunodeficient BALB/c-scid mice, which was associated with increased cell infiltration. Our results indicate that sponge matrices carrying bioluminescent mammalian cells are a valid model system to study immune rejection in situ.

Saving death: apoptosis for intervention in transplantation and autoimmunity.

Long considered immunologically "bland," apoptotic cells are now recognized as important modulators of immune responses. The role of apoptosis in immunological homeostasis has been inferred from several findings, for example, induction of tolerance after injection of apoptotic cells and the capacity of APCs like macrophages and DCs to induce and maintain tolerance after phagocytosis of dead cells. Processing of apoptotic cells by DCs is of particular interest, because DCs are the only known APCs capable of activating naïve T lymphocytes to become effector or regulatory cells. In that regard, recent evidence suggests that phagocytosis of apoptotic cells by DCs can induce Tregs, a finding that has significant implications for the treatment of a variety of immune-mediated inflammatory disorders. Here, we review the relationship between apoptotic cells, DCs, and Tregs, and its impact on prevention of transplant rejection and treatment of autoimmune diseases.

Pro-apoptotic DNA vaccination ameliorates new onset of autoimmune diabetes in NOD mice and induces foxp3+ regulatory T cells in vitro.

We have shown previously that incorporation of a cDNA coding for the pro-apoptotic protein BAX into plasmid DNA coding for a secreted form of the pancreatic beta-cell antigen glutamic acid decarboxylase (GAD) promotes prevention of type 1 diabetes in non-obese diabetic (NOD) mice. Here we present evidence indicating that injection of the same vaccine at time of early diabetes onset could ameliorate the disease with efficacy, with 42% of mice overtly diabetic by 40 weeks of age compared to 92% in control groups. In addition, immunological analysis revealed that the DNA vaccine induced CD4(+)CD25(+) T cells cultured from draining lymph nodes that had immunosuppressive function in vitro. The induced regulatory T cells (Tregs) expressed the foxp3 gene and showed cell-contact-dependent as well as TGF-beta- and IL-10-independent immunosuppressive activity. Data also revealed that CD4(+)CD25(-) T cells from mice immunized with the DNA vaccine yielded a cell population that was foxp3(+), showed increased expression of CD25 compared to control, and had immunosuppressive function in vitro, indicating that Tregs could have developed from antigen-induced, peripheral T lymphocytes. In contrast, injection of DNA coding for SGAD55 or BAX alone did not induce Tregs. Altogether, our data confirm that pro-apoptotic DNA vaccination can be used as an immunosuppressive strategy and demonstrate its potential for therapy of pathological autoimmunity.

Co-delivery of pro-apoptotic BAX with a DNA vaccine recruits dendritic cells and promotes efficacy of autoimmune diabetes prevention in mice.

Genetic vaccines encoding pancreatic beta cell antigens can prevent autoimmune (type 1) diabetes when delivered into murine model systems, but there is a need to improve their efficacy. Here, we investigated the effects of intramuscular delivery of DNA coding for the pro-apoptotic protein BAX together with an intracellular or a secreted form of the beta cell antigen glutamic acid decarboxylase (GAD) on diabetes onset and immune responses in non-obese diabetic (NOD) mice. We hypothesized that induction of apoptosis in vaccine-containing cells could lead to GAD tolerance and disease suppression. Remarkably, monitoring of spontaneous diabetes onset indicated that only delivery of DNA coding for secreted GAD and BAX resulted in significant prevention of the disease. Using GFP as a model plasmid-encoded antigen revealed that co-delivery of BAX resulted in the recruitment of GFP-containing dendritic cells (DCs) in the draining lymph nodes and spleen of NOD mice. Furthermore, data indicated that subcellular localization of GAD had an effect on both the number and function of antigen presenting cells (APCs) recruited by BAX as well as on IFN-gamma secretion, and that diabetes suppression was unlikely to be caused by increased T helper 2 (Th2)-like activity. Our results indicate that, under certain conditions, co-delivery of DNA encoding BAX can improve the efficacy of genetic vaccination for prevention of pathogenic autoimmunity via a mechanism likely to involve modulation of antigen presenting cell function. In addition, our data also suggest that properties associated with subcellular localization of an antigen in apoptotic cells can have a significant effect on induced immune responses.

Intradermal or oral delivery of GAD-encoding genetic vaccines suppresses type 1 diabetes.

Genetic vaccines are promising candidates for prevention of type 1 diabetes, an autoimmune disease resulting from cell-mediated destruction of pancreatic beta cells. It is known that the prophylactic effect and immune responses induced by administration of a genetic vaccine can depend on site of delivery. In the work presented here, we used the NOD mouse model for type 1 diabetes to evaluate different routes of delivery for DNA vaccines coding for the beta-cell antigen glutamic acid decarboxylase (GAD). Plasmid DNA coding for intracellular or secreted GAD was given via either the intramuscular (i.m.), intradermal (i.d.), or oral route, using, respectively, 300, 100, or 300 micro g DNA per mouse. Results indicated that both i.d. and oral delivery of GAD-encoding DNA were more effective than i.m. delivery for disease suppression. In addition, cytokine-specific ELISpot analysis indicated that immune responses induced by the different immunization protocols were more dependent on the cellular localization of GAD antigen than on the delivery route, while ELISA of anti-GAD serum antibody isotypes indicated that i.d. delivery of DNA was most likely to induce a Th2-like response. Our results suggest that i.d. or oral delivery of a genetic vaccine for type 1 diabetes might be preferable over the i.m. route in a future clinical setting.

Decreased insulitis and blood glucose levels after injection of GAD-transduced lymphocytes into NOD mice.

We used NOD mice to investigate the effects of injecting transduced lymphocytes on insulitis, nonfasting blood glucose levels, and immune responses. Syngeneic splenocytes were transduced with retroviral particles carrying a cDNA construct encoding the beta cell antigen glutamic acid decarboxylase (GAD65), a secreted form of GAD65 (SGAD55), or secreted alkaline phosphatase (SEAP) as a control antigen. Different multiplicities of infection (m.o.i.) were used with different constructs. Four-week-old NOD mice received intravenous injection of CD4(+) cells isolated from transduced splenocytes, and insulitis and blood glucose levels were determined at 10 weeks of age. No significant effects were observed with lymphocytes transduced with gad65 and sgad55 constructs at low m.o.i. By contrast, at high m.o.i., lymphocytes transduced with the sgad55 and seap constructs caused a decrease in insulitis and blood glucose levels and in insulitis alone, respectively. ELISA of anti-GAD antibody isotypes indicated that GAD-transduced lymphocytes induced similar Th2-like responses at all m.o.i. These results suggest that retroviral particles carrying sgad55 can be used for engineering cell vaccines for type 1 diabetes and provide further evidence that Th2-like responses induced by immunization may not always be a primary cause of diabetes suppression in NOD mice.