DNA vaccine encoding CD40 targeted to dendritic cells in situ prevents the development of Heymann nephritis in rats
Authors: Ya Wang, Yuan Min Wang, Yiping Wang, Guoping Zheng, Geoff Yu Zhang, Jimmy Jianheng Zhou, Thian Kui Tan, Qi Cao, Min Hu, Debbie Watson, Huiling Wu, Dong Zheng, ChangQi Wang, Mireille H. Lahoud, Irina Caminschi, David C. Harris, Stephen I. Alexander
Journal: Kidney International (2012)

The CD40-CD154 costimulatory pathway has been shown to be critical for both T- and B-cell activation in autoimmune disease. Here, we assessed the effects of blocking this pathway using CD40 DNA vaccine enhanced by dendritic cell targeting on the development of active Heymann nephritis, a rat model of human membranous nephropathy. DNA vaccination delivers plasmid DNA encoding the target antigen, either alone or in combination with enhancing elements, to induce both humoral and cellular immune responses. To determine whether CD40 DNA vaccine targeting the encoded CD40 directly to dendritic cells would improve the efficacy of the vaccination against self-protein CD40, we utilized a plasmid encoding a single-chain Fv antibody specific for the dendritic cell–restricted antigen-uptake receptor DEC205 (scDEC), the target gene CD40, and the adjuvant tetanus sequence p30. This vaccine plasmid was compared to a control plasmid without scDEC. Rats vaccinated with scDEC-CD40 had significantly less proteinuria and renal injury than did rats receiving scControl-CD40 and were protected from developing Heymann nephritis. Thus, CD40 DNA vaccination targeted to dendritic cells limits the development of Heymann nephritis. The CD40-CD154 costimulatory pathway has been shown to be critical for both T- and B-cell activation in autoimmune disease. Here, we assessed the effects of blocking this pathway using CD40 DNA vaccine enhanced by dendritic cell targeting on the development of active Heymann nephritis, a rat model of human membranous nephropathy. DNA vaccination delivers plasmid DNA encoding the target antigen, either alone or in combination with enhancing elements, to induce both humoral and cellular immune responses. To determine whether CD40 DNA vaccine targeting the encoded CD40 directly to dendritic cells would improve the efficacy of the vaccination against self-protein CD40, we utilized a plasmid encoding a single-chain Fv antibody specific for the dendritic cell–restricted antigen-uptake receptor DEC205 (scDEC), the target gene CD40, and the adjuvant tetanus sequence p30. This vaccine plasmid was compared to a control plasmid without scDEC. Rats vaccinated with scDEC-CD40 had significantly less proteinuria and renal injury than did rats receiving scControl-CD40 and were protected from developing Heymann nephritis. Thus, CD40 DNA vaccination targeted to dendritic cells limits the development of Heymann nephritis. Membranous nephropathy, a major autoimmune cause of chronic renal failure, is an immune disease driven in many cases by the recently described cognate antigen PLA2R expressed on the glomerular epithelium.1.Fogo A.B. Mechanisms of progression of chronic kidney disease.Pediatr Nephrol. 2007; 22: 2011-2022Crossref PubMed Scopus (180) Google Scholar,2.Beck Jr L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1501) Google Scholar CD40 expressed by B cells and other antigen-presenting cells and its ligand CD154 on T cells are critical costimulatory pathways required for T-cell activation and for B-cell differentiation and class switching.3.Diehl L. Den Boer A.T. van der Voort E.I. et al.The role of CD40 in peripheral T cell tolerance and immunity.J Mol Med (Berl). 