**3.1. Insulin DNA vaccines**

Thus far, the only DNA vaccine that has been tested in both preclinical and clinical trials is a plasmid DNA construct coding for intracellular proinsulin, which is a partially processed nonfunctional form of insulin. Insulin is not only the hormone produced by beta-cells that controls carbohydrate and fat metabolism in the body, it is also a main target autoantigen in autoim‐ mune diabetes and the presence of anti-insulin autoantibodies can be an indication of disease initiation [59]. DNA vaccines coding for different forms of insulin have been investigated for type 1 diabetes immunotherapy since the late 1990's. The first report to demonstrate efficacy used a virus-induced diabetic mouse model system, and showed that intramuscular injection of plasmid DNA encoding the insulin B chain reduces the incidence of diabetes (blood glucose > 350 mg/dL) from 100% down to 50% [60]. The DNA vaccine induced insulin B-chain specific CD4+ T regulatory cells that secreted interleukin-4, and locally reduced autoreactive activity of cytotoxic T lymphocytes in the pancreatic draining lymph nodes. Further work showed that co-delivery of interleukin-4 was required to prevent diabetes onset in male nonobese diabetic mice [61].

that a single amino acid substitution in B7-1 (denoted B7-1wa) could abrogate specific binding to CD28 but not to CTLA-4. Co-delivery of B7-1wa and preproinsulin I encoded by plasmid DNA abrogated reactivity to insulin and ameliorated type 1 diabetes in nonobese diabetic mice, although delivery of either preproinsulin I or B7-1wa alone did not suppress the disease [65]. Interferon-gamma and interleukin-4 were both depressed, arguing against a T helper-2 bias, but reactivity to glutamic acid decarboxylase 65 was not altered. Suppressor cells were not identified, suggesting induction of tolerance to insulin by either T cell anergy or deletion in

DNA Immunotherapies for Type 1 Diabetes http://dx.doi.org/10.5772/55727 545

Among the most promising reports of insulin DNA vaccination is a plasmid DNA construct encoding mouse proinsulin II that could reduce the incidence of diabetes in nonobese diabetic mice when administered intramuscularly to prediabetic 8-week old mice (prophylactic setting), and to diabetic mice older than 12-week (therapeutic setting) with blood glucose > 170 mg/dL [66, 67]. The efficacy of the vaccine was improved by increasing the level of expression of insulin, frequency of dosing, dosage, and subcelullar localization modification of the autoantigen to the intracellular compartment instead of being secreted. In the prophylactic setting, 8 weekly injections of 50 microgram of the DNA vaccine decreased the incidence of diabetes from 80% in the control group down to 45% in 25-week old mice. The treatment caused increased numbers of interferon-gamma secreting cells and a decrease in insulin autoantibod‐ ies. In the therapeutic setting, the DNA vaccine reduced diabetes from 100% in the control groups down to 25% in treated mice 25 weeks post treatment initiation. The treatment induced increased numbers of insulin-specific interferon-gamma-producing T cells and levels of interleukin-10, which suggested the possible induction of T regulatory-1 cells. Adoptive transfer experiments indicated that the protection was not mediated by the induction of

Most important, a similar DNA vaccine encoding human proinsulin was used in phase I/II clinical trials and has been the only human trial of a DNA vaccine for diabetes conducted to date [68, 69]. Construct BHT-3021 was delivered intramuscularly using four doses of plasmid DNA, i.e., 0.3, 1, 3 and 6 milligram, administered once a week for 12 weeks. The interim results for the 1 milligram dose showed pancreatic beta-cell preservation, demonstrated by a mean 17% increase in C-peptide levels with BHT-3021 by week 15 after enrollment, whereas placebo patients experienced a mean 42% decrease in C-peptide. Evidence for immune tolerance was suggested by a mean 17% reduction in anti-insulin antibodies, and 25% reduction in antiglutamic acid decarboxylase 65 antibodies by week 15 after enrollment, whereas placebo patients experienced a mean 6% and 4% increase, respectively. The most recent report of the trial claimed that BHT-3021 could preserve C-peptide levels for at least six months and one year in some of the patients from the point of initiation of the therapy [15]. These results together with its favorable side-effects profile appear to be somewhat comparable to those reported with anti-CD3 monoclonal antibody, and the glutamic acid decarboxylase 65 protein

However, thus far, no immunotherapy alone has been reported to cause long term remission of clinical type 1 diabetes. This was confirmed with the announcements in 2011 of the failure of three phase III clinical trials for type 1 diabetes that tested two anti-CD3 monoclonal

this model.

CD4+

CD25+

vaccines for type 1 diabetes.

T regulatory cells.

Two isoforms of insulin are synthesized in rodent animals, insulin I in islets and insulin II in both islets and thymus while humans have only one form of insulin. The pancreatic beta cells synthesize proinsulin before converting it to functional insulin. In that regard, intranasal delivery of plasmid DNA encoding mouse proinsulin II together with injection of an anti-CD154 (also named CD40 ligand) antibody to prevent T cell activation was reported to prevent type 1 diabetes in nonobese diabetic mice [62]. Delivery of 300 microgram DNA and 50 microgram antibody over a 2-week interval at 4 weeks of age synergistically prevented diabetes, reducing disease incidence from 100% diabetic down to 0% in 40-week old mice. Injection of the anti-CD154 antibody alone reduced the incidence to 50%. However, delivery of the DNA vaccine alone did not reduce diabetes incidence, even though it could induce T regulatory cells and reduce insulitis.

Another report has shown that co-delivery of 50 microgram plasmid DNA encoding human proinsulin together with 100 microgram insulin peptide twice over a 2-week interval could prevent diabetes until 24 weeks of age in 6 week old nonobese diabetic mice. In contrast, DNA or peptide alone did not prevent disease [63]. Results also indicated induction of CD4+ CD25 islet specific T regulatory cells producing transforming growth factor-beta only in the coimmunization group.

In another study, a DNA vaccine encoding proinsulin and pancreatic regenerating (Reg) III protein resulted in a significant reduction of hyperglycemia and diabetes incidence with increased serum insulin in a streptozotocin- induced mice model [64]. The treatment also restored the balance of T helper-1/T helper-2 cytokines, expanded CD4+ CD25+ Foxp3+ T regulatory cells, and attenuated insulitis by inhibiting activation of nuclear factor-kappa B (NFκB) in the pancreas, which is thought to promote the regeneration of islet beta cells.

