DNA Vaccination with CCL2 DNA Modified by the Addition of an Adjuvant Epitope Protects against "Nonimmune" Toxic Renal Injury
Guoping Zheng*,
Yiping Wang*,
Shi-Hua Xiang,
Yuet-Ching Tay*,
Huiling Wu,,
Debbie Watson,
Jason Coombes*,
Gopala K. Rangan*,
Stephen I. Alexander and
David C.H. Harris*
* Centre for Transplantation and Renal Research, the University of Sydney at Westmead Millenium Institute; Centre for Kidney Research, the Children’s Hospital at Westmead; Department of Renal Medicine, Royal Prince Alfred Hospital, University of Sydney, Sydney, New South Wales, Australia; and Department of Cancer Immunology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
Address correspondence to: Dr. Guoping Zheng, Centre for Transplantation and Renal Research, University of Sydney at Westmead Millenium Institute, Westmead, Sydney, NSW 2145 Australia. Phone: +61-2-9845-8906; Fax: +61-2-9633-9351; guoping_zheng{at}wmi.usyd.edu.au
Received for publication February 13, 2005.
Accepted for publication November 25, 2005.
CC-chemokine-encoding DNA vaccine has been reported to be capableof inducing immunologic memory to corresponding pathogenic selfCC-chemokines in animal models of autoimmune disease. This studyinvestigated whether introduction of a foreign T helper epitopeinto monocyte chemoattractant protein 1 (CCL2) DNA vaccine couldboost its immunogenicity by inducing strong neutralizing autoantibodyagainst the pathogenic chemokine CCL2 sufficiently to be protectivein a classically nonimmune model of disease, Adriamycin nephropathy(AN). Modification of the CCL2 DNA vaccine by replacing a surfaceloop region of CCL2 sequence with tetanus toxoid T helper epitopeP30 elicited a strong self-specific CCL2 autoantibody production,as well as an IFN-–producing T cell cellular response.The increased immunogenicity of modified CCL2 DNA vaccinationbut not unmodified CCL2 DNA vaccination was protective againstfunctional and structural renal injury in rat AN. The protectiveeffect of the modified CCL2 DNA vaccine was associated withblockade of glomerular and interstitial macrophage recruitmentby neutralizing autoantibody against CCL2, which plays a criticalrole in eliciting renal injury in AN. Therefore, modificationwith a foreign T helper epitope breaks self-tolerance by inducinga cellular and humoral response against self-protein and providesa strategy to increase the potency of DNA vaccination sufficientlyto afford protection in toxin-induced chronic renal disease.
Adriamycin nephropathy (AN) is an experimental analogue of humanfocal glomerular sclerosis, characterized by severe nephroticsyndrome, focal segmental glomerulosclerosis, tubular atrophyand prominent infiltration of macrophages and CD4+ and CD8+T cells (1–5). AN is a toxic response to Adriamycin (ADR)that can occur in the absence of cognate immune responses. Overtchronic proteinuria develops shortly after ADR administration,and glomerulosclerosis and tubulointerstitial injury becomeobvious by week 4 (2,6). CC-chemokines, particularly CCL2, playa crucial role in attracting mononuclear inflammatory cellsto the interstitium and glomerulus and in modulating interactionsbetween resident and inflammatory cells and consequent renalinjury (7–10).
Several pieces of evidence have demonstrated the importanceof CCL2 in causing renal interstitial and glomerular inflammation.We showed previously that NF-B–dependent CCL2 transcriptionin rat renal cortex was increased 1 wk after ADR administrationand peaked at week 2 (11), coincident with the peak of interstitialmacrophage infiltration (3,6). In vitro studies revealed thatCCL2 expression in rat proximal tubular epithelial cells wasinduced by LPS and by albumin and transferrin (12,13). Deletionof CCL2 dramatically reduced macrophage and T cell recruitmentto kidney and consequent tubular injury (10). Blockade of chemokinesand their receptors using specific antibody (Ab) has been shownto be only partially effective or ineffective (14,15) in modelsof renal disease, which might be a consequence of developmentof host Ab to the therapeutic Ab after repeated administration.
DNA vaccination represents a novel immunization strategy thatuses in vivo antigen expression to induce both humoral and cellularimmune responses. DNA vaccines encoding CC-chemokines have beenreported to be capable of breaking host immune tolerance topathogenic chemokines that are produced under disease conditionssuch as experimental autoimmune encephalomyelitis and adjuvantarthritis (16,17). The advantages of DNA vaccination in theseautoimmune diseases are its long-lasting protective immunityagainst target antigen and its ability to break immune toleranceto pathologic self-proteins. However, the potency of DNA vaccinationremains a challenge to its application (18,19). We demonstratedrecently that DNA vaccination using vaccines encoding both CCL2and CCL5 is protective against renal injury in AN (20). Blockadeof CCL2 alone using dominant negative mutant CCL2 interferencehas been shown to be sufficient to attenuate renal injury inducedby protein-overload proteinuria (21) and prevent renal fibrosis(8). Nevertheless, redundancy of chemokine networks suggeststhat more than one chemokine is likely to be operative and thattargeting a single chemokine by DNA vaccination may not be capableof affording protection. In this study, modification of wild-typeCCL2 gene by insertion of a foreign T helper epitope, P30 fromtetanus toxoid, into surface loop region to enhance the potencyof the DNA vaccine was tested for its efficacy in the classicallynonimmune model, AN.
DNA is known to activate antigen-presenting cells through theToll receptors. However the mechanism of DNA vaccination ininducing responses against self-antigens is still not clear.Adjuvants including CpG motifs, complement factors, and co-stimulatorymolecules have been incorporated into naked DNA vaccines toenhance efficacy. As compared with viral vaccine strategies,naked DNA is restricted in its actions, which may be of benefitin not inducing pathogenic autoreactivity. We have previouslyshown that DNA vaccination with the addition of CpG adjuvantcan induce inhibition of TCR V by production of antibodies againstthe V (22). Here we show that the addition of P30 induces acellular as well as a humoral response that is sufficient toprotect against CCL2-mediated macrophage-driven tissue injury.