2000; 78: 363-371Crossref PubMed Scopus (63) Google Scholar CD40-CD154 blockade has been shown to be protective in a number of models of renal disease, including rodent membranous glomerulonephritis and Adriamycin nephropathy.4.Biancone L. Andres G. Ahn H. et al.Inhibition of the CD40-CD40 ligand pathway prevents murine membranous glomerulonephritis.Kidney Int. 1995; 48: 458-468Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 5.Kairaitis L. Wang Y. Zheng L. et al.Blockade of CD40-CD40 ligand protects against renal injury in chronic proteinuric renal disease.Kidney Int. 2003; 64: 1265-1272Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 6.Reynolds J. Khan S.B. Allen A.R. et al.Blockade of the CD154-CD40 costimulatory pathway prevents the development of experimental autoimmune glomerulonephritis.Kidney Int. 2004; 66: 1444-1452Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar CD154 blocking antibodies were shown to be highly effective in primate studies on transplantation and are used clinically. However, their effect in activating CD154 on platelets and subsequent thrombosis has limited their clinical use.7.Boumpas D.T. Furie R. Manzi S. et al.A short course of BG9588 (anti-CD40 ligand antibody) improves serologic activity and decreases hematuria in patients with proliferative lupus glomerulonephritis.Arthritis Rheum. 2003; 48: 719-727Crossref PubMed Scopus (528) Google Scholar, 8.Davis Jr J.C. Totoritis M.C. Rosenberg J. et al.Phase I clinical trial of a monoclonal antibody against CD40-ligand (IDEC-131) in patients with systemic lupus erythematosus.J Rheumatol. 2001; 28: 95-101PubMed Google Scholar, 9.Sidiropoulos P.I. Boumpas D.T. Lessons learned from anti-CD40L treatment in systemic lupus erythematosus patients.Lupus. 2004; 13: 391-397Crossref PubMed Scopus (159) Google Scholar CD40 antibodies can block this pathway without associated thrombosis and are in preclinical testing, although some are associated with depletion of CD40-expressing cells including B cells.10.Pearson T.C. Trambley J. Odom K. et al.Anti-CD40 therapy extends renal allograft survival in rhesus macaques.Transplantation. 2002; 74: 933-940Crossref PubMed Scopus (149) Google Scholar, 11.Haanstra K.G. Ringers J. Sick E.A. et al.Prevention of kidney allograft rejection using anti-CD40 and anti-CD86 in primates.Transplantation. 2003; 75: 637-643Crossref PubMed Scopus (117) Google Scholar, 12.Haanstra K.G. Sick E.A. Ringers J. et al.Costimulation blockade followed by a 12-week period of cyclosporine A facilitates prolonged drug-free survival of rhesus monkey kidney allografts.Transplantation. 2005; 79: 1623-1626Crossref PubMed Scopus (48) Google Scholar, 13.Aoyagi T. Yamashita K. Suzuki T. et al.A human anti-CD40 monoclonal antibody, 4D11, for kidney transplantation in cynomolgus monkeys: induction and maintenance therapy.Am J Transplant. 2009; 9: 1732-1741Crossref PubMed Scopus (96) Google Scholar, 14.Imai A. Suzuki T. Sugitani A. et al.A novel fully human anti-CD40 monoclonal antibody, 4D11, for kidney transplantation in cynomolgus monkeys.Transplantation. 2007; 84: 1020-1028Crossref PubMed Scopus (77) Google Scholar Recent studies suggest that blockade of this pathway using antibodies generates antigen-specific Tregs and limits antigen-specific CD8 expansion.15.Ferrer I.R. Wagener M.E. Song M. et al.Antigen-specific induced Foxp3+ regulatory T cells are generated following CD40/CD154 blockade.Proc Nat Acad Sci USA. 2011; 108: 20701-20706Crossref PubMed Scopus (73) Google Scholar DNA vaccination delivers plasmid DNA encoding the target antigen, either alone or in combination with enhancing elements, to induce both humoral and cellular immune responses. Characteristically, DNA vaccines have been used to target foreign antigens; however, we have shown significant benefits in different models of renal disease targeting self-antigens including chemokines CCL2 (MCP-1) and specific TCRs using this strategy, including the benefits of T cell–directed adjuvants in therapeutic plasmids.16.Wu H. Walters G. Knight J.F. et al.DNA vaccination against specific pathogenic TCRs reduces proteinuria in active Heymann nephritis by inducing specific autoantibodies.J Immunol. 2003; 171: 4824-4829Crossref PubMed Scopus (21) Google Scholar,17.Zheng G. Wang Y. Xiang S.H. et al.DNA vaccination with CCL2 DNA modified by the addition of an adjuvant epitope protects against 'nonimmune' toxic renal injury.J Am Soc Nephrol. 2006; 17: 465-474Crossref PubMed Scopus (30) Google Scholar The use of DNA vaccination has been limited by poor immune responses and this is a particular problem while generating responses to self-antigens where there is intrinsic self-tolerance.18.Ada G. Ramshaw I. DNA vaccination.Expert Opin Emerg Drugs. 