Cytotoxic T lymphocyte antigen 4 (CTLA-4 or CD152) is a strong negative regulator of T cell activity and another example of an immunomodulator that can be co-delivered with an autoantigen. Like CD28 (a positive regulator), CTLA-4 binds to B7-1 and B7-2. It was found that a single amino acid substitution in B7-1 (denoted B7-1wa) could abrogate specific binding to CD28 but not to CTLA-4. Co-delivery of B7-1wa and preproinsulin I encoded by plasmid DNA abrogated reactivity to insulin and ameliorated type 1 diabetes in nonobese diabetic mice, although delivery of either preproinsulin I or B7-1wa alone did not suppress the disease [65]. Interferon-gamma and interleukin-4 were both depressed, arguing against a T helper-2 bias, but reactivity to glutamic acid decarboxylase 65 was not altered. Suppressor cells were not identified, suggesting induction of tolerance to insulin by either T cell anergy or deletion in this model.

**3.1. Insulin DNA vaccines**

CD4+

544 Type 1 Diabetes

mice [61].

regulatory cells and reduce insulitis.

immunization group.

Thus far, the only DNA vaccine that has been tested in both preclinical and clinical trials is a plasmid DNA construct coding for intracellular proinsulin, which is a partially processed nonfunctional form of insulin. Insulin is not only the hormone produced by beta-cells that controls carbohydrate and fat metabolism in the body, it is also a main target autoantigen in autoim‐ mune diabetes and the presence of anti-insulin autoantibodies can be an indication of disease initiation [59]. DNA vaccines coding for different forms of insulin have been investigated for type 1 diabetes immunotherapy since the late 1990's. The first report to demonstrate efficacy used a virus-induced diabetic mouse model system, and showed that intramuscular injection of plasmid DNA encoding the insulin B chain reduces the incidence of diabetes (blood glucose > 350 mg/dL) from 100% down to 50% [60]. The DNA vaccine induced insulin B-chain specific

 T regulatory cells that secreted interleukin-4, and locally reduced autoreactive activity of cytotoxic T lymphocytes in the pancreatic draining lymph nodes. Further work showed that co-delivery of interleukin-4 was required to prevent diabetes onset in male nonobese diabetic

Two isoforms of insulin are synthesized in rodent animals, insulin I in islets and insulin II in both islets and thymus while humans have only one form of insulin. The pancreatic beta cells synthesize proinsulin before converting it to functional insulin. In that regard, intranasal delivery of plasmid DNA encoding mouse proinsulin II together with injection of an anti-CD154 (also named CD40 ligand) antibody to prevent T cell activation was reported to prevent type 1 diabetes in nonobese diabetic mice [62]. Delivery of 300 microgram DNA and 50 microgram antibody over a 2-week interval at 4 weeks of age synergistically prevented diabetes, reducing disease incidence from 100% diabetic down to 0% in 40-week old mice. Injection of the anti-CD154 antibody alone reduced the incidence to 50%. However, delivery of the DNA vaccine alone did not reduce diabetes incidence, even though it could induce T

Another report has shown that co-delivery of 50 microgram plasmid DNA encoding human proinsulin together with 100 microgram insulin peptide twice over a 2-week interval could prevent diabetes until 24 weeks of age in 6 week old nonobese diabetic mice. In contrast, DNA or peptide alone did not prevent disease [63]. Results also indicated induction of CD4+

islet specific T regulatory cells producing transforming growth factor-beta only in the co-

In another study, a DNA vaccine encoding proinsulin and pancreatic regenerating (Reg) III protein resulted in a significant reduction of hyperglycemia and diabetes incidence with increased serum insulin in a streptozotocin- induced mice model [64]. The treatment also

regulatory cells, and attenuated insulitis by inhibiting activation of nuclear factor-kappa B (NF-

Cytotoxic T lymphocyte antigen 4 (CTLA-4 or CD152) is a strong negative regulator of T cell activity and another example of an immunomodulator that can be co-delivered with an autoantigen. Like CD28 (a positive regulator), CTLA-4 binds to B7-1 and B7-2. It was found

restored the balance of T helper-1/T helper-2 cytokines, expanded CD4+

κB) in the pancreas, which is thought to promote the regeneration of islet beta cells.

CD25-

Foxp3+ T

CD25+

Among the most promising reports of insulin DNA vaccination is a plasmid DNA construct encoding mouse proinsulin II that could reduce the incidence of diabetes in nonobese diabetic mice when administered intramuscularly to prediabetic 8-week old mice (prophylactic setting), and to diabetic mice older than 12-week (therapeutic setting) with blood glucose > 170 mg/dL [66, 67]. The efficacy of the vaccine was improved by increasing the level of expression of insulin, frequency of dosing, dosage, and subcelullar localization modification of the autoantigen to the intracellular compartment instead of being secreted. In the prophylactic setting, 8 weekly injections of 50 microgram of the DNA vaccine decreased the incidence of diabetes from 80% in the control group down to 45% in 25-week old mice. The treatment caused increased numbers of interferon-gamma secreting cells and a decrease in insulin autoantibod‐ ies. In the therapeutic setting, the DNA vaccine reduced diabetes from 100% in the control groups down to 25% in treated mice 25 weeks post treatment initiation. The treatment induced increased numbers of insulin-specific interferon-gamma-producing T cells and levels of interleukin-10, which suggested the possible induction of T regulatory-1 cells. Adoptive transfer experiments indicated that the protection was not mediated by the induction of CD4+ CD25+ T regulatory cells.

Most important, a similar DNA vaccine encoding human proinsulin was used in phase I/II clinical trials and has been the only human trial of a DNA vaccine for diabetes conducted to date [68, 69]. Construct BHT-3021 was delivered intramuscularly using four doses of plasmid DNA, i.e., 0.3, 1, 3 and 6 milligram, administered once a week for 12 weeks. The interim results for the 1 milligram dose showed pancreatic beta-cell preservation, demonstrated by a mean 17% increase in C-peptide levels with BHT-3021 by week 15 after enrollment, whereas placebo patients experienced a mean 42% decrease in C-peptide. Evidence for immune tolerance was suggested by a mean 17% reduction in anti-insulin antibodies, and 25% reduction in antiglutamic acid decarboxylase 65 antibodies by week 15 after enrollment, whereas placebo patients experienced a mean 6% and 4% increase, respectively. The most recent report of the trial claimed that BHT-3021 could preserve C-peptide levels for at least six months and one year in some of the patients from the point of initiation of the therapy [15]. These results together with its favorable side-effects profile appear to be somewhat comparable to those reported with anti-CD3 monoclonal antibody, and the glutamic acid decarboxylase 65 protein vaccines for type 1 diabetes.