Construction and Modification of the CCL2 DNA Vaccine and Vaccination
Wild-type rat CCL2 cDNA was amplified by reverse transcription–PCR(RT-PCR) from RNA extracts from kidney of AN rats using thefollowing primers: CCL2 sense 5'-ccactatgcaggtctctgtc-3' andantisense 5'-atcacattccaaatcacactag-3'. Rat CCL2 gene was modifiedby replacing a surface loop region 109 to 138 (37 to 46 aminoacids) by P30 tetanus toxoid helper epitope sequence FNNFTVSFWLRVPKVSASHLEusing sequence overlapping primer extension PCR. Rat CCL2 primersin combination with overlapping primers were used for this modification.The overlapping primers were as follows: Overlap primer 1 (antisense)GACCTTGGGCACGCGCAGCCAGAAGCTGACGGTGAAGTTGGTGAAgtagcagcaggtgagtggand overlap primer 2 (sense) TGGCTGCGCGTGCCCAAGGTCAGCGCCAGCCACCTGGAGatgagtcggctggagaacta.
CCL2 5' primer and overlap primer 1 were used to amplify thefirst fragment of modified CCL2 gene, whereas overlap primer2 and CCL2 3' primer were used to amplify the second fragmentof modified CCL2. The first fragment annealed to the secondfragment at overlap region, then the whole modified CCL2 genewas extended further and amplified with 5' and 3' CCL2 primers.The modified CCL2 PCR product was cloned into pTarget vector(Promega, Madison, WI) to make the modified CCL2 DNA vaccine.The modified CCL2 (CCL2) vaccine sequence was verified by DNAsequencing after cloning. Plasmid DNA was prepared in large-scaleusing Qiagen Plasmid Mega Kit (Qiagen, Hilden, Germany).
In Vitro Expression of Modified CCL2 Gene and Three-Dimensional Prediction of Modified CCL2 Protein
HeLa cells, which do not express CCL2, were transiently transfectedwith plasmid DNA of pTarget vector, pTarget/CCL2, and pTarget/CCL2using lipofectamine 2000 (Invitrogen, Carlsbad, CA). Cell lysateswere immunoblotted with rabbit anti-rat CCL2 (Chemicon, Temecula,CA), followed by goat anti-rabbit Ig:horseradish peroxidase,and detected with a chemiluminescent ECL detection system (Chemicon).The three-dimensional structure of modified rat CCL2 proteinwas predicted and compared with wild-type using SWISS-MODELonline (http://swissmodel.expasy.org/).
DNA Vaccination and Induction of AN
Male Wistar rats that were approximately 4 to 5 wk of age andweighed 90 to 110 g were purchased from the Australian ResearchCouncil and maintained under standard sterile conditions inthe Department of Animal Care at Westmead Hospital. Experimentswere carried out in accordance with protocols approved by theAnimal Ethics Committee of Sydney West Area Health Service.Animals were divided randomly into five groups: Normal control(n = 4), ADR control (n = 6), vector control (n = 6), unmodifiedCCL2 vaccine (n = 6), and modified CCL2 vaccine (n = 7). Ratswere pretreated with 0.75% bupivacaine (1 µl/g body wt;Sigma, St. Louis, MO) by intramuscular injection into tibialisanterior muscle 1 wk before plasmid DNA vaccination. PlasmidDNA (300 µg) was injected weekly on four occasions intothe same site as bupivacaine. One week after the fourth DNAvaccination, AN was induced by a single tail-vein injectionof ADR (5 mg/kg; David Bull Labs, Victoria, Australia; Figure 1).
Figure 1. Experimental protocol for assessment of DNA vaccination.
Evaluation of Antichemokine Ab Titer in Sera of DNA-Vaccinated Rats
A direct ELISA assay as described (16) was used to determinethe anti-CCL2 or anti-CCL3 (MIP-1) Ab titer in sera from DNA-vaccinatedrats. Recombinant rat CCL2 and CCL3 were coated onto 96-wellELISA plates (Titertek, Horsham, PA) at a concentration of 50ng/well in 100 µl of coating buffer. Rat sera, in serialdilution from 26 to 220, were added to coated ELISA plates.Goat anti-rat IgG alkaline phosphatase-conjugated Ab (Sigma)and p-Nitrophenol phosphate (Sigma) as substrate were used sequentiallyfor the Ab titer analysis. Absorbance at 405 nm was read byan ELISA reader (Bio-Rad, Oakland, CA). The results are expressedas Ab titer dilution ± SEM.
Detection of CCL2 Expression in AN Kidney
The level of CCL2 mRNA expressed in glomeruli and tubules ofAN rats was analyzed by Laser Capture MicroDissection (PALM;Microlaser Technologies AG, Bernried, Germany) of OCT (Tissue-Tek,Sakura Rinetek, Torrance, CA) frozen tissue sections and RT-PCR.OCT-embedded frozen kidney cortex was cut in 10-µm sectionsand mounted on DEPC-pretreated glass slide, fixed in 70% ethanoland briefly stained in Mayers Hematoxylin, then laser capturedimmediately into lysis buffer (Absolutely RNA Nanoprep Kit;Stratagene, La Jolla, CA) under x200 magnification. Total RNAfrom microdissected glomerular or tubular samples was isolatedusing the Absolutely RNA Nanoprep Kit. RT-PCR was performedusing Superscript III CellsDirect cDNA Synthesis Kit (Invitrogen),then amplified for CCL2 with primers described previously andglyceraldehyde-3-phosphate dehydrogenase with the followingprimers: Sense 5'-aatggggtgatgctggtgctga-3' and antisense 5'-tgggggctgagttgggatgg-3'.The level of CCL2 expression was quantified by the densitometricratio of CCL2/glyceraldehyde-3-phosphate dehydrogenase.