2003; 8: 27-35Crossref PubMed Scopus (46) Google Scholar,19.Ada G. Vaccines and vaccination.N Engl J Med. 2001; 345: 1042-1053Crossref PubMed Scopus (226) Google Scholar A recent study by Steinman and others has shown the benefits of targeting the DNA-encoded plasmids to DCs using scFv antibodies directed at DEC205 on the DC surface and we have used this approach in targeting CD40 to DCs.20.Nchinda G. Kuroiwa J. Oks M. et al.The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells.J Clin Invest. 2008; 118: 1427-1436Crossref PubMed Scopus (154) Google Scholar The ability to direct antigens to DCs has the potential to enhance immunogenicity, which is crucial for DNA vaccination and for generating responses against self-antigens. Active Heymann nephritis (HN), an experimental rat model of human autoimmune-mediated membranous nephritis, is induced in Lewis rats by immunization with a crude renal tubular antigen (RTA/Fx1A) and reproduces clinical features of human idiopathic membranous glomerulonephritis.21.Heymann W. Hackel D.B. Harwood S. et al.Production of nephrotic syndrome in rats by Freund's adjuvants and rat kidney suspensions.Proc Soc Exp Biol Med. 1959; 100: 660-664Crossref PubMed Scopus (427) Google Scholar,22.Salant D.J. Quigg R.J. Cybulsky A.V. Heymann nephritis: mechanisms of renal injury.Kidney Int. 1989; 35: 976-984Abstract Full Text PDF PubMed Scopus (95) Google Scholar Both T cell– and B cell–generated antibodies have been shown to have a role in its etiology, and thus CD40/CD154, a key costimulatory pathway between T and B cells, is an attractive target for blockade by DNA vaccination. Here we assess the role of the CD40 DNA vaccine targeting an encoded CD40 fused to a tetanus toxoid T-helper epitope P30 to dendritic cells and evaluate its efficacy in protecting against renal disease, the level of antibody produced, and its functional characteristics in vivo and in vitro. To generate DNA vaccine targeting the encoded CD40 to DCs, the open reading frame for mouse CD40 extracellular domain was cloned into pSC-DEC-OLLA vector (scDEC), a modified pcDNA3.1 vector that contains a gene encoding a single-chain antibody specific for mouse DEC205.20.Nchinda G. Kuroiwa J. Oks M. et al.The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells.J Clin Invest. 2008; 118: 1427-1436Crossref PubMed Scopus (154) Google Scholar We therefore generated a fusion DNA construct with the CD40 gene fused in frame to the C-terminus of scDEC as shown in Figure 1a. Our previous studies showed that introduction of the P30 epitope increased immunogenicity of CCL2 DNA vaccination in a murine model of Adriamycin Nephropathy.17.Zheng G. Wang Y. Xiang S.H. et al.DNA vaccination with CCL2 DNA modified by the addition of an adjuvant epitope protects against 'nonimmune' toxic renal injury.J Am Soc Nephrol. 2006; 17: 465-474Crossref PubMed Scopus (30) Google Scholar We therefore included the P30 epitope at the C-termimus of scDEC-CD40. As a control, CD40 DNA vaccine that does not target the encoded CD40 to DCs (scControl-CD40) was generated as described above using pSC-GL117-OLLA (scControl) vector instead of scDEC vector. The expression of the DNA constructs was confirmed by immunoblotting of cell lysates from cells transfected with respective plasmid DNA (Figure 1b). We further demonstrated that scDEC-CD40 but not scControl-CD40 fusion protein was able to bind to DEC205-expressing CHO-K1 cells (Figure 1c), demonstrating the targeting of scDEC-CD40 to DCs. We measured the serum level of the anti-CD40 autoantibody using the enzyme-linked immunosorbent assay (ELISA) to assess the potency of the different vaccines. Rats were injected intramuscularly (i.m.) twice with scDEC-CD40 or scControl-CD40 DNA vaccines within a 3-week interval. One week after the second vaccination, sera were collected and subjected to ELISA analyses. Serum collected from nonvaccinated rats was used as control. As shown in Figure 2a, there was a fourfold increase in anti-CD40 autoantibody level in scDEC-CD40-vaccinated rats as compared with scControl-CD40-vaccinated ones (0.5238±0.2216 vs. 0.1163±0.0724, P<0.01). The serum level of the anti-CD40 autoantibody in scControl-CD40-vaccinated rats was not significantly increased