However, thus far, no immunotherapy alone has been reported to cause long term remission of clinical type 1 diabetes. This was confirmed with the announcements in 2011 of the failure of three phase III clinical trials for type 1 diabetes that tested two anti-CD3 monoclonal antibodies and a GAD65 protein vaccine. The need to increase therapeutic efficacy has generated an increased interest in combination immunotherapies [70-72]. Indeed, it is reason‐ able to anticipate that a variety of synergistic and additive effects may be induced by combining different agents. For example, combination treatment with anti-CD3 epsilon specific antibody and intranasal delivery of proinsulin peptide could reverse recent onset diabetes in nonobese diabetic mice as well as in a virus-induced diabetic mouse model with much higher efficacy than monotherapy using anti-CD3 or peptide alone [71]. Protection was associated with expansion of CD25+ Foxp3+ and insulin specific T regulatory cells producing protective cytokines, such as interleukin-10, transforming growth factor-beta, and interleukin-4. In addition, these cells can transfer dominant tolerance to recent onset diabetic recipients, and suppress heterologous autoaggressive CD8 T cell responses. While animal studies do provide a rationale for combining therapies, there are hurdles that still need to be overcome for translation to the clinic. For example, the possibility of unforeseen drug interactions is not in the interest of pharmaceutical and biotech companies and could stop them from evaluating their drugs in combination trials [72].

cell population in the spleen, with higher production of interleukin-10 in the spleen and islets, and with a decreased infiltration of CD8 T lymphocytes in the islets. The same DNA vaccine with the same dose and delivery reduced the occurrence of diabetes from 100% to 33% in 28 week old nonobese diabetic mice when injected at 4-week of age, and was associated with a reduction in CD4 and CD8 T cells infiltration, appearance of CD25 cells, and increased levels

DNA Immunotherapies for Type 1 Diabetes http://dx.doi.org/10.5772/55727 547

Glutamic acid decarboxylase 65 is an enzyme that catalyzes the synthesis of gamma-amino‐ butyric acid (GABA) which acts as a neuroinhibitor as well as an immunoregulatory molecule. Evidence indicates that glutamic acid decarboxylase 65 may have a critical early role in mediating islet beta cell destruction and is an important target autoantigen in type 1 diabetes. Detection of anti-glutamic acid decarboxylase antibodies in the sera of prediabetic patients is a reliable predictive marker for the progression to overt diabetes, and anti-glutamic acid decarboxylase reactivity can be detected in nonobese diabetic mice model early in the disease

Plasmid DNA vaccines coding for glutamic acid decarboxylase 65 are currently at the preclin‐ ical stage. The first report of a beneficial effect in nonobese diabetic mice showed that plasmid DNA encoding wild-type intracellular or engineered secreted glutamic acid decarboxylase, i.e., a fusion of the interleukin-2 signal peptide with a truncated form of human glutamic acid decarboxylase 65, caused decreased insulitis compared to plasmid vector alone when deliv‐ ered intramuscularly, and was accompanied by elevated secretion of interleukin-4 by spleno‐ cytes [84]. A subsequent report indicated that only the DNA vaccine encoding secreted glutamic acid decarboxylase could suppress cyclophosphamide-accelerated diabetes in 4 week old female nonobese diabetic mice with a tendency to increase T helper-2 like activity

A report published the same year corroborated the notion that secretion of glutamic acid decarboxylase encoded by a DNA vaccine is important to ameliorate diabetes in mice [86]. In this report, plasmid DNA was engineered to encode a secreted fusion protein of a truncated form of glutamic acid decarboxylase 65 and an IgG Fc fragment as well as interleukin-4. Intramuscular injection of 50 microgram of the vaccine effectively prevented diabetes in nonobese diabetic mice treated at early (4-week old, 3 times weekly) or late (12-week old, 4 times weekly) preclinical stages of diabetes. Diabetic onset reduction went from 75% in controls to 25% in treated animals at week 50. Protection was dependent on interleukin-4 as well as endogenous interleukin-4, and associated with the induction of glutamic acid decarboxylase 65 specific regulatory T helper-2 cells [87]. In addition, the same strategy was used with insulin as the target autoantigen. In this case, the DNA vaccine encoding an insulin B chain/IgG Fc fusion protein and interleukin-4 caused accelerated progression of insulitis and diabetes, which correlated with increased numbers of interferon-gamma secreting T cells in response to insulin B chain specific peptides. On the other hand, a group reported that a DNA vaccine encoding full-length intracellular human glutamic acid decarboxylase 65 alone could prevent spontaneous diabetes in nonobese diabetic mice when delivered at 4 or 10 weeks of age using

when two times 400 microgram were delivered intramuscularly over 3 days [85].

of interleukin-10 in pancreatic islets [81].

process [82, 83].

**3.3. Glutamic acid decarboxylase DNA vaccines**

#### **3.2. Heat shock protein 60 /65 DNA vaccines**

Heat shock protein 60 (HSP60) is a 60-kilodalton mammalian protein that promotes the proper folding of mitochondrial proteins and a possible autoantigen in type 1 diabetic children and murine models [73, 74]. Epitope scanning of heat shock protein 60 with antibodies identified peptide DiaPep277 with the amino sequence VLGGGVALLRVIPALDSLTPANED as an immunodominant epitope in type 1 diabetic child patients [75, 76]. From an immunological standpoint, mammalian heat shock proteins can also act as damage-associated molecular patterns that are released or presented by dying cells and can activate antigen-presenting cells of the innate immune system [77]. Heat shock proteins activate macrophages and dendritic cells through Toll-like receptor 4, which belongs to a class of membrane-bound proteins that act as sensors for immune cell activation, and promote proinflammatory effector immune responses. Paradoxically, heat shock proteins can also mediate anti-inflammatory response through Toll-like receptor 2 on T cells, and DiaPep277 peptide functions through a Toll-like receptor 2-mediated mechanism [78].