Renal Function
Blood and 16-h urine samples were collected weekly after ADRadministration. Urine protein concentrations were determinedby colorimetric assay (Bio-Rad). Blood and urine creatininewere determined as described previously (6). Creatinine clearancewas calculated as creatinine excretion divided by serum creatinineconcentration.
Histopathology
Four weeks after ADR administration, animals were killed fortissue collection. A coronal slice of kidney from each animalwas fixed in 10% neutral-buffered formalin for 24 h and thendehydrated in graded alcohols and embedded in paraffin. Tissueswere cut at 5 µM and stained with periodic acid-Schiffand hematoxylin. Glomerulosclerosis, tubular atrophy, and interstitialexpansion were measured using a quantitative method (5). Digitalimages of cortical sections were analyzed using image software(Image J; National Institutes of Health, Bethesda, MD). Glomerularcapillary tuft was outlined and computed as a measure of totalglomerular area. Glomerulosclerosis was quantified as percentageof periodic acid-Schiff–positive staining area in totalglomerular area of the same glomerulus. The mean of 20 randomlyselected glomeruli was determined for each section. Tubularatrophy was defined by low cell height with absence of brushborder. The degree of interstitial expansion was determinedby relative interstitial volume. The mean value of five corticalfields was determined for each section. Random cortical fieldswere viewed by an observer who was blinded to group identificationsat a magnification of x200. The areas for morphometric analysiswere anatomically identical for each section and positionedbefore microscopic visualization.
Immunohistochemistry
Frozen sections were cut at 6 µM from cortical slicesof kidneys that were embedded in OCT compound, fixed with acetoneat 4°C for 10 min, and immersed in 0.3% H2O2 for 10 minto eliminate endogenous peroxidase and blocked with BackgroundBuster (AXELL, Westbury, NY) at 10 min each to minimize backgroundand nonspecific Ab binding. Sections were incubated individuallywith mAb against macrophages (CD68+) and CD8+ and CD4+ T cells(Serotec, Oxford, UK) for 90 min, followed by goat anti-mouseIgG:horseradish peroxidase–conjugated secondary Ab (Serotec)and visualized by addition of freshly prepared 3,3-diaminobenzidinetetrahydrochloride. The number of macrophages and CD8+ and CD4+T cells was quantified in 10 nonoverlapping cortical fields(x400), expressed as number of cells per x400 field per animal.
Enzyme-Linked Immunospot Assay
Enzyme-linked immunospot (ELISpot) assay was performed accordingBD ELISpot protocol. The 96-well multiscreen plates (Millipore,Bedford, MA) were coated with capture Ab, mouse anti-rat IFN-(Biosource, Camarillo, CA; 25 ng/well). Lymphocytes from poplitealdraining lymph nodes (DLN) of DNA-vaccinated rats were isolatedand added in triplicate to the ELISpot plate at 3 x 105 cells/wellin RPMI 1640 with 5% FCS, then stimulated with recombinant CCL2(rCCL2) at concentrations of 0, 25, 50, and 100 ng/well andincubated at 37°C in 5% CO2 for 24 h. The detection Ab,biotin-conjugated mouse anti-rat IFN- (Biosource; 25 ng/well),was added, then kept at 4°C overnight, incubated with streptavidinalkaline phosphatase (Becton Dickinson, San Jose, CA) at roomtemperature for 2 h, and then developed with BCIP/NBT kit (Bio-Rad).Spots were counted under a dissecting microscope. The resultsare expressed as number of spot-forming cells/106 DLN cells.
In Vitro Chemotaxis Assay
The chemotaxis assay was performed according to Youssef’smethod (16) with modification. Rat rCCL2 (10 ng/ml; Chemicon)in RPMI 1640 was added to lower wells of six-well plates withor without preincubation with the sera of DNA-vaccinated rats.Monocytes that were prepared by Lymphoprep (Axia-Shield PoCAS, Oslo, Norway) from rat splenocytes were seeded onto upperwells (insert with polycarbonate membrane of 8 µM poresize; Nunc, Roskilde, Denmark) at 4 x 106 cells/ml in RPMI 1640enriched with 1% BSA and incubated at 37°C, 5% CO2 for 2h. Cells that migrated to lower wells were harvested by trypsinization,and the total number of migrated cells in each well was countedby hemocytometer. The results are expressed as total numberof migrated cells after subtracting the number of cells in controlwells without rCCL2.
Statistical Analyses
Data are presented as mean ± SEM. Significance of differenceswas examined using one-way multiple range ANOVA test by posthoc Fisher PLSD analysis to compare means among groups. P <0.05 was considered significant.
Modification of Wild-Type CCL2 DNA Vaccine to Express a Hybrid Protein In Vitro that Could be Recognized by Anti-CCL2 Ab
DNA sequencing and three-dimensional structure prediction revealedthat modification of the CCL2 DNA vaccine replaced a surfaceloop region of the wild-type CCL2 with foreign T helper epitopeP30 while maintaining its natural folding and presenting theP30 peptide at surface loop region (Figure 2) to induce an autoimmuneresponse against the self-antigen rat CCL2. In vitro expressiondemonstrated that modified CCL2 DNA vaccine expressed a hybridprotein with a slightly higher molecular weight (15 kD) thanthe wild type (14 kD) and could be recognized by anti-rat CCL2Ab by Western blot (Figure 3).
Figure 2. Three-dimensional structure prediction of modified CCL2 protein by SWISS-MODEL. (A) Wild-type CCL2: Peptide in yellow is targeted for modification. (B) Modified CCL2: Peptide in yellow is the replacing P30 sequence.
