Prevention Of Type 1 Diabetes With DNA Vaccine Encoding Glutamic Acid Decarboxylase

Type 1 diabetes mellitus (T1DM) is an autoimmune diseasecharacterized by selective destruction of the insulin-producingβcells inthe islets of Langerhans. A variety of immune effector cells, includingCD4⁺ T cells, CD8⁺ T cells, and macrophages have been implicated asmediators of pancreatic isletsβcells destruction in T1DM. Previousresearches indicated that programmed cell death, or apoptosis is the mainform ofβcells death responsible for the development of T1DM in animalmodels. Although the etiology of autoimmune diabetes is complex andremain largely unknown, accumulating experimental evidences suggestedthat the insulin-secreting pancreaticβ-cells were damaged by cytotoxicThl cells and their cytokines. Hence, a fine tuning of the immune systemtoward Th2-like immunity may represent an attractive and reasonablepreventive and therapeutic strategy for T1DM. Additionally, CD4⁺CD25⁺regulatory T cells have described recently as an important powerfulmediator of peripheral tolerance in autoimmune diabetes, and couldefficiently protect and cure of animal model from diabetes development.Many previous studies suggested that individuals could have a pre-clinical period for a long time before it finally progressed to overtT1DM, and overt T1DM could been delayed or prevented by efficientimmune intervention at the early stage. Thus, an increasing number ofresearchers put their interests in finding efficient therapies to cure T1DM.Re-establish the tolerance to autoantigen is one of the important mannersto prevent T1DM. Glutamic acid decarbocylase (GAD) 65 has beenrecognized as a key antigen among islets autoantigens in the progressionof T1DM, and has been shown to lessen insulitis and prevent T1DM byinduction of immune tolerance or other mechanisms in previous studies.DNA vaccine has been applied to treat infectious disease and cancer,and promising effects have been observed in recent years. The establishedand potential advantages of DNA vaccination over other methods ofimmunization have been recognized. The fact that DNA vaccine is safety,and can be constructed easily and produced with low cost and also onlyrequires convenient condition for storage and transportation. Moreover,GAD65 DNA vaccines have been reported recently to prevent T1DM inanimal models. However, there are inconsistent results in those previousstudies. More importantly, our previous study showed that incidence ofspontaneous autoimmune diabetes only decreased by thirty percent byDNA vaccination encoding full-length GAD65 gene in that study in anexcellent animal model of human T1DM, the non-obese diabetic (NOD)mice. Therefore, preventive efficacy of GAD65 DNA vaccine should to be improved.In conclusions, the aims of present study are to optimize the DNAvaccine containing full-length GAD65 gene for prevention of T1DM, andto investigate the roles of different optimized GAD65 DNA vaccines indiabetes onset and insulitis development in NOD mice, and also toexplore the possible mechnisms of optimized GAD65 DNA vaccines inpreventing T1DM in NOD mice, which provide an experimentalfoundation for prevention of human T1DM.In this study, we firstly optimize DNA vaccine encoding full-lengthGAD65 gene by means of autoantigen geng sequence modification, andaddition of signal peptide sequence, and co-expression with protectivecytokine or other autoantigen gene, to construct and identify eight newGAD65 fragment DNA vaccines. Moreover, we investigate the effects ofdifferent optimized GAD65 fragment DNA vaccines on development ofdiabetes and insulitis in NOD mice. Finally, we examine the roles ofdifferent optimized GAD65 fragment DNA vaccines in apoptosis ofβcells, immune tolerance to GAD65, Th1/Th2 immune