Plasmid DNA coding for heat shock protein 60 has been shown to prevent type 1 diabetes in mice. For example, two times 100 microgram intramuscular injections of plasmid DNA coding for mammalian heat shock protein 60 into 4-week old nonobese diabetic mice suppressed cyclophosphamide-accelerated diabetes, with 30% of treated mice developing diabetes compared with 60% in vector treated controls [79]. Disease prevention was associated with reduced T cell proliferation, increased in interleukin-10 and interleukin-5 secretion, and decreased interferon-gamma secretion, which suggested a shift from a T helper-1 like toward a T helper-2 like immune response.

In addition, plasmid DNA encoding mycobacterial 65-kilodalton heat shock protein could also affect diabetes by causing decreased insulitis when injected intramuscularly in three doses (100 microgram each) at 2-week intervals into 6- to 8-week-old, streptozotocin-induced diabetic C57BL/6 mice [80]. The treatment was associated with the appearance of a regulatory cell population in the spleen, with higher production of interleukin-10 in the spleen and islets, and with a decreased infiltration of CD8 T lymphocytes in the islets. The same DNA vaccine with the same dose and delivery reduced the occurrence of diabetes from 100% to 33% in 28 week old nonobese diabetic mice when injected at 4-week of age, and was associated with a reduction in CD4 and CD8 T cells infiltration, appearance of CD25 cells, and increased levels of interleukin-10 in pancreatic islets [81].

### **3.3. Glutamic acid decarboxylase DNA vaccines**

antibodies and a GAD65 protein vaccine. The need to increase therapeutic efficacy has generated an increased interest in combination immunotherapies [70-72]. Indeed, it is reason‐ able to anticipate that a variety of synergistic and additive effects may be induced by combining different agents. For example, combination treatment with anti-CD3 epsilon specific antibody and intranasal delivery of proinsulin peptide could reverse recent onset diabetes in nonobese diabetic mice as well as in a virus-induced diabetic mouse model with much higher efficacy than monotherapy using anti-CD3 or peptide alone [71]. Protection was associated with

cytokines, such as interleukin-10, transforming growth factor-beta, and interleukin-4. In addition, these cells can transfer dominant tolerance to recent onset diabetic recipients, and suppress heterologous autoaggressive CD8 T cell responses. While animal studies do provide a rationale for combining therapies, there are hurdles that still need to be overcome for translation to the clinic. For example, the possibility of unforeseen drug interactions is not in the interest of pharmaceutical and biotech companies and could stop them from evaluating

Heat shock protein 60 (HSP60) is a 60-kilodalton mammalian protein that promotes the proper folding of mitochondrial proteins and a possible autoantigen in type 1 diabetic children and murine models [73, 74]. Epitope scanning of heat shock protein 60 with antibodies identified peptide DiaPep277 with the amino sequence VLGGGVALLRVIPALDSLTPANED as an immunodominant epitope in type 1 diabetic child patients [75, 76]. From an immunological standpoint, mammalian heat shock proteins can also act as damage-associated molecular patterns that are released or presented by dying cells and can activate antigen-presenting cells of the innate immune system [77]. Heat shock proteins activate macrophages and dendritic cells through Toll-like receptor 4, which belongs to a class of membrane-bound proteins that act as sensors for immune cell activation, and promote proinflammatory effector immune responses. Paradoxically, heat shock proteins can also mediate anti-inflammatory response through Toll-like receptor 2 on T cells, and DiaPep277 peptide functions through a Toll-like

Plasmid DNA coding for heat shock protein 60 has been shown to prevent type 1 diabetes in mice. For example, two times 100 microgram intramuscular injections of plasmid DNA coding for mammalian heat shock protein 60 into 4-week old nonobese diabetic mice suppressed cyclophosphamide-accelerated diabetes, with 30% of treated mice developing diabetes compared with 60% in vector treated controls [79]. Disease prevention was associated with reduced T cell proliferation, increased in interleukin-10 and interleukin-5 secretion, and decreased interferon-gamma secretion, which suggested a shift from a T helper-1 like toward

In addition, plasmid DNA encoding mycobacterial 65-kilodalton heat shock protein could also affect diabetes by causing decreased insulitis when injected intramuscularly in three doses (100 microgram each) at 2-week intervals into 6- to 8-week-old, streptozotocin-induced diabetic C57BL/6 mice [80]. The treatment was associated with the appearance of a regulatory

and insulin specific T regulatory cells producing protective

expansion of CD25+ Foxp3+

546 Type 1 Diabetes

their drugs in combination trials [72].

receptor 2-mediated mechanism [78].

a T helper-2 like immune response.

**3.2. Heat shock protein 60 /65 DNA vaccines**

Glutamic acid decarboxylase 65 is an enzyme that catalyzes the synthesis of gamma-amino‐ butyric acid (GABA) which acts as a neuroinhibitor as well as an immunoregulatory molecule. Evidence indicates that glutamic acid decarboxylase 65 may have a critical early role in mediating islet beta cell destruction and is an important target autoantigen in type 1 diabetes. Detection of anti-glutamic acid decarboxylase antibodies in the sera of prediabetic patients is a reliable predictive marker for the progression to overt diabetes, and anti-glutamic acid decarboxylase reactivity can be detected in nonobese diabetic mice model early in the disease process [82, 83].

Plasmid DNA vaccines coding for glutamic acid decarboxylase 65 are currently at the preclin‐ ical stage. The first report of a beneficial effect in nonobese diabetic mice showed that plasmid DNA encoding wild-type intracellular or engineered secreted glutamic acid decarboxylase, i.e., a fusion of the interleukin-2 signal peptide with a truncated form of human glutamic acid decarboxylase 65, caused decreased insulitis compared to plasmid vector alone when deliv‐ ered intramuscularly, and was accompanied by elevated secretion of interleukin-4 by spleno‐ cytes [84]. A subsequent report indicated that only the DNA vaccine encoding secreted glutamic acid decarboxylase could suppress cyclophosphamide-accelerated diabetes in 4 week old female nonobese diabetic mice with a tendency to increase T helper-2 like activity when two times 400 microgram were delivered intramuscularly over 3 days [85].