Figure 3. Expression of modified CCL2 protein in vitro. HeLa cells were transiently transfected with plasmid constructs of modified and unmodified CCL2 DNA vaccines as well as pTarget vector control. Lysates of transfected HeLa cells were probed with rabbit anti-rat CCL2 polyclonal antibodies (Ab). Lane 1, HeLa cells that were transfected with modified CCL2 DNA vaccine; lane 2, HeLa cells that were transfected with unmodified CCL2 DNA vaccine; lane 3, HeLa cells that were transfected with pTarget vector control; lane 4, HeLa cell lysate control.
Production of Autoantibodies against CCL2 and CCL3 by Vaccination with Modified CCL2 DNA
Self-specific Ab against CCL2 was produced in sera of rats aftervaccination with modified CCL2 DNA as well as unmodified CCL2DNA. However, the Ab titer in the sera of rats that were vaccinatedwith modified CCL2 was significantly higher than that of ratsthat were vaccinated with unmodified CCL2 at week 2 of AN (Figure 4A),the time of peak interstitial macrophage infiltration.High-level autoantibody was sustained throughout the diseasecourse until week 4 (Figure 4A). Ab titer of normal rats thatwere vaccinated with modified CCL2 was significantly lower thanthat of AN rats that were vaccinated with modified or unmodifiedCCL2 but not different from that of AN rats that were vaccinatedwith vector control and normal control.
Figure 4. Self-specific Ab titer to CCL2 (A) and CCL3 (B) determined by ELISA using serial dilution of sera of DNA-vaccinated rats. Results are means ± SEM. (A) The anti-CCL2 Ab titer in sera of rats that were vaccinated with modified CCL2 DNA was significantly higher at weeks 2 and 4 compared with that of rats that were vaccinated with unmodified CCL2 at weeks 2 and 4 (#P < 0.05) and with each other group at weeks 0, 2, and 4 (**P < 0.001). The anti-CCL2 Ab titer in sera of modified CCL2-vaccinated rats without Adriamycin (ADR) was not different from that of normal control at weeks 0, 2, and 4. (B) High levels of anti-CCL3 Ab were produced by rats that were vaccinated with modified or unmodified CCL2 DNA at 2 and 4 wk after ADR (*P < 0.05 versus vector control, modified CCL2 vaccine alone, and Adriamycin nephropathy [AN] alone).
As cross-reactivity of Ab against CC-chemokine DNA vaccine towardhomologous CC-chemokines has been reported elsewhere (17), anti-serafrom rats that were vaccinated with modified CCL2 was testedalso for Ab titer to CCL3 and CCL5 (RANTES). Ab titer to rCCL3was significantly higher in rats that were vaccinated with modifiedCCL2 as well as unmodified CCL2 compared with that of the vectorcontrol group at weeks 2 and 4 (Figure 4B) but not so for rCCL5(data not shown).
Glomerular and Tubular CCL2 Expression in AN
CCL2 expression was detected in both glomeruli and tubules thatwere isolated by laser capture microdissection. Significantlyhigher levels of CCL2 expression were detected in tubules 1wk after ADR compared with normal control (Figure 5). The highlevel of CCL2 expression in tubules was also observed at weeks2, 3, and 4. Compared with tubular expression, glomerular expressionof CCL2 was much lower but significantly increased from week1 to week 4 after ADR in comparison with its normal control(Figure 5).
Figure 5. CCL2 expression in diseased kidney of AN rats was upregulated in both tubules and glomeruli that were isolated by microdissection. Values presented in the graph are densitometric ratios of CCL2 to glyceraldehyde-3-phosphate dehydrogenase control from three independent experiments. *P < 0.05 and **P < 0.01 versus normal control.
Protection against Kidney Injury in AN by DNA Vaccination with Modified CCL2 Vaccine
Rats that were vaccinated with modified CCL2 DNA vaccine developedmuch less severe renal structural injury at week 4 of AN. Glomerularand tubular damage was only mild in rats that were vaccinatedwith modified CCL2 DNA, in comparison with very severe glomerularand tubular injury in rats from ADR control, vector control,and unmodified CCL2 vaccine groups (Figure 6). Morphometricanalysis (Table 1) showed significantly less glomerular injurywith respect to total glomerular area, percentage of glomerulosclerosis,and number of nuclei per glomerulus in rats that were vaccinatedwith modified CCL2 compared with that of rats that were vaccinatedwith vector control or unmodified CCL2. Tubular cell height—butnot tubular diameter—and interstitial volume were alsosignificantly less in the modified CCL2 group compared withunmodified CCL2 and vector control groups.
Figure 6. Protection against renal structural injury in AN rats by vaccination with modified CCL2 DNA vaccine. Kidney tissues that were harvested 4 wk after ADR administration were analyzed under periodic acid-Schiff stain. Representative histology of normal control (A), rats that were vaccinated with vector control (B), rats that were vaccinated with modified CCL2 DNA (C), and rats that were vaccinated with unmodified CCL2 DNA (D).
Table 1. Morphometric quantification of histologic changes in AN rats 4 wk after Adriamycin
Renal function also was protected significantly by vaccinationwith modified CCL2 vaccine. Creatinine clearance was significantlyhigher in rats that were vaccinated with modified CCL2 thanthat of ADR control, vector control, and unmodified CCL2 vaccinegroups at weeks 2 and 4 (Figure 7) but not different from normalcontrols. There were no statistical differences in creatinineclearance among groups at weeks 1 and 3, although the mean valueof the modified CCL2 group was higher than all other groupsthat received ADR. Urine protein/creatinine ratio was lowerin rats that received modified CCL2 vaccination than in groupsthat received vector control and ADR only at week 3 and allother ADR groups at week 4 (Figure 8).
Figure 7. Protection of renal function (creatinine clearance) in AN rats by vaccination with modified CCL2 DNA vaccine. Creatinine clearance of AN rats (n = 7) that were vaccinated with modified CCL2 was significantly higher at weeks 2 and 4 compared with that of vector control rats (n = 6) and rats that were vaccinated with unmodified CCL2 (n = 6) and ADR alone (n = 6; 2.62 ± 0.13 versus 1.79 ± 0.24 and 1.47 ± 0.26 ml/min, *P < 0.05; 2.65 ± 0.04 versus 1.57 ± 0.04, 1.19 ± 0.12, and 1.54 ± 0.01 ml/min, *P < 0.05) but not different from normal control (n = 4; 2.12 ± 0.09 and 2.97 ± 0.4, P = 0.20 and 0.25, respectively). Values are means ± SEM.
