Research Article Designation of a Novel DKK1 Multiepitope...

Research ArticleDesignation of a Novel DKK1 Multiepitope DNA Vaccine andInhibition of Bone Loss in Collagen-Induced Arthritic Mice

Xiaoqing Zhang12 Sibo Liu1 Shentao Li1 Yuxuan Du1 Yunpeng Dou1

Zhanguo Li3 Huihui Yuan1 and Wenming Zhao1

1Department of Immunology School of Basic Medical Sciences Capital Medical University No 10 Xitoutiao You An MenFengtai Beijing 100069 China2Department of Immunology College of Medical Sciences China Medical University No 77 Puhe Road Shenyang North AreaShenyang Liaoning 110122 China3Department of Rheumatology amp Immunology Clinical Immunology Center Peking University Peoplersquos HospitalNo 11 Xizhimen South Street Beijing 100044 China

Correspondence should be addressed to Huihui Yuan huihui yuan163com and Wenming Zhao zhao-wenming163com

Received 28 November 2014 Accepted 5 January 2015

Academic Editor Long Shen

Copyright copy 2015 Xiaoqing Zhang et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Dickkopf-1 (DKK1) a secretory inhibitor of canonical Wnt signaling plays a critical role in certain bone loss diseases Studies haveshown that serum levels ofDKK1 are significantly higher in rheumatoid arthritis (RA) patients and are correlatedwith the severity ofthe disease which indicates the possibility that bone erosion in RAmay be inhibited by neutralizing the biological activity of DKK1In this study we selected a panel of twelve peptides using the softwareDNASTAR 71 and screened high affinity and immunogenicityepitopes in vitro and in vivo assays Furthermore we optimized four B cell epitopes to design a novel DKK1 multiepitope DNAvaccine and evaluated its bone protective effects in collagen-induced arthritis (CIA) a mouse model of RA High level expressionof the designed vaccine was measured in supernatant of COS7 cells In addition intramuscular immunization of BALBc mice withthis vaccine was also highly expressed and sufficient to induce the production of long-term IgG which neutralized natural DKK1in vivo Importantly this vaccine significantly attenuated bone erosion in CIA mice compared with positive control mice Theseresults provide evidence for the development of a DNA vaccine targeted against DKK1 to attenuate bone erosion

1 Introduction

Rheumatoid arthritis (RA) a chronic symmetrical autoim-mune disease is characterized by synovial inflammation andproliferation accompanied by cartilage erosion and bone loss[1] More than one-third of patients eventually experienceemployment disability and lower quality of life becauseof this disease which is largely responsible for the highsocioeconomic burden of RA [2] Furthermore mortalityrates in RA patients are higher than in the healthy population[3]

The aim of RA treatment is the achievement of remis-sion [4] but many RA patients who are judged by theirconsulting rheumatologist to be in remission after receiving

conventional therapy still show structural deterioration [5]Therefore the long-term goals of treatment are to preventjoint destruction and the comorbidities of the disease [3]A number of reports have suggested that modern therapydoes not inhibit joint damage satisfactorily despite achievingclinical remission [6]Therefore the development of effectiveagents to inhibit bone erosion in RA patients is urgent

The canonical Wnt signaling pathway promotes boneformation not only by stimulating the differentiation ofosteoblasts increasing the growth rate of osteoblasts andreducing their apoptosis but also by inhibiting osteoclasto-genesis [7] The canonical Wnt signaling pathway is trig-gered by the association of secretory Wnt with its frizzled(Fz) receptors and low density lipoprotein receptor-related

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 765490 9 pageshttpdxdoiorg1011552015765490

2 BioMed Research International

protein 56 (LRP56) on the cell surface This stabilizes120573-catenin which is eventually translocated into the nucleusand activates the TCFLEF-mediated transcription of targetgenes that elicit a variety of effects including the inductionof osteoblast differentiation and proliferation [8] Dickkopf-1 (DKK1) a soluble and natural inhibitor of the canonicalWnt signaling pathway may play an active role in inhibitingosteogenesis by binding the ligands ofWnt proteins [9 10] Inaddition to LPR56 DKK1 was also found to bind to Kremenanother cell surface coreceptor forming a ternary complexthat is rapidly endocytosed resulting in the depletion ofcell surface LRP56 [11] Studies have demonstrated that thelevels of DKK1 in serum were significantly higher in RApatients and were correlated with the severity of the disease[12] In a mouse model of RA treatment with an anti-DKK1 antibody has attenuated bone erosion [9] ThereforeDKK1 may be a promising therapeutic target for RA boneloss