A report published the same year corroborated the notion that secretion of glutamic acid decarboxylase encoded by a DNA vaccine is important to ameliorate diabetes in mice [86]. In this report, plasmid DNA was engineered to encode a secreted fusion protein of a truncated form of glutamic acid decarboxylase 65 and an IgG Fc fragment as well as interleukin-4. Intramuscular injection of 50 microgram of the vaccine effectively prevented diabetes in nonobese diabetic mice treated at early (4-week old, 3 times weekly) or late (12-week old, 4 times weekly) preclinical stages of diabetes. Diabetic onset reduction went from 75% in controls to 25% in treated animals at week 50. Protection was dependent on interleukin-4 as well as endogenous interleukin-4, and associated with the induction of glutamic acid decarboxylase 65 specific regulatory T helper-2 cells [87]. In addition, the same strategy was used with insulin as the target autoantigen. In this case, the DNA vaccine encoding an insulin B chain/IgG Fc fusion protein and interleukin-4 caused accelerated progression of insulitis and diabetes, which correlated with increased numbers of interferon-gamma secreting T cells in response to insulin B chain specific peptides. On the other hand, a group reported that a DNA vaccine encoding full-length intracellular human glutamic acid decarboxylase 65 alone could prevent spontaneous diabetes in nonobese diabetic mice when delivered at 4 or 10 weeks of age using intramuscular injections of two times 50 microgram DNA [88]. Notably, disease prevention was associated with CD28/B7 costimulation because co-expression of B7-1 or B7-2 and glutamic acid decarboxylase 65 by the same DNA vaccine abrogated protection. Another study investigated the relationship between endogenous expression levels of glutamic acid decar‐ boxylase in beta-cells and the efficacy of DNA vaccination [89]. Injection of plasmid DNA coding for glutamic acid decarboxylase into mice with lower expression levels of the autoan‐ tigen resulted in the induction of autosuppressive regulatory cells characterized by increased interleukin-4 production (T helper-2 like). In contrast, higher levels of the autoantigen favored T helper-1-like autoaggressive responses characterized by increased the interferon-gamma generation. Immunization with a DNA vaccine coding the glutamic acid decarboxylase and interleukin-4 reduced the risk of augmenting autoaggression and thus increased the safety of this immune-based therapy.

65/IgG Fc fusion polypeptide into 10-week old nonobese diabetic mice was compared with intramuscular injection of 50 microgram of the same vaccine [92]. Results indicated that, in both cases, gene expression peaked at week 8 post deliveries and was maintained until at least week 35 with more than 40% higher expression from the gene-gun delivery. However, only gene-gun delivery protected mice from diabetes with 90% diabetic animals in controls down to 50% diabetic mice at 35 weeks of age. In contrast, gene-gun administration of plasmid DNA encoding intracellular glutamic acid decarboxylase 65 to 3-week old nonobese diabetic mice did not suppress diabetes in nonobese diabetic mice [93]. The different results might be

DNA Immunotherapies for Type 1 Diabetes http://dx.doi.org/10.5772/55727 549

As mentioned earlier, combination therapy is being increasingly considered as a means to improve efficacy of immunotherapy for type 1 diabetes. Combining a DNA vaccine coding for intracellular GAD65 with an anti-CD3 monoclonal antibody has been investigated in two different mouse model systems for that purpose [94]. Results indicated that successful treatment was observed in a virus-induced diabetic model (the RIP-LCMV-GP model) but not the nonobese diabetic mouse. Efficacy was associated with an expansion of bystander sup‐ pressor T regulatory cells recognizing the C-terminal region of GAD65 and secreting interleu‐ kin-10, transforming growth factor-beta and interferon-gamma. These results also showed that efficacy was associated with numbers of antigen-specific T cells available at time of treatment, which was different beween the two animal models. The findings hold important implications to predict the success of antigen-based clinical trials where responsiveness to immunotherapy

Thus far, we have described in this section how DNA vaccines can be engineered to enhance tolerogenic-like immune responses by co-delivering cytokine-encoding DNA, an antibody, engineering subcellular localization of a target autoantigen, and choosing an effective route and method of delivery. These results obtained by different laboratories illustrate the prom‐ ising potential of DNA vaccination as a safe, low-cost and patient-friendly means to treat autoimmune diabetes and other immune-mediated inflammatory disorders. Yet, as with all immunotherapies that seek means of improving the life of diabetic individuals, there is a pressing need to improve treatment efficacy through the identification of novel molecular

Ideally, these adjuvants should attempt to mimic how immune tolerance is maintained in steady state. Here, we briefly discuss plasmid-induced apoptosis as a possible means to mimic physiological immune tolerance and to approach the "Holy Grail" of immunotherapy, namely, the ability to suppress inflammation in a homeostatic manner. Apoptosis is a constantly ongoing form of cell death that produces fifty to seventy billion dead cells on a daily basis in an average human adult [95]. Upon a given intrinsic or extrinsic signal, cells initiate the process of apoptosis and become membrane-bound cellular fragments, or apoptotic bodies, which are rapidly engulfed and processed by surrounding living cells. For many years, it was believed that these apoptotic bodies had little effect on the immune system. Today, it has become clear that apoptosis is an important physiological means to establish and maintain immune tolerance in peripheral tissues. Apoptotic cells play a fundamental role as they not only serve as a source of self-antigens, but also recruit antigen-presenting cells, secrete anti-inflammatory

attributed to the different subcellular localizations of the autoantigen.

may vary from patient to patient.

adjuvants for the safe induction of immune tolerance.

DNA vaccines encoding secreted glutamic acid decarboxylase combined with anti-inflamma‐ tory interleukins have also been applied to pancreatic transplant for type 1 diabetes. Survival of syngeneic neonatal pancreata transplanted under the kidney capsule of nonobese diabetic mice was promoted by intramuscular injection of a DNA vaccine encoding the secreted glutamic acid decarboxylase 65/IgG Fc fusion and interleukin-4 plus interleukin-10 [90]. The treatment consisted of 50 microgram of the vaccine delivered weekly for four weeks from the age of 10 weeks with transplantation performed one week after the final DNA vaccination. DNA vaccination protected syngeneic islet in transplanted mice, with 100% diabetic mice in controls compared to 20% diabetes incidence in treated animals at 30 weeks of age and 15 weeks post transplant. Increased islet survival required co-delivery of both interleukin-4 and interleukin-10 and correlated with a marked reduction in interferon-gamma reactivity as well as an increase in interleukin-10 secreting T cells. These results made apparent the increased difficulty in protecting exogenous syngeneic islet and the need for more stringent conditions of vaccination in the transplantation setting.