Figure 8. Urine protein/creatinine ratio taken from 16-h urine collection. The urine protein/creatinine ratio of AN rats that were vaccinated with modified CCL2 was significantly lower at week 3 compared with that of vector control and ADR alone groups (4438.3 ± 259.7 versus 5679.6 ± 401.9 and 5471.7 ± 347.8; *P < 0.05) and at week 4 compared with all ADR groups (4573.7 ± 167.7 versus 5856.3 ± 318.9, 6181.3 ± 356.2, and 6897.6 ± 617.3; **P < 0.01). Values are means ± SEM.
Protection of AN Was Associated with Reduced Glomerular and Interstitial Macrophage Infiltration
Immunohistochemical staining of inflammatory cells revealedthat modified CCL2 DNA vaccination significantly reduced glomerularmacrophage recruitment compared with unmodified CCL2 DNA vaccine,vector control, and ADR control. There was an even greater reductionin interstitial macrophage infiltration (Figure 9). There wereno differences among unmodified CCL2, vector control, and ADRcontrol groups. There was no difference in numbers of interstitialCD8+ and CD4+ T cells among the groups (Figure 9).
Figure 9. Number of interstitial inflammatory cells in glomeruli (Glom) and tubules (Tub), determined by immunohistochemistry. Results are the average number of cells counted from 10 of x400 fields per animal. Values are means ± SEM. *P < 0.05, **P < 0.001 versus the other three groups (unmodified CCL2, vector control, and ADR alone).
Increased IFN-–Producing T Cells from DLN after Modified CCL2 DNA Vaccination
To assess the cellular response of modified CCL2 DNA vaccinein comparison with unmodified vaccine and vector control, thenumbers of IFN-–producing T cells from DLN of vaccinatednormal rats were assessed by ELISpot assay. Rats that were vaccinatedwith modified CCL2 DNA had more T cells from DLN that producedIFN- when stimulated with 100 ng/ml rCCL2 in vitro than ratsthat were vaccinated with unmodified CCL2 or vector controlrats. The number of IFN-–producing T cells of rats thatwere vaccinated with unmodified CCL2 vaccine was not differentfrom that of vector control (Figure 10).
Figure 10. IFN-–producing T cells (from draining lymph nodes [DLN]) as determined by enzyme-linked immunospot assay in response to rCCL2 stimulation in vitro. Results are the number of spot-forming cells (SFC) per 106 DLN cells, expressed as mean ± SEM of three different samples. aP = 0.06 versus unmodified CCL2 and vector vaccination; bP < 0.001 versus unmodified CCL2 and vector vaccination and normal control.
Antisera Neutralization of Chemoattractive Effect of CCL2 toward Monocytes In Vitro
To explore the mechanism whereby modified CCL2 DNA vaccinationreduced glomerular and interstitial macrophages, the effectof antisera was tested in vitro for its blocking effect againstCCL2-induced monocyte chemotaxis. The total number of migratedmonocytes was significantly lower under antisera of rats thatwere vaccinated with modified CCL2 than under antisera fromrats that were vaccinated with unmodified CCL2 DNA or vectorcontrol or with rCCL2 only control wells (Figure 11).
Figure 11. Blockade of monocyte chemotaxis by sera of DNA-vaccinated rats, as assessed by total number of cells that migrated to the lower chamber through an 8-µM membrane in response to rCCL2. Values are means ± SEM of three different serum samples. **P < 0.01 versus unmodified CCL2 and vector control.
DNA vaccination is a new approach in which genes that encodetarget antigens are delivered and expressed in vivo within hostcells. Although underlying mechanisms to explain both humoraland cellular immune responses to DNA vaccination are not fullyunderstood, the presence of stimulatory CpG motifs within theexpression plasmid backbone of bacterial origin is thought toact as adjuvant for stimulation of specific host immune responsetoward the antigen encoded by the DNA vaccine (23–25).Although DNA vaccines have shown broad promise in models ofinfectious diseases, cancer, and autoimmune diseases, low potencyremains a hurdle to its clinical application (26). Approachesto increase potency of DNA vaccine include improved vaccinedelivery and increased immunogenicity of expressed antigensby the manipulation of their encoding genes. The foreign T helperepitope has been reported to break B cell tolerance to the highlyconserved self-protein ubiquitin when expressed as a hybridprotein with ubiquitin (27,28). P30 from tetanus toxoid wasalso reported to bypass immunologic tolerance in a vaccine againstIL-5 (29). T cell help has been considered crucial in breakingB cell tolerance to permit development of autoantibodies againstself-proteins (29,30). In this study, we sought to enhance immunogenicityof DNA vaccine against self-protein CCL2 by modification ofits gene. Modification of the CCL2 gene, by insertion of a foreignT helper epitope P30 in the surface loop region, enhanced productionof autoantibody against native CCL2 protein, as well as thenumber of IFN-–producing T cells from DLN. Modified CCL2hybrid protein was designed to maintain the tertiary structureof native CCL2 as much as possible, to induce cross-reactiveantibodies against self CCL2 protein.