Over the last 20 years great progress has been made indeveloping DNA vaccines [13] DNA vaccines have manyadvantages over conventional chemical agents biologicalagents and protein vaccines in the treatment of diseasesFirst plasmid preparation is rapid and cost effective anddoes not suffer from problems such as improper proteinfolding Plasmid DNA is highly stable and flexible allowingfor the modification of plasmid sequences [14] In additionthe antigen presenting cells (APCs) process and present theepitopes from antigens on MHC I and II molecules therebyinducing both humoral immunity and cellular immunityActive immunotherapy is feasible to reverse the disorders ofautoimmune diseases DNA vaccines have proven effective inanimal models including RA Crohnrsquos disease systemic ery-thematosus lupus (SLE) and infectious diseases [15] and havebeen extensively evaluated in humans The advancementsof bioinformation software and molecular immunology pro-moted the development of DNA vaccine [16] Currently 72Phase I 20 Phase II and 2 Phase III clinical trials have beenidentified [17]

In this study we selected a panel of twelve peptides usingthe software DNASTAR 71 and screened high affinity andimmunogenicity epitopes in vitro and in vivo assays Thenwe optimized four B cell epitopes and designed a novel DKK1multiepitope DNA vaccine determined its immunogenicityand evaluated its protective effects Our data demonstratedthat this DNA vaccine ameliorated bone erosion significantlyin mice with collagen-induced arthritis (CIA)

2 Materials and Methods

21 Cells and Mice COS7 cells were grown in Dulbeccorsquosmodified Eaglersquos medium (DMEM) supplemented with 5fetal bovine serum (FBS) in a humidified 5 CO

2incubator

at 37∘CSix-week-old female BALBc (H-2d) mice and 5-week-

old male DBA1 mice (a CIA-susceptible mouse strain H-2q) were both purchased from HFK Biotechnology Co LtdAll mice were maintained in a specific pathogen-free envi-ronment All animal experiments were performed according

to the guidelines of the Animal Care and Use Committee ofCapital Medical University

22 Construction and Preparation of the DNA Vaccine B cellepitopes in the amino acid sequence of human DKK1 wereanalyzed using the software DNASTAR 71 The separatedepitopes were synthesis from Invitrogen (Life TechnologiesCalifornia USA) Subsequently the indirect ELISA wasutilized to detect the affinity of separated epitopes Simply96-well plates were coated with peptides (1 120583gmL) overnightat 4∘C The DKK1 polyclonal antibodies (diluted 1 1000200120583L RampD Systems Minneapolis MN USA) were addedto wells and incubated overnight at 4∘C After washing theHRP conjugate goat anti-human DKK1 secondary antibody(100 120583L diluted 1 2000 Southern Biotech BirminghamAL USA) was added and the plates were incubated for1 h at 37∘C Then the peptides were immunized BALBcmice and the immunogenicity was measured as previouslydescribed [18] The reactions were stopped by the addition of50 120583L of Stop Solution (RampD Systems) and OD450 readingswere measured with an ELISA plate reader To reduce thebioactivity of DKK1 vaccine the peptides (100 120583ginjectionintraperitoneally) were applied for a week The tibias werecollected and an acid phosphatase kit (Sigma-Aldrich StLouis MO USA) was used to demonstrate osteoclasts [19]

Four fragments in the DKK1 sequence (110ndash144aa 153ndash181aa 182ndash216aa and 228ndash253aa) with high affinity andimmunogenicity were selected to construct the multiepitopeDNA vaccine The DNA sequences encoding four aminoacids (R203 H204 K211 and R236) in DKK1 were mutatedto glutamate to reduce the biological activity of DKK1 [20]AAY spacers between two adjacent epitope fragments wereused to link the four selected epitopes A Th2 cell epitopewas added at both sequence termini Finally the signalpeptide of human DKK1 was added at the N-terminus of thesequenceThe synthetic nucleotide sequence named DP wasincorporated into the expression vector pCMV6-XL5 usinga standard DNA recombination procedure resulting in therecombinant plasmid pCMV-DP Plasmids for immunizationwere extracted and purified from transformed Escherichiacoli strain DH5120572 using an endotoxin-free plasmid extractionkit (Roche Mannheim Germany) according to the manu-facturerrsquos instructions The purified plasmids were adjustedto a concentration of 1mgmL in sterile saline and stored atminus80∘C