compared with that in nonvaccinated ones. We assessed the longevity of antibody production, the elevation of anti-CD40, and measured anti-CD40 autoantibody levels at 4, 6, 8, 10, and 12 weeks after induction of HN. Rats were divided into four groups as described in Materials and Methods: DEC-CD40-HN, con-CD40-HN, HN, and complete Freund's adjuvant (CFA). There was a persistence of significantly elevated levels of CD40 antibody throughout the time course of HN in the DEC-CD40-HN group (Figure 2b). The anti-CD40 autoantibody levels in the con-CD40-HN group and HN group were low and not significantly different throughout the time course of HN. To assess the effects of CD40 vaccination on the development of HN, we measured total urinary protein over 16h, urine creatinine, serum albumin, and creatinine at 6, 8, 10, 12 weeks post Fx1A/CFA immunization. The DEC-CD40-HN group did not develop proteinuria throughout the 12-week period. Proteinuria (urine protein/creatinine) of the DEC-CD40-HN group was equivalent to that of the control CFA group throughout the time course (Figure 3a). In the con-CD40-HN group, the onset of proteinuria was delayed by 2 weeks as compared with the HN group, demonstrating an effect, although less potent compared with the DEC-modified vaccine. At week 12, the DEC-CD40-HN group had significantly higher serum levels of albumin compared with the con-CD40-HN and HN groups (Figure 3b) and the serum creatinine level at week 12 was significantly lower in the DEC-CD40-HN group than in the HN group (Figure 3c). No significant differences were observed in terms of creatinine clearance among all four groups at week 12 (data not shown). Our results showed that both scControl-CD40 vaccination and scDEC-CD40 vaccination protected HN rats with a reduction in proteinuria; however, scDEC-CD40 vaccination was significantly more protective compared with scControl-CD40 vaccination. Renal structural injury was assessed histologically by evaluating glomerulosclerosis and tubular atrophy (Figure 4a). Both con-CD40-HN and DEC-CD40-HN groups had significantly less glomerulosclerosis and tubular atrophy compared with the HN group (Figure 4b). Immune cell infiltrations were assessed by immunohistochemical staining of kidney sections with antibodies specific for CD4, CD8, and macrophages at week 12 post Fx1A/CFA immunization. As shown in Figure 5a, the DEC-CD40-HN group had significantly less CD4+ and macrophage cell infiltrates compared with the HN group, whereas the con-CD40-HN group was not significantly different compared with the HN group. CD8+ cell infiltration did not differ significantly among the four groups. Figure 5b shows representative histology from each group. Furthermore, glomerular immunoglobulin G (IgG) deposition (one of the major characteristics of HN) in the DEC-CD40-HN group was less dense compared with the other HN groups (Figure 5c). Instead of a continuous ribbon-like staining of IgG, as seen in HN rats, rats in the DEC-CD40-HN group displayed reduced deposition of IgG along the GBM, whereas IgG deposition in the con-CD40-HN group was equivalent to that in the HN group (Figure 5d). To determine the mechanism underlying the protective effects of CD40 DNA vaccination, we examined the expression of several cytokines and costimulatory molecules in draining lymph nodes (DLNs) at week 12 post Fx1A/CFA immunization by semiquantitative real-time PCR. CTLA-4 mRNA level was upregulated in the DEC-CD40-HN group, and this was not because of changes in CD40 mRNA, which remained unchanged (Figure 6). No significant changes were detected for other cytokines and costimulatory molecules (data not shown). To determine whether the protective effects of scDEC-CD40 vaccination on HN are due to changes in B-cell activation, we assessed the in vitro effects of serum on spleen cells from normal Lewis rats. Sera from the DEC-CD40-HN or CFA group collected at week 4 post Fx1A/CFA immunization were used. For control purpose, an agonist monoclonal antibody against CD40 was used simultaneously in this experiment. B-cell activation was determined by CD86 expression. As compared with agonist CD40 Ab, CD86 was not induced by serum from DEC-CD40-HN and