Using this DNA vaccination strategy, we demonstrated that modifiedCCL2 DNA vaccination is protective against both renal functionaland structural injury in AN. Given the concurrence of elevatedCCL2 expression with macrophage infiltration and renal damagein AN (3,11) and the widely known role of CCL2 in recruitmentand activation of monocytes, it is likely that CCL2 is responsiblefor recruitment and activation of macrophages to the interstitiumand glomerulus and consequent renal injury. The pathogenic roleof CCL2 has been proved in other models of renal disease bythe use of neutralizing antibodies against CCL2 (14,31) andby blockade of CCL2/CCR2 using mutant CCL2 gene therapy (21,32).Consistent with these findings, our study showed that blockadeof CCL2 by autoantibodies that are produced through DNA vaccinationwith modified CCL2 protected the chemokine-mediated renal injuryof AN. This contention is strongly supported by reduced interstitialand glomerular macrophage infiltration in rats that were vaccinatedwith modified CCL2, high titers of anti-CCL2 autoantibodiesin their sera, and the neutralizing effect of the anti-serain blocking CCL2-mediated monocyte chemotaxis in vitro.
Glomerular expression of CCL2 has been reported in other modelsof kidney disease to be responsible for recruitment of macrophages,which in turn are responsible for triggering production of inflammatorycytokines and other chemokines that lead to tissue injury andconsequent glomerulosclerosis (10,14,33,34). In this study,we demonstrated that although its expression was predominantlytubular, CCL2 was also expressed by glomeruli in AN. Blockadeof CCL2-induced macrophage recruitment and activation withinglomeruli may well explain the protection of glomerulosclerosis,as well as tubulointerstitial injury, by modified CCL2 DNA vaccination.Consistent with this effect on glomerulosclerosis, proteinuriawas also reduced by vaccination with modified CCL2 DNA. Proteinuriaitself may be injurious to glomerular and tubular epithelium(35–38) and lead to local upregulation of chemokines,particularly CCL2 (13,37,39).
Only modified CCL2 DNA vaccine but not unmodified CCL2 DNA vaccinewas found to be protective against both functional and structuralrenal injury in AN, and anti-CCL2 Ab titers and number of IFN-–producingT cells from DLN were greater with modified than unmodifiedDNA vaccination. Thus, the potency of unmodified DNA vaccine,with the adjuvant effect of stimulatory CpG motif within backboneto enhance its immunogenicity, was insufficient to block theeffect of CCL2, whereas its modification with foreign T helperepitope P30 to greatly enhance its immunogenicity rendered itsufficiently potent to block the effect of CCL2. It is interestingthat, in vaccinated rats, not only the titer of anti-CCL2 antibodywas significantly increased but also that of anti-CCL3. Thismay be due to the structural similarities of these chemokinesleading to cross-reactivity. As multiple chemokines such asCCL2, CCL3, and CCL5 have been reported to be implicated inglomerular and tubulointerstitial inflammation (40), particularlyin rat AN (11,20), it is possible that the additional inhibitoryeffect against closely related chemokines such as CCL3 in thisstudy may have helped to overcome redundancy and provide greaterclinical efficacy.
Another important finding in this study was that the anti-CCL2autoantibody level in normal rats that were vaccinated withmodified CCL2 was much lower than in vaccinated rats with ANand not different from that of vector control vaccination, indicatingthe disease specificity of autoantibody production and educationof host immune system against the pathogenic self-protein. Thisinteresting feature of DNA vaccination responses that are limitedto inflamed tissues is intriguing and suggests the possibilitythat innate danger signals may play a role in priming the autoantibodyresponses while limiting these in noninflamed tissues. The presenceof cellular immunity of a Th1 type found in the DLN with theuse of the modified vaccine was shown by the presence of IFN-–producingT cells from DLN of vaccinated rats, specific for rCCL2. TheseIFN-–producing T cells include both CD4+ and CD8+ T cells(41,42). CD4+ IFN-–producing T cells represent helperand memory T cells that assist B cell antibody production andlong-lasting memory, whereas CD8+ IFN-–producing T cellsare effector or cytotoxic T cells whose role in modified CCL2DNA vaccination requires further investigation.
Modification of CCL2 DNA vaccine by the addition of P30 enhancedits immunogenicity, leading to a novel cellular response andan increased humoral response to CCL2 chemokine. The effectof the inhibition of this crucial pathway is shown by the inhibitionof interstitial and glomerular macrophage infiltration and consequentrenal injury in a toxin-induced model of chronic renal disease.
Acknowledgments
This study was supported by a project grant (249414) from NH&MRCof Australia.
A portion of this work was presented at the American Societyof Nephrology Meeting; October 29 to November 1, 2004; St. Louis,MO.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Bertani T, Rocchi G, Sacchi G, Mecca G, Remuzzi G: Adriamycin-induced glomerulosclerosis in the rat.
Am J Kidney Dis 7
: 12
–19, 1986
Rangan GK, Wang Y, Harris DC: Induction of proteinuric chronic glomerular disease in the rat (Rattus norvegicus) by intracardiac injection of doxorubicin hydrochloride.
Contemp Top Lab Anim Sci 40
: 44
–49, 2001[Medline]
Wang Y, Wang YP, Tay YC, Harris DC: Progressive Adriamycin nephropathy in mice: Sequence of histologic and immunohistochemical events.
Kidney Int 58
: 1797
–1804, 2000[CrossRef][Medline]
Wang Y, Wang YP, Tay YC, Harris DC: Role of CD8(+) cells in the progression of murine Adriamycin nephropathy.
Kidney Int 59
: 941
–949, 2001[CrossRef][Medline]
Wang Y, Feng X, Bao S, Yi S, Kairaitis L, Tay YC, Rangan GK, Harris DC: Depletion of CD4(+) T cells aggravates glomerular and interstitial injury in murine Adriamycin nephropathy.
Kidney Int 59
: 975
–984, 2001[CrossRef][Medline]
Rangan GK, Wang Y, Tay YC, Harris DC: Inhibition of nuclear factor-kappaB activation reduces cortical tubulointerstitial injury in proteinuric rats.
Kidney Int 56
: 118
–134, 1999[CrossRef][Medline]
Strutz F, Neilson EG: New insights into mechanisms of fibrosis in immune renal injury.