balance and statusof CD4⁺CD25⁺ regulatory T cells in this model. Part I Construction, Identification and Optimization of GlutamicAcid Decarbocylase (GAD) 65 DNA VaccineObjective To construct and optimize DNA vaccines containingdifferent GAD65 gene fragments for prevention of type 1 diabetes.Methods The cDNAs of GAD_190-315 (GAD1) and GAD_490-570(GAD2) fragments were amplified from GAD65 plasmid, and linked withIL-2 signal peptide cDNA respectively through overlap PCR. And thefusion genes, SGAD1 and SGAD2, were inserted into pGEM-T vectors.IL-4, IL-10, insulin B chain (InsB) genes were amplified from IL-4,IL-10, proinsulin plasmids and cloned into pGEM-T vectors, respectively.Then, pBudCE4.1-SGAD1 and pBudCE4.1-SGAD1 were constructedwhen SGAD1 and SGAD2 were cloned into an eukaryotic expressionvector pBudCE4.1, which contains two multiple cloning sites under thecontrol of different promoters. Moreover, IL-4, IL-10, InsB genes werecloned respectively after the other promoter into pBudCE4.1-SGAD 1 andpBudCE4.1-SGAD2 plasmid to establish six recombinant eukaryoticexpression vectors, pBud-SGAD 1/IL-4, pBud-SGAD 1/IL-10,pBud-SGAD1/InsB, pBud-SGAD2/IL-4, pBud-SGAD2/IL-10 andpBud-SGAD2/InsB. After being identified by DNA sequencing, all of therecombinant eukaryotic expression vectors were transfected into COS-7cells respectively by liposome in vitro. The expression products in theCOS-7 cells transfected with recombinants were detected using Westernblot or ELISA. Results(1) DNA sequencing of the target genes cloned into recombinanteukaryotic expression vectors were in accordance with the reportedsequence, with correct open reading frame.(2) Western blot showed that the products of those recombinanteukaryotic expression vectors containing SGAD1 or SGAD2 gene wereexpressed and secreted. InsB gene was also expressed in COS-7 cells invitro.(3) The expression products of IL-4, IL-10 gene in COS-7 cellstransfected with those recombinant eukaryotic expression vectors containcytokines gene were determined by ELISA.Conclusions The pBudCE4.1-SGAD1, pBudCE4.1-SGAD1,pBud-SGAD1/IL-4, pBud-SGAD1/IL-10, pBud-SGAD1/InsB,pBud-SGAD2/IL-4, pBud-SGAD2/IL-10 and pBud-SGAD2/InsB DNAvaccines were constructed, being a foundation for further development ofDNA vaccine against type 1 diabetes. Partâ…¡Effects of GAD65 DNA Vaccines on Development ofAutoimmune Diabetes and Insulitis in NOD MiceObjective To investigate the roles of different GAD65 fragmentDNA vaccines in diabetes onset and insulitis severity in NOD mice.Methods Female NOD mice at 3-4 weeks of age were randomlydivided into eleven groups and received intramuscular injection of eitherpBudCE4.1 blank vector alone (n=18) or pBudCE4.1 carrying theSGAD1 (n=17), SGAD2 (n=18), SGAD1/IL-4 (n=19), SGAD2/IL-4(n=17), SGAD1/IL-10 (n=20), SGAD2/IL-10 (n=18), SGAD1/InsB(n=17), SGAD2/InsB (n=19) construct, or pcDNA3.1(+) vector encodingfull-length GAD65 gene (n=18), and mice received injection of PBS(n=20) as control. All mice were injected with 75μg DNA vaccine or PBSinto the tibialis anterior muscle and an identical set of injection wereperformed 1 week later, for a total of 150μg of DNA vaccine per mouse.The incidence of spontaneous diabetes was monitored up to 30 weeks ofage. From 10 weeks of age, mice were determined once a week forglycosuria, and diabetes was diagnosed when the blood glucose levelsexceeded 16.7mmol/L on two consecutive readings. Six to eight ofnon-diabetic animals from each group were killed at 12 weeks of age andpancreas were removed to score insulitis by routine H&E staining.Results(1) Compared with PBS group, there was a significant reduction ofdiabetes onset in NOD mice treated with either pBud-SGAD1,pBud-SGAD1/IL-4, or pBud-SGAD1/IL-10 DNA vaccine (P＜0.05 or P＜0.01).