CFA rats (Figure 7a). To determine whether anti-CD40 autoantibody, generated as a result of CD40 vaccination, can inhibit T-cell proliferation by costimulatory blockade, we assessed in vitro effects of serum on a mixed lymphocyte reaction. Carboxyfluorescein diacetate succinamidyl ester (CFSE)–labeled responder cells were cocultured with stimulator cells (irradiated Wistar rat lymphocytes) in the presence or absence of 5% serum. We observed a significant reduction of CD8+ T-cell proliferation in the DEC-CD40-HN serum–treated group compared with the HN serum group (Figure 7b). A representative plot is shown in Figure 7c. The protective effects of anti-CD40 therapy on allograft survival in nonhuman primates have been associated with B-cell depletion.10.Pearson T.C. Trambley J. Odom K. et al.Anti-CD40 therapy extends renal allograft survival in rhesus macaques.Transplantation. 2002; 74: 933-940Crossref PubMed Scopus (149) Google Scholar We therefore assessed B-cell numbers in the spleens of all four groups. As shown in Figure 8a, B-cell numbers were not significantly altered by CD40 vaccination. Figure 8b shows representative immunohistochemical staining of B cells in each group. Our current study demonstrates the therapeutic potential of CD40 DNA vaccine in treating autoimmune renal disease. Our results show that an induction of anti-CD40 antibody by DNA vaccination is enhanced by DC targeting. This antibody induction was persistent in the disease model. CD40 vaccination alone had some protective effect against proteinuria but was exceedingly limited. However, by targeting the CD40 to DCs, the effect was greatly enhanced, with a prevention of the development of proteinuria, an improvement in renal function, and a reduction in histological injury, glomerular antibody deposition, and immune infiltration in this group. As compared with other antibodies, the antibodies generated by DNA vaccination were specific, potent, and nondepleting. Clinically, the costimulatory molecules have been an obvious target for therapeutics with successful introduction clinically of agents such as belatacept tailored to inhibit the B7-CD28 pathway in clinical use in transplantation and other areas.23.Snanoudj R. Frangie C. Deroure B. et al.The blockade of T-cell co-stimulation as a therapeutic stratagem for immunosuppression: Focus on belatacept.Biologics: Targets Ther. 2007; 1: 203-213PubMed Google Scholar Agents targeted at the CD40-CD154 pathway had initially reached clinical use when targeted at CD154 but were limited by side effects.24.Kawai T. Andrews D. Colvin R.B. et al.Thromboembolic complications after treatment with monoclonal antibody against CD40 ligand.Nat Med. 2000; 6: 114Crossref Scopus (543) Google Scholar Newer agents targeting CD40 do not seem to have the thrombotic risk of the previous agents, which would be in keeping with the likely cause of thrombosis in the CD154 studies being due to CD154 expressed on platelets.13.Aoyagi T. Yamashita K. Suzuki T. et al.A human anti-CD40 monoclonal antibody, 4D11, for kidney transplantation in cynomolgus monkeys: induction and maintenance therapy.Am J Transplant. 2009; 9: 1732-1741Crossref PubMed Scopus (96) Google Scholar Currently, CD40 blocking reagents have been applied in large animal studies and seem much more promising.13.Aoyagi T. Yamashita K. Suzuki T. et al.A human anti-CD40 monoclonal antibody, 4D11, for kidney transplantation in cynomolgus monkeys: induction and maintenance therapy.Am J Transplant. 2009; 9: 1732-1741Crossref PubMed Scopus (96) Google Scholar,14.Imai A. Suzuki T. Sugitani A. et al.A novel fully human anti-CD40 monoclonal antibody, 4D11, for kidney transplantation in cynomolgus monkeys.Transplantation. 