Springer Semin Immunopathol 24
: 459
–476, 2003[CrossRef][Medline]
Wada T, Furuichi K, Sakai N, Iwata Y, Kitagawa K, Ishida Y, Kondo T, Hashimoto H, Ishiwata Y, Mukaida N, Tomosugi N, Matsushima K, Egashira K, Yokoyama H: Gene therapy via blockade of monocyte chemoattractant protein-1 for renal fibrosis.
J Am Soc Nephrol 15
: 940
–948, 2004[Abstract/Free Full Text]
Natori Y, Sekiguchi M, Ou Z: Gene expression of CC chemokines in experimental crescentic glomerulonephritis (CGN).
Clin Exp Immunol 109
: 143
–148, 1997[CrossRef][Medline]
Tesch GH, Maifert S, Schwarting A, Rollins BJ, Kelley VR: Monocyte chemoattractant protein 1-dependent leukocytic infiltrates are responsible for autoimmune disease in MRL-Fas(lpr) mice.
J Exp Med 190
: 1813
–1824, 1999[Abstract/Free Full Text]
Rangan GK, Wang Y, Tay YC, Harris DC: Cytokine gene expression in Adriamycin nephropathy: Effects of antioxidant nuclear factor kappaB inhibitors in established disease.
Nephron 86
: 482
–490, 2000[CrossRef][Medline]
Wang Y, Rangan GK, Goodwin B, Tay YC, Harris DC: Lipopolysaccharide-induced MCP-1 gene expression in rat tubular epithelial cells is nuclear factor-kappaB dependent.
Kidney Int 57
: 2011
–2022, 2000[CrossRef][Medline]
Wang Y, Chen J, Chen L, Tay YC, Rangan GK, Harris DC: Induction of monocyte chemoattractant protein-1 in proximal tubule cells by urinary protein.
J Am Soc Nephrol 8
: 1537
–1545, 1997[Abstract]
Fujinaka H, Yamamoto T, Takeya M, Feng L, Kawasaki K, Yaoita E, Kondo D, Wilson CB, Uchiyama M, Kihara I: Suppression of anti-glomerular basement membrane nephritis by administration of anti-monocyte chemoattractant protein-1 antibody in WKY rats.
J Am Soc Nephrol 8
: 1174
–1178, 1997[Abstract]
Wu X, Dolecki GJ, Sherry B, Zagorski J, Lefkowith JB: Chemokines are expressed in a myeloid cell-dependent fashion and mediate distinct functions in immune complex glomerulonephritis in rat.
J Immunol 158
: 3917
–3924, 1997[Abstract]
Youssef S, Wildbaum G, Maor G, Lanir N, Gour-Lavie A, Grabie N, Karin N: Long-lasting protective immunity to experimental autoimmune encephalomyelitis following vaccination with naked DNA encoding C-C chemokines.
J Immunol 161
: 3870
–3879, 1998[Abstract/Free Full Text]
Youssef S, Maor G, Wildbaum G, Grabie N, Gour-Lavie A, Karin N: C-C chemokine-encoding DNA vaccines enhance breakdown of tolerance to their gene products and treat ongoing adjuvant arthritis.
J Clin Invest 106
: 361
–371, 2000[Medline]
Kutzler MA, Weiner DB: Developing DNA vaccines that call to dendritic cells.
J Clin Invest 114
: 1241
–1244, 2004[CrossRef][Medline]
Muthumani K, Kudchodkar S, Zhang D, Bagarazzi ML, Kim JJ, Boyer JD, Ayyavoo V, Pavlakis GN, Weiner DB: Issues for improving multiplasmid DNA vaccines for HIV-1.
Vaccine 20
: 1999
–2003, 2002[CrossRef][Medline]
Wu H, Wang Y, Tay YC, Zheng G, Zhang C, Alexander SI, Harris DC: DNA vaccination with naked DNA encoding MCP-1 and RANTES protects against renal injury in Adriamycin nephropathy.
Kidney Int 67
: 2178
–2186, 2005[CrossRef][Medline]
Shimizu H, Maruyama S, Yuzawa Y, Kato T, Miki Y, Suzuki S, Sato W, Morita Y, Maruyama H, Egashira K, Matsuo S: Anti-monocyte chemoattractant protein-1 gene therapy attenuates renal injury induced by protein-overload proteinuria.
J Am Soc Nephrol 14
: 1496
–1505, 2003[Abstract/Free Full Text]
Wu H, Walters G, Knight JF, Alexander SI: DNA vaccination against specific pathogenic TCRs reduces proteinuria in active Heymann nephritis by inducing specific autoantibodies.
J Immunol 171
: 4824
–4829, 2003[Abstract/Free Full Text]
Davis HL: CpG motifs for optimization of DNA vaccines.
Dev Biol (Basel) 104
: 165
–169, 2000[Medline]
Encke J, zu Putlitz J, Stremmel W, Wands JR: CpG immuno-stimulatory motifs enhance humoral immune responses against hepatitis C virus core protein after DNA-based immunization.
Arch Virol 148
: 435
–448, 2003[CrossRef][Medline]
Liu MA: DNA vaccines: A review.
J Intern Med 253
: 402
–410, 2003[CrossRef][Medline]
Donnelly J, Berry K, Ulmer JB: Technical and regulatory hurdles for DNA vaccines.
Int J Parasitol 33
: 457
–467, 2003[CrossRef][Medline]
Dalum I, Jensen MR, Hindersson P, Elsner HI, Mouritsen S: Breaking of B cell tolerance toward a highly conserved self protein.
J Immunol 157
: 4796
–4804, 1996[Abstract]
Dalum I, Jensen MR, Gregorius K, Thomasen CM, Elsner HI, Mouritsen S: Induction of cross-reactive antibodies against a self protein by immunization with a modified self protein containing a foreign T helper epitope.
Mol Immunol 34
: 1113
–1120, 1997[CrossRef][Medline]
Hertz M, Mahalingam S, Dalum I, Klysner S, Mattes J, Neisig A, Mouritsen S, Foster PS, Gautam A: Active vaccination against IL-5 bypasses immunological tolerance and ameliorates experimental asthma.