Most DNA vaccines for type 1 diabetes have been delivered into muscle tissue. The main rationale for using this route of delivery is that it permits administration of larger amounts of DNA, but other routes may be more advantageous to induce tolerogenic responses. In that regard, a report compared intramuscular, intradermal, and oral delivery of plasmid DNA coding for the intracellular or secreted form of glutamic acid decarboxylase for prevention of diabetes in 4-week-old nonobese diabetic mice [91]. Results indicated that both intradermal and oral deliveries were more effective than intramuscular delivery for delaying the disease. Cytokine-specific ELISpot analysis indicated that immune responses induced by the DNA vaccination were generally more dependent on the cellular localization of glutamic acid decarboxylase antigen than on the delivery route, although ELISA indicated that intradermal delivery of DNA is most likely to induce a T helper-2 like response.

In addition to route of delivery, the method used to administer a DNA vaccine can be beneficial by increasing efficacy of DNA uptake and improving immune responses. For example, dermal delivery of plasmid DNA using gene gun technology, which consists in shooting gold microparticles covered with DNA, can improve protection from diabetes. In this regard, gene-gun delivery of 1 microgram of a DNA vaccine encoding the secreted glutamic acid decarboxylase 65/IgG Fc fusion polypeptide into 10-week old nonobese diabetic mice was compared with intramuscular injection of 50 microgram of the same vaccine [92]. Results indicated that, in both cases, gene expression peaked at week 8 post deliveries and was maintained until at least week 35 with more than 40% higher expression from the gene-gun delivery. However, only gene-gun delivery protected mice from diabetes with 90% diabetic animals in controls down to 50% diabetic mice at 35 weeks of age. In contrast, gene-gun administration of plasmid DNA encoding intracellular glutamic acid decarboxylase 65 to 3-week old nonobese diabetic mice did not suppress diabetes in nonobese diabetic mice [93]. The different results might be attributed to the different subcellular localizations of the autoantigen.

intramuscular injections of two times 50 microgram DNA [88]. Notably, disease prevention was associated with CD28/B7 costimulation because co-expression of B7-1 or B7-2 and glutamic acid decarboxylase 65 by the same DNA vaccine abrogated protection. Another study investigated the relationship between endogenous expression levels of glutamic acid decar‐ boxylase in beta-cells and the efficacy of DNA vaccination [89]. Injection of plasmid DNA coding for glutamic acid decarboxylase into mice with lower expression levels of the autoan‐ tigen resulted in the induction of autosuppressive regulatory cells characterized by increased interleukin-4 production (T helper-2 like). In contrast, higher levels of the autoantigen favored T helper-1-like autoaggressive responses characterized by increased the interferon-gamma generation. Immunization with a DNA vaccine coding the glutamic acid decarboxylase and interleukin-4 reduced the risk of augmenting autoaggression and thus increased the safety of

DNA vaccines encoding secreted glutamic acid decarboxylase combined with anti-inflamma‐ tory interleukins have also been applied to pancreatic transplant for type 1 diabetes. Survival of syngeneic neonatal pancreata transplanted under the kidney capsule of nonobese diabetic mice was promoted by intramuscular injection of a DNA vaccine encoding the secreted glutamic acid decarboxylase 65/IgG Fc fusion and interleukin-4 plus interleukin-10 [90]. The treatment consisted of 50 microgram of the vaccine delivered weekly for four weeks from the age of 10 weeks with transplantation performed one week after the final DNA vaccination. DNA vaccination protected syngeneic islet in transplanted mice, with 100% diabetic mice in controls compared to 20% diabetes incidence in treated animals at 30 weeks of age and 15 weeks post transplant. Increased islet survival required co-delivery of both interleukin-4 and interleukin-10 and correlated with a marked reduction in interferon-gamma reactivity as well as an increase in interleukin-10 secreting T cells. These results made apparent the increased difficulty in protecting exogenous syngeneic islet and the need for more stringent conditions

Most DNA vaccines for type 1 diabetes have been delivered into muscle tissue. The main rationale for using this route of delivery is that it permits administration of larger amounts of DNA, but other routes may be more advantageous to induce tolerogenic responses. In that regard, a report compared intramuscular, intradermal, and oral delivery of plasmid DNA coding for the intracellular or secreted form of glutamic acid decarboxylase for prevention of diabetes in 4-week-old nonobese diabetic mice [91]. Results indicated that both intradermal and oral deliveries were more effective than intramuscular delivery for delaying the disease. Cytokine-specific ELISpot analysis indicated that immune responses induced by the DNA vaccination were generally more dependent on the cellular localization of glutamic acid decarboxylase antigen than on the delivery route, although ELISA indicated that intradermal

In addition to route of delivery, the method used to administer a DNA vaccine can be beneficial by increasing efficacy of DNA uptake and improving immune responses. For example, dermal delivery of plasmid DNA using gene gun technology, which consists in shooting gold microparticles covered with DNA, can improve protection from diabetes. In this regard, gene-gun delivery of 1 microgram of a DNA vaccine encoding the secreted glutamic acid decarboxylase

this immune-based therapy.

548 Type 1 Diabetes

of vaccination in the transplantation setting.

delivery of DNA is most likely to induce a T helper-2 like response.

As mentioned earlier, combination therapy is being increasingly considered as a means to improve efficacy of immunotherapy for type 1 diabetes. Combining a DNA vaccine coding for intracellular GAD65 with an anti-CD3 monoclonal antibody has been investigated in two different mouse model systems for that purpose [94]. Results indicated that successful treatment was observed in a virus-induced diabetic model (the RIP-LCMV-GP model) but not the nonobese diabetic mouse. Efficacy was associated with an expansion of bystander sup‐ pressor T regulatory cells recognizing the C-terminal region of GAD65 and secreting interleu‐ kin-10, transforming growth factor-beta and interferon-gamma. These results also showed that efficacy was associated with numbers of antigen-specific T cells available at time of treatment, which was different beween the two animal models. The findings hold important implications to predict the success of antigen-based clinical trials where responsiveness to immunotherapy may vary from patient to patient.