2007; 84: 1020-1028Crossref PubMed Scopus (77) Google Scholar Thus, our studies appear in keeping with the research demonstrating a key role of CD40-CD154 in immune activation in autoimmune disease and the success in therapeutically blocking this pathway. The antibodies generated by the modified vaccine appear to be blocking and nondepleting, both of which are crucial clinically and may potentially function at the site of T cell-DC/APC interaction in the DLN where we find an increase in expression of CTLA-4, a negative regulator of T-cell function. The efficacy in larger animal models for human testing remains to be studied. Interestingly, despite no proteinuria, the CD40-DEC-205-treated rats had a slightly lower level of serum albumin compared with CFA-treated rats and a lower level of serum creatinine, possibly suggesting some constitutional change in the treated rats. An effect from the CD40 vaccine alone–treated group was suggested by histology with a slight improvement in proteinuria, although both cellular infiltration and immune deposits were not significantly improved in this group. In conclusion, CD40 appears to be an excellent therapeutic target in autoimmune renal disease. The scDEC-CD40 vaccine was more effective compared with the scControl-CD40 vaccine in attenuating progression of HN. This suggests that DNA vaccination enhanced by DC targeting can be used therapeutically to block specific immune pathways that are involved in renal disease progression. Inbred male Lewis rats (aged 6 weeks and weighing 180–200g), inbred male Wistar rats, and outbred male Sprague–Dawley rats (aged 8 weeks and weighing 200–220g) were purchased from the Animal Resources Centre in Perth, Australia, and maintained under standard sterile conditions in the Department of Animal Care at Westmead Hospital. Experiments were carried out in accordance with the protocols approved by the Animal Ethics Committee of Sydney West Area Health Service. Antibodies used in immunohistochemical studies included the following: mouse anti-rat CD4 (OX35) (Serotec, Oxford, UK), mouse anti-rat CD8a (OX8) (eBioscience, San Diego, CA), mouse anti-rat CD68 (ED1) (Serotec), mouse anti-rat CD45RA (OX33) (BD Pharmingen, San Diego, CA), and biotinylated goat anti-mouse immunoglobulin (Zymed Laboratories, San Francisco, CA). FITC-goat anti-rat IgG used for immunofluorescence staining was from Zymed Laboratories. APC-mouse anti-rat CD4 and PE-mouse anti-rat CD8 used for flow cytometry staining were purchased from BD Pharmingen. APC-mouse anti-rat CD45RA (OX33) used for flow cytometry staining was from eBioscience. Anti-mouse/rat CD40 antibody (clone: HM40-3) (eBioscience) was used for in vitro stimulation experiments. PE anti-mouse CD40 antibody (FGK45.5) used for the binding assay was kindly provided by Dr Irina Caminschi from Burnet Institute, Melbourne, Victoria, Australia. Propidium iodide used for flow cytometry analyses was from Merck Millipore Biosciences (Darmstadt, Germany). pSC-DEC-OLLA and pSC-GL117-OLLA vectors were kindly provided by Dr Godwin Nchinda from the USA.20.Nchinda G. Kuroiwa J. Oks M. et al.The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells.J Clin Invest. 2008; 118: 1427-1436Crossref PubMed Scopus (154) Google Scholar Mouse cDNA for amplification of CD40 was reverse-transcribed from total RNA extracted from the spleen of Adriamycin Nephropathy mouse. Total RNA was extracted using an RNeasy Mini kit (Qiagen, Hilden, Germany) and reverse transcription was performed using a SuperScript III First-Strand Synthesis system (Invitrogen, Carlsbad, CA). The gene encoding the extracellular domain of CD40 was modified by fusing in frame to a sequence encoding a P30 epitope (FNNFTVSFWLRVPKVSASHLE,17.Zheng G. Wang Y. Xiang S.H. et al.DNA vaccination with CCL2 DNA modified by the addition of an adjuvant epitope protects against 'nonimmune' toxic renal injury.J Am Soc Nephrol. 2006; 17: 465-474Crossref PubMed Scopus (30) Google Scholar), with P30 at the C-terminus of CD40. Construction of CD40-P30 fusion DNA was performed using sequence overlapping primer extension PCR with specific primers: CD40-Nterm-FW: 5′-CGTGGCGGCCGCCTAGGGCAGTGTGTTACGTGC-3′; CD40-P30-Rev: 5′-GGGCACGCGCAGCCAGAAGCTGACGGTGAAGTTGTTGAATCGCATCCGGGACTTTAAACC-3′; P30-Cterm template: 5′-AGCTTCTGGCTGCGCGTGCCCAAGGTCAGCGCCAGCCACCTGGAG-3′; and P30-Cterm-Rev: 5′-CAGTCCGCGGCTCCAGGTGGCTGGCGCTGAC-3′. The CD40-P30 fusion DNA fragment was then cloned into pSC-DEC-OLLA or pSC-GL117-OLLA vector to generate scDEC-CD40 or scControl-CD40 DNA vaccines. The sequences of the two CD40 vaccines were confirmed by DNA sequencing using specific primers: FW 5′-GCGAATGAATTGGGACCT-3′ and Rev 