J Immunol 167
: 3792
–3799, 2001[Abstract/Free Full Text]
Wenzel U, Schneider A, Valente AJ, Abboud HE, Thaiss F, Helmchen UM, Stahl RA: Monocyte chemoattractant protein-1 mediates monocyte/macrophage influx in anti-thymocyte antibody-induced glomerulonephritis.
Kidney Int 51
: 770
–776, 1997[Medline]
Furuichi K, Wada T, Iwata Y, Kitagawa K, Kobayashi K, Hashimoto H, Ishiwata Y, Tomosugi N, Mukaida N, Matsushima K, Egashira K, Yokoyama H: Gene therapy expressing amino-terminal truncated monocyte chemoattractant protein-1 prevents renal ischemia-reperfusion injury.
J Am Soc Nephrol 14
: 1066
–1071, 2003[Abstract/Free Full Text]
Anders HJ, Vielhauer V, Kretzler M, Cohen CD, Segerer S, Luckow B, Weller L, Grone HJ, Schlondorff D: Chemokine and chemokine receptor expression during initiation and resolution of immune complex glomerulonephritis.
J Am Soc Nephrol 12
: 919
–931, 2001[Abstract/Free Full Text]
Tang WW, Qi M, Warren JS: Monocyte chemoattractant protein 1 mediates glomerular macrophage infiltration in anti-GBM Ab GN.
Kidney Int 50
: 665
–671, 1996[Medline]
Ruggenenti P, Perna A, Mosconi L, Pisoni R, Remuzzi G: Urinary protein excretion rate is the best independent predictor of ESRF in non-diabetic proteinuric chronic nephropathies. "Gruppo Italiano di Studi Epidemiologici in Nefrologia" (GISEN).
Kidney Int 53
: 1209
–1216, 1998[CrossRef][Medline]
Eddy AA: Experimental insights into the tubulointerstitial disease accompanying primary glomerular lesions.
J Am Soc Nephrol 5
: 1273
–1287, 1994[Abstract]
Zoja C, Donadelli R, Colleoni S, Figliuzzi M, Bonazzola S, Morigi M, Remuzzi G: Protein overload stimulates RANTES production by proximal tubular cells depending on NF-kappa B activation.
Kidney Int 53
: 1608
–1615, 1998[CrossRef][Medline]
Abbate M, Zoja C, Corna D, Capitanio M, Bertani T, Remuzzi G: In progressive nephropathies, overload of tubular cells with filtered proteins translates glomerular permeability dysfunction into cellular signals of interstitial inflammation.
J Am Soc Nephrol 9
: 1213
–1224, 1998[Abstract]
Wang Y, Rangan GK, Tay YC, Harris DC: Induction of monocyte chemoattractant protein-1 by albumin is mediated by nuclear factor kappaB in proximal tubule cells.
J Am Soc Nephrol 10
: 1204
–1213, 1999[Abstract/Free Full Text]
Anders HJ, Vielhauer V, Schlondorff D: Chemokines and chemokine receptors are involved in the resolution or progression of renal disease.
Kidney Int 63
: 401
–415, 2003[CrossRef][Medline]
Kaech SM, Wherry EJ, Ahmed R: Effector and memory T-cell differentiation: Implications for vaccine development.
Nat Rev Immunol 2
: 251
–262, 2002[CrossRef][Medline]
Combadiere B, Boissonnas A, Carcelain G, Lefranc E, Samri A, Bricaire F, Debre P, Autran B: Distinct time effects of vaccination on long-term proliferative and IFN-gamma-producing T cell memory to smallpox in humans.
J Exp Med 199
: 1585
–1593, 2004[Abstract/Free Full Text]
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Abstract
Introduction
–producing T cell cellular response. The increased immunogenicity of modified CCL2 DNA vaccination but not unmodified CCL2 DNA vaccination was protective against functional and structural renal injury in rat AN. The protective effect of the modified CCL2 DNA vaccine was associated with blockade of glomerular and interstitial macrophage recruitment by neutralizing autoantibody against CCL2, which plays a critical role in eliciting renal injury in AN. Therefore, modification with a foreign T helper epitope breaks self-tolerance by inducing a cellular and humoral response against self-protein and provides a strategy to increase the potency of DNA vaccination sufficiently to afford protection in toxin-induced chronic renal disease. 
B–dependent CCL2 transcription in rat renal cortex was increased 1 wk after ADR administration and peaked at week 2 (11), coincident with the peak of interstitial macrophage infiltration (3,6). In vitro studies revealed that CCL2 expression in rat proximal tubular epithelial cells was induced by LPS and by albumin and transferrin (12,13). Deletion of CCL2 dramatically reduced macrophage and T cell recruitment to kidney and consequent tubular injury (10). Blockade of chemokines and their receptors using specific antibody (Ab) has been shown to be only partially effective or ineffective (14,15) in models of renal disease, which might be a consequence of development of host Ab to the therapeutic Ab after repeated administration. 
by production of antibodies against the V
CCL2) vaccine sequence was verified by DNA sequencing after cloning. Plasmid DNA was prepared in large-scale using Qiagen Plasmid Mega Kit (Qiagen, Hilden, Germany). 
) Ab titer in sera from DNA-vaccinated rats. Recombinant rat CCL2 and CCL3 were coated onto 96-well ELISA plates (Titertek, Horsham, PA) at a concentration of 50 ng/well in 100 µl of coating buffer. Rat sera, in serial dilution from 26 to 220, were added to coated ELISA plates. Goat anti-rat IgG alkaline phosphatase-conjugated Ab (Sigma) and p-Nitrophenol phosphate (Sigma) as substrate were used sequentially for the Ab titer analysis. Absorbance at 405 nm was read by an ELISA reader (Bio-Rad, Oakland, CA). The results are expressed as Ab titer dilution ± SEM. 