Thus far, we have described in this section how DNA vaccines can be engineered to enhance tolerogenic-like immune responses by co-delivering cytokine-encoding DNA, an antibody, engineering subcellular localization of a target autoantigen, and choosing an effective route and method of delivery. These results obtained by different laboratories illustrate the prom‐ ising potential of DNA vaccination as a safe, low-cost and patient-friendly means to treat autoimmune diabetes and other immune-mediated inflammatory disorders. Yet, as with all immunotherapies that seek means of improving the life of diabetic individuals, there is a pressing need to improve treatment efficacy through the identification of novel molecular adjuvants for the safe induction of immune tolerance.

Ideally, these adjuvants should attempt to mimic how immune tolerance is maintained in steady state. Here, we briefly discuss plasmid-induced apoptosis as a possible means to mimic physiological immune tolerance and to approach the "Holy Grail" of immunotherapy, namely, the ability to suppress inflammation in a homeostatic manner. Apoptosis is a constantly ongoing form of cell death that produces fifty to seventy billion dead cells on a daily basis in an average human adult [95]. Upon a given intrinsic or extrinsic signal, cells initiate the process of apoptosis and become membrane-bound cellular fragments, or apoptotic bodies, which are rapidly engulfed and processed by surrounding living cells. For many years, it was believed that these apoptotic bodies had little effect on the immune system. Today, it has become clear that apoptosis is an important physiological means to establish and maintain immune tolerance in peripheral tissues. Apoptotic cells play a fundamental role as they not only serve as a source of self-antigens, but also recruit antigen-presenting cells, secrete anti-inflammatory cytokines, and display tolerogenic molecules [96]. The remarkable capacity of apoptotic cells to induce either tolerogenic immune responses or immunogenic responses depending on signals received makes them attractive candidates to intervene in many disorders like infectious diseases, cancer, and autoimmune diseases.

with interleukin 4/MCP1, and animals in both groups had fewer CD8 T lymphocyte counts in

In another example, a DNA vaccine treatment strategy was designed to target an antigenic peptide (glucose-6-phosphate isomerase, GPI) to the lysosomal compartment. A specific T cell

BDC-2.5 in the NOD mouse [102]. Lysosome targeting of single peptide epitope was sufficient to induce protection against type 1 diabetes which was not the result of antigen-specific T cell anergy. Typical T helper-2 cytokines like interleukin-10 or -4 were undetectable in 2.5mi+ T cells, arguing against a mechanism of immune deviation. Instead, the expanded 2.5mi+

population produced interferon-gamma similar to 2.5mi+ T cells from naive mice. Protection against diabetes induced by DNA vaccination was completely lost in NOD.CD28-/- mice, which are largely deficient of natural T regulatory cells. Furthermore, diabetes onset was delayed in T regulatory-reconstituted and DNA-treated NOD.SCID mice, although antigen-specific

DNA vaccination has been tested in clinical trials for treatment of new-onset type 1 diabetes with encouraging results, but efficacy must be improved. The number and variety of strategies that have been developed to improve efficacy of DNA vaccination for autoimmune diabetes

**4. Conclusion: Plasmid DNA as a promising immunotherapy for Type 1**

Plasmid DNA is a versatile vector platform permitting the seamless integration of different immune modulators into a product that can be manufactured in a generic manner. As we have seen in this chapter, plasmid DNA has been extensively investigated for the prevention and treatment of type 1 diabetes in different animal model systems. Plasmid DNA-based gene immunotherapies do not encode an autoantigen and act systemically to different degrees, which could result in serious adverse events if used over time. Nevertheless, gene immuno‐ therapies could still be utilized as molecular adjuvants with DNA vaccines that target pan‐ creatic beta cell autoantigens. It is possible that different stages during progression of disease will require different therapeutic agents or combinations thereof according to immune responses to therapy. It is also anticipated that some strategies will be safer and more robust than others, but there is unfortunately no animal model that can predict successful bench-tobedside translation of a given strategy. In that regard, immunological biomarkers and their pre-clinical and clinical correlates will be needed to determine which strategies are most likely to be effective in humans, and to what extent different immunotherapies might be combined. Combinatorial therapies include co-delivery of DNA vaccines with gene therapy, peptide

is a testament to the flexibility and potential of DNA vaccine immunotherapy.

 cells did not expand in response to DNA treatment. These findings indicated a T regulatory-mediated protective mechanism that was independent of the expansion or de novo

T cells is known to share reactivity with the diabetogenic T cell clone

T cell

551

DNA Immunotherapies for Type 1 Diabetes http://dx.doi.org/10.5772/55727

spleen.

Foxp3+

**diabetes**

population termed 2.5mi+

**3.5. Summary of section 3**

generation of antigen-specific T regulatory cells.

The first report of DNA vaccines designed for pro-apoptotic immunoregulation, i.e., antiinflammatory, used plasmid DNA coding for the pro-apoptotic BAX protein and intracel‐ lular or secreted glutamic acid decarboxylase, to prevent diabetes in the nonobese diabetic mouse [97]. Results indicated that intramuscular injection of the BAX cDNA recruited dendritic cells carrying vaccine-encoded protein in both spleen and lymph nodes. Further‐ more, delivery of two times 150 microgram plasmid DNA coding for secreted glutamic acid decarboxylase and BAX at 3 days interval into 4-week old mice prevented diabetes, i.e, reduced the incidence from 93% in controls down to 47% in treated animals. In contrast, the vaccines coding for BAX DNA alone or intracellular glutamic acid decarboxylase and BAX did not prevent diabetes. Notably, ELISA results suggested that co-delivery of BAX suppressed T helper-2 like activity, which indicated that another cell type was responsi‐ ble for disease suppression. Indeed, a subsequent report showed that delivery of both secreted glutamic acid decarboxylase and BAX were required to induce CD4+ CD25+ FoxP3+ cells with contact dependent regulatory activity [98].