23 Transfection of Plasmid pCMV-DP into COS7 CellsCOS7 cells were cultured in a 6-well tissue culture plateuntil the cells reached approximately 60 to 80 confluenceThe cells were transfected with the purified plasmid DNAusing TurboFect in vitro Transfection Reagent (FermentasThermoScientific Pittsburgh PA USA) according to themanufacturerrsquos instructions Briefly 4 120583L of plasmid DNA(1mgmL) was diluted in 390120583L of serum-free DMEMTurboFect reagents (6 120583L) were added to the diluted DNAAfter immediate mixing and incubation for 20min at roomtemperature 400 120583L of the mixture was distributed dropwiseinto culturedCOS7 cells in a 6-well plateThe transfected cells

BioMed Research International 3

were cultured for 72 h Cell lysates and culture supernatantswere collected for further analyses

24 Western Blotting of the Expressed Multiepitope Proteinin Cell Lysates The proteins in the COS7 cell lysates wereseparated on a 12 SDS-PAGE gel and transferred onto anitrocellulose membrane by electroblotting The membranewas blocked with 5 nonfat dry milk in TBST (TBS with005 Tween-20) for 1 h at room temperature and then incu-bated with a polyclonal rabbit anti-DKK1 antibody (1 1000Millipore) in TBST containing 025 bovine serum albuminovernight at 4∘C After washing three times with TBSTthe membrane was incubated with horseradish peroxidase-(HRP-) conjugated goat anti-rabbit IgG (1 10000 Protein-Tech Group Chicago USA) for 1 h at room temperatureAfter washing three times the membrane was exposed toa SuperSignal West Pico stable peroxide solution (PierceThermoScientific Pittsburgh PA USA)

25 Enzyme-Linked ImmunosorbentAssay (ELISA)Analysis ofthe Expressed Multiepitope Protein in the Cell Culture Super-natant The protein secreted in the cell culture supernatantwas detected with ELISA Briefly 96-well plates were coatedwith a goat anti-human DKK1 antibody (200 ngmL RampDSystems Minneapolis MN USA) overnight at 4∘C The cellculture supernatant (200120583L) was added to the wells and wasincubated overnight at 4∘C After incubating with 100120583L of agoat anti-human DKK1 antibody (50 ngmL RampD Systems)for 2 h at 37∘C 100 120583L of a working dilution of Streptavidin-HRP was added to each well Color was developed by theaddition of a substrate solution of orthophenylene diamine(OPD) for 20min at 37∘C The reaction was stopped by theaddition of 50 120583L of 2M H

2SO4 The absorbance was read at

450 nm by a microplate reader (Thermomax Technologies)

26 Immunohistochemical Analysis of theMultiepitope ProteinIn Vivo BALBc mice were injected intramuscularly with100 120583g of plasmid pCMV-DP or empty vector pCMV Sevendays later the injected muscles were surgically removedand frozen sections were prepared The sections were driedfor 45min at room temperature fixed with anhydrous ace-tone and then incubated with 005 H

2O2for 20min to

quench the endogenous peroxidase After blocking with 5horse serum the sections were incubated with a goat anti-human DKK1 antibody (5 120583gmL RampD Systems) overnightat 4∘C The next day the sections were incubated with aHRP-conjugated donkey anti-goat IgG (1 1000 ProteinTechGroup) for 1 h at room temperatureThe positive signals weredetected with 3 31015840 diaminobenzidine (DAB RampD Systems)

27 Immunization and Serum Collection For DNA immu-nization experiments 6-week-old female BALBc mice (119899 =6) were injected intramuscularly (im) with plasmid pCMV-DP three times at weeks 0 2 and 4 Mice (119899 = 6)immunized with empty vector pCMV served as negativecontrols Intramuscular injection of plasmid DNA followedby electroporation (DNA + EP) was performed as previ-ously described [21] Briefly 100 120583g of plasmid pCMV-DP

was injected intramuscularly in one tibialis anterior muscleusing a 27-gauge needle Immediately after the injectionelectroporation with 6 electric pulses was applied througha pair of silver electrodes spaced 3mm apart covering theim injection site The electric pulses were 