(2) pBud-SGAD1/IL-4 and pBud-SGAD1/IL-10 DNA vaccinationdecreased the incidence of diabetes in NOD mice, compared with eitherGAD65 group or SGAD1 group (P＜0.05 or P＜0.01).(3) No significant difference of diabetes onset was observed amongthe PBS, pBudCE, GAD65, SGAD2, SGAD1/InsB, SGAD2/IL-4,SGAD2/IL-10 groups (all P＞0.05).(4) Insulitis scoring showed that mice received either pBud-SGAD1,pBud-SGAD1/IL-4, or pBud-SGAD1/IL-10 DNA vaccine significantlysuppressed islet inflammation as compared to mice received PBS andpcDNA3.1(+)/GAD65 DNA vaccine (all P＜0.01).Conclusion Immunization with optimized GAD65 fragment DNAvaccines, pBud-SGAD1, pBud-SGAD1/IL-4, and pBud-SGAD1/IL-10,prevents diabetes onset and lessens insulitis in NOD mice, which is moreeffective than former DNA vaccine containing full-length GAD65 gene. Partâ…¢The Preventive Mechanisms of GAD65 DNA Vaccines onAutoimmune Diabetes in NOD MiceObjective To explore the mechanisms of optimized GAD65fragment DNA vaccines in preventing autoimmune diabetes and insulitisin NOD mice.Methods Six to eight of non-diabetic mice from either PBS,pBudCE, GAD65, SGAD1, SGAD1/IL-4, or SGAD1/IL-10 group, weresacrificed at 12 weeks of age. Firstly, the apoptotic isletsβcells wereevaluated by TUNEL and immunohistochemistry. Moreover, splenocyteswere isolated and cultured in vitro with the rGAD65, the proliferationwas quantified by determining [3H]-thymidine incorporation. Thepopulation of CD4⁺CD25⁺ regulatory T cells was analyzed by flowcytometry. Furthermore, cytokine IFN-γ, IL-4 and IL-10 releasestimulated by rGAD65 were measured by ELISA. Finally, total RNA wasisolated from fresh spleen tissue of NOD mice with or without treatmentof GAD65 DNA fragment vaccines. The levels of cytokine IFN-γ, IL-4,IL-10 and transcription factor Foxp3 mRNA expression in spleen weredetected by RT-PCR.Results(1) Isletβcells apoptosis rates decreased in NOD mice receivedeither SGAD1, SGAD1/IL-4, or SGAD1/IL-10 DNA vaccine treatmentcompared with mice received PBS injection (all P＜0.05).(2) Splenocyte proliferation response to autoantigen GAD65 inSGAD1, SGAD1/IL-4, or SGAD1/IL-10 DNA vaccine treated mice were suppressed as compared to control mice (all P＜0.01).(3) The release of IFN-γin supematants of splenocytes in eitherSGAD1, SGAD1/IL-4, or SGAD1/IL-10 DNA vaccine treated micegroup, were lower than those in PBS (all P＜0.01) and GAD65 group (allP＜0.01). The similar difference was observed in level of IFN-γ, mRNAexpression.(4) The secretion of IL-4 in supematants of splenocytes in eitherSGAD1, SGAD1/IL-4, or SGAD1/IL-10 DNA vaccine treated micegroup, were higher than those in PBS group (P＜0.05 or P＜0.01). Theonly SGAD1/IL-4 group showed significant difference compared toGAD65 group (all P＜0.05). And IL-4 mRNA expression were increasedin either SGAD1, SGAD1/IL-4, or SGAD1/IL-10 DNA vaccine treatedmice compared with PBS and DNA vaccine containing full-lengthGAD65 gene treated mice (P＜0.05 or P＜0.01).(5) SGAD1/IL-10 DNA vaccine treatment increased IL-10 levels insupematants of splenocytes in mice as compared to those in PBS group(P＜0.05) or GAD65 group (P＜0.05), and IL-10 mRNA expressionincreased in mice received either SGAD1/IL-4 or SGAD1/IL-10 DNAvaccine treatment compared with mice in PBS (P＜0.05 or P＜0.01) orDNA vaccine containing full-length GAD65 gene injection (all P＜0.05).(6) No significant differences in both CD4⁺CD25⁺ regulatory T cellpopulation and Foxp3 mRNA expression were found between the DNAvaccine treated mice and the untreated mice group. (all P＞0.05).Conclusion A shift to Th2 immune response, inhibition of the apoptosis of isletβcells, and induction of tolerance of autoreactive Tcells against autoantigen GAD65, are found as the preventivemechanisms in immunization with the optimized GAD65 DNA fragmentvaccines.