Importantly, additional studies revealed that increased CpG methylation of plasmid DNA together with delivery of secreted glutamic acid decarboxylase and BAX DNA could act synergistically to ameliorate recent onset diabetes in nonobese diabetic mice [99]. A weekly intradermal injection of 50 microgram of the vaccine over eight weeks following early hyperglycemia ameliorated diabetes at 40 weeks of age, from 90% diabetic mice in controls down to 20% in treated mice. Remarkably, DNA hypermethylation caused increased numbers of tolerogenic-like plasmacytoid dendritic cells in lymph nodes. It is hypothesized that increased CpG methylation of plasmid DNA makes the DNA vaccine appear more mamma‐ lian-like to the immune system, as it is known that bacterial DNA has low levels of CpG methylation that can act as an inflammatory signal [100]. Taken together these results indicate that apoptosis-inducing DNA vaccination is a promising approach for treatment of type 1 diabetes.

#### **3.4. Other DNA vaccines**

DNA vaccines encoding less studied autoantigens have also been investigated. For example, insulinoma-associated protein 2 (IA-2), which is expressed in islets, brain, and neuro-endocrine cells, is a member of the protein tyrosine phosphatase family and targeted by autoimmune T cells in type 1 diabetes. A DNA vaccine encoding insulinoma-associated protein 2 with or without the combination of DNA coding for interleukin-4 and monocyte chemoattractant protein-1 (MCP-1) was injected intramuscularly into pre-diabetic non-obese diabetic mice using 3 x 100 microgram DNA delivered over four weeks [101]. The treatment could protect mice from diabetes, from 60% in controls to 10-20% diabetic animals at 30 weeks of age. There was no difference in efficacy between groups treated with the DNA vaccine alone or combined with interleukin 4/MCP1, and animals in both groups had fewer CD8 T lymphocyte counts in spleen.

In another example, a DNA vaccine treatment strategy was designed to target an antigenic peptide (glucose-6-phosphate isomerase, GPI) to the lysosomal compartment. A specific T cell population termed 2.5mi+ T cells is known to share reactivity with the diabetogenic T cell clone BDC-2.5 in the NOD mouse [102]. Lysosome targeting of single peptide epitope was sufficient to induce protection against type 1 diabetes which was not the result of antigen-specific T cell anergy. Typical T helper-2 cytokines like interleukin-10 or -4 were undetectable in 2.5mi+ T cells, arguing against a mechanism of immune deviation. Instead, the expanded 2.5mi+ T cell population produced interferon-gamma similar to 2.5mi+ T cells from naive mice. Protection against diabetes induced by DNA vaccination was completely lost in NOD.CD28-/- mice, which are largely deficient of natural T regulatory cells. Furthermore, diabetes onset was delayed in T regulatory-reconstituted and DNA-treated NOD.SCID mice, although antigen-specific Foxp3+ cells did not expand in response to DNA treatment. These findings indicated a T regulatory-mediated protective mechanism that was independent of the expansion or de novo generation of antigen-specific T regulatory cells.

#### **3.5. Summary of section 3**

cytokines, and display tolerogenic molecules [96]. The remarkable capacity of apoptotic cells to induce either tolerogenic immune responses or immunogenic responses depending on signals received makes them attractive candidates to intervene in many disorders like

The first report of DNA vaccines designed for pro-apoptotic immunoregulation, i.e., antiinflammatory, used plasmid DNA coding for the pro-apoptotic BAX protein and intracel‐ lular or secreted glutamic acid decarboxylase, to prevent diabetes in the nonobese diabetic mouse [97]. Results indicated that intramuscular injection of the BAX cDNA recruited dendritic cells carrying vaccine-encoded protein in both spleen and lymph nodes. Further‐ more, delivery of two times 150 microgram plasmid DNA coding for secreted glutamic acid decarboxylase and BAX at 3 days interval into 4-week old mice prevented diabetes, i.e, reduced the incidence from 93% in controls down to 47% in treated animals. In contrast, the vaccines coding for BAX DNA alone or intracellular glutamic acid decarboxylase and BAX did not prevent diabetes. Notably, ELISA results suggested that co-delivery of BAX suppressed T helper-2 like activity, which indicated that another cell type was responsi‐ ble for disease suppression. Indeed, a subsequent report showed that delivery of both

secreted glutamic acid decarboxylase and BAX were required to induce CD4+

Importantly, additional studies revealed that increased CpG methylation of plasmid DNA together with delivery of secreted glutamic acid decarboxylase and BAX DNA could act synergistically to ameliorate recent onset diabetes in nonobese diabetic mice [99]. A weekly intradermal injection of 50 microgram of the vaccine over eight weeks following early hyperglycemia ameliorated diabetes at 40 weeks of age, from 90% diabetic mice in controls down to 20% in treated mice. Remarkably, DNA hypermethylation caused increased numbers of tolerogenic-like plasmacytoid dendritic cells in lymph nodes. It is hypothesized that increased CpG methylation of plasmid DNA makes the DNA vaccine appear more mamma‐ lian-like to the immune system, as it is known that bacterial DNA has low levels of CpG methylation that can act as an inflammatory signal [100]. Taken together these results indicate that apoptosis-inducing DNA vaccination is a promising approach for treatment of type 1

DNA vaccines encoding less studied autoantigens have also been investigated. For example, insulinoma-associated protein 2 (IA-2), which is expressed in islets, brain, and neuro-endocrine cells, is a member of the protein tyrosine phosphatase family and targeted by autoimmune T cells in type 1 diabetes. A DNA vaccine encoding insulinoma-associated protein 2 with or without the combination of DNA coding for interleukin-4 and monocyte chemoattractant protein-1 (MCP-1) was injected intramuscularly into pre-diabetic non-obese diabetic mice using 3 x 100 microgram DNA delivered over four weeks [101]. The treatment could protect mice from diabetes, from 60% in controls to 10-20% diabetic animals at 30 weeks of age. There was no difference in efficacy between groups treated with the DNA vaccine alone or combined

CD25+

FoxP3+

infectious diseases, cancer, and autoimmune diseases.

cells with contact dependent regulatory activity [98].

diabetes.

550 Type 1 Diabetes

**3.4. Other DNA vaccines**

DNA vaccination has been tested in clinical trials for treatment of new-onset type 1 diabetes with encouraging results, but efficacy must be improved. The number and variety of strategies that have been developed to improve efficacy of DNA vaccination for autoimmune diabetes is a testament to the flexibility and potential of DNA vaccine immunotherapy.