5′-CTTCTGAGATGAGTTTTTGTTCG-3′. Plasmid DNA was prepared in large scale using Plasmid Maxi Prep (Qiagen). Plasmid DNAs (scControl-CD40, scDEC-CD40, scControl vector, and scDEC vector) were transiently transfected into HeLa cells, and 24h post transfection total cell lysates were immunoblotted with goat anti-mouse CD40, followed by donkey anti-goat Ig-horseradish peroxidase, and detected with a chemiluminescent ECL detection system (Chemicon, Temeculat, CA). Full-length mouse DEC-205 cDNA was expressed in CHO-K1 cells, and DEC205-expressing CHO-K1 cells were sorted by flow cytometry (CHO-DEC-205 transfectants). Parental CHO-K1 cells or CHO-DEC-205 transfectants were incubated (30min; 4°C) with supernatant from nontransfected cells (N/T sup) or with supernatant from cells transfected with scControl-CD40 or scDEC-CD40 constructs. Cells were washed and stained with PE rat anti-CD40 mAb (FGK4.5). Propidium iodide (0.5mg/ml) was added to the final cell wash and cells were subjected to flow cytometry analyses. Propidium iodide–positive dead cells were excluded from the analyses. Rats were divided into four groups: the DEC-CD40-HN group (n=5), comprising rats vaccinated with scDEC-CD40, followed by Fx1A immunization; the con-CD40-HN group (n=5), comprising rats vaccinated with scControl-CD40, followed by Fx1A immunization; the HN group (n=4), comprising nonvaccinated rats with Fx1A immunization; and the CFA group (n=3), comprising nonvaccinated rats with CFA immunization. Rats were immunized with DNA by i.m. injection in conjunction with electroporation in the anterior tibialis (TA) muscles of the right hind leg. One week before DNA immunization, rats were injected i.m. with 0.75% bupivacaine (1μl/g body wt; Sigma, St Louis, MO), which was followed by two i.m. DNA injections (3 weeks apart with 300μg/150μl sterile H2O/rat/injection) at the same location as the bupivacaine injection. Each DNA injection was followed immediately by square wave electroporation at the injection site using BTX830 two-needle array electrodes (BTX Harvard Apparatus, San Diego, CA). The distance between the electrodes was 10mm and the array was inserted longitudinally relative to the muscle fibers. In vivo electroporation parameters were as follows: 100V/cm; 50-msec pulse length; six pulses with reversal of polarity after three pulses. Two weeks after the second DNA injection, HN was induced. To induce HN, Lewis rats were immunized subcutaneously in both hind footpads. Each footpad was injected with 100μl of emulsion containing 15mg of Fx1A, 1mg mycobacterium tuberculosis HRa37 (Difco, Detroit, MI), 100μl of IFA (Sigma-Aldrich, St Louis, MO), and 100μl of PBS. The CFA control rats were immunized with emulsion without Fx1A. Fx1A was extracted from outbred male Sprague–Dawley rats as described previously.16.Wu H. Walters G. Knight J.F. et al.DNA vaccination against specific pathogenic TCRs reduces proteinuria in active Heymann nephritis by inducing specific autoantibodies.J Immunol. 2003; 171: 4824-4829Crossref PubMed Scopus (21) Google Scholar All procedures were performed with rats under isoflurane anesthesia. Serum anti-CD40 antibody level was evaluated using an ELISA 'Ensemble' kit for the detection of rat primary antibody (Alpha Diagnostic International, San Antonio, TX) according to the manufacturer's protocols. C96 Maxisorp MicroWell™ (NUNC, Thermo Fisher Scientific, Waltham, MA) plates were used and recombinant mouse CD40 protein (R&D system, Minneapolis, MN) was used for coating of the plates at a concentration of 1μg/ml in 100μl of coating buffer. All sera were diluted 1:10 with a sample diluent as provided in the kit. Absorbance was measured at 450nm with a Multiskan EX Microplate photometer (Thermo Fisher Scientific). Absorbance at 570nm was used as background correction. Blood and 16-h urine samples were collected every 2 weeks after the induction of HN. Urine protein concentration was measured using a colorimetric assay (Bio-Rad, Hercules, CA) based on the method of Bradford. Urine creatinine, serum albumin, and serum creatinine were analyzed using an automated chemistry analyzer VITROS (Ortho Clinical Diagnostics, Johnson & Johnson, New Brunswick, NJ) by staff at the Institute of Clinical Pathology and Medical Research at Westmead Hospital. Coronal sections of the kidney were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections of paraffin block measuring 4μm in thickness were stained with periodic acid–Schiff's reagent and counterstained with hematoxylin. Renal histopathology was graded as previously described.25.Wang Y.M. Zhang G.Y. Wang Y. et al.Foxp3-transduced polyclonal regulatory T cells protect against chronic renal injury from adriamycin.J Am Soc Nephrol. 2006; 17: 697-706Crossref PubMed Scopus (74) Google Scholar Immunohistochemical staining was performed to determine the infiltrations of CD4+, CD8+ T cells, and macrophages in the kidney. For staining of CD4+ T cells, frozen sections (cut at 5μm from kidney tissues embedded in OCT compound) were used. Further, for staining of CD8+ T cells and macrophages, paraffin sections were used. Sections were incubated with primary antibody (16h, 4°C), followed by secondary antibody incubation (30min, RT) as previously described.25.Wang Y.M. Zhang G.Y. Wang Y. et al.Foxp3-transduced polyclonal regulatory T cells protect against chronic renal injury from adriamycin.J Am Soc Nephrol. 2006; 17: 697-706Crossref PubMed Scopus (74) Google Scholar Immune cell infiltration was quantified by counting 10 consecutive high-power fields per animal and expressed as cells per × 200 field. The number of OX33-positive B cells in the spleen was quantified by counting three random high-power fields per animal and expressed as cells per × 400 field. Slides were scanned using a ScanScope digital slide scanner (Aperio Technologies, Vista, CA). Image analysis was performed using Image J software (NIH, Bethesda, MD). Immunofluorescence staining of IgG in kidney sections was performed to assess IgG deposition on glomeruli. Frozen sections were incubated with goat serum (15min, RT), followed by FITC-goat anti-rat IgG antibody (2h, RT). Images (magnification × 400) were taken using a DeltaVision core microscope (Applied Precision, Washington). Fluorescence density was quantified using Image J software (NIH). Lewis rat lymphocytes from the spleen were labeled with CFSE according to the manufacturer's protocol (Invitrogen). CFSE-labeled Lewis lymphocytes (4 × 105) (responder cells) were stimulated with irradiated (25Gy 137Cs) unlabeled Wistar lymphocytes (4 × 105) in flat-bottomed 96-well plates. Cells were cultured in RPMI1640 medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum, 100μ/ml penicillin/streptomycin, 10mmol/l Hepes, sodium pyruvate, nonessential amino acids (NEAA), 50μmol/l 2-ME, and 2mmol/l l-glutamine (Invitrogen) at 37°C, 5% CO2. After 5 days of culture, cells were harvested and subjected to flow cytometry analysis. Single cell suspension was directly stained with APC- and PE-conjugated mouse anti-rat mAb to cell surface antigens. All samples were analyzed on a FACSCanto or FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA). Flowjo software was used for analysis (Tree Star, Ashland, Oregon). Total RNA was isolated from rat DLNs and reverse transcribed (as above). cDNA was subjected to quantitative PCR analysis using Taqman Gene Expression Assays specific for the genes of interest (Applied Biosystem, Melbourne, VIC, Australia). PCR reaction mix was subjected to a Rotor-Gene 3000 thermal cycler (Corbett Life Science, Brisbane, QLD, Australia) for acquisition and analysis. Statistical analysis was performed using one-way analysis of variance for multiple comparisons. For comparison between two groups, the Student t-test was performed. Results are expressed as the group mean±s.d. Differences were considered significant at P<0.05. Statistical analysis was performed using Prism 5 (GraphPad Software, La Jolla, CA). This work was supported by research grants from the National Health and Medical Research Council of Australia (NHMRC grant 571343 and 632517). We thank the animal house staff in the Westmead Hospital Animal House Facility for care of the animals. We also thank Godwin Nchinda (The Rockefeller University) for kindly providing the DEC205 vectors and Virginia James (Westmead Millennium Institute, Australia) for her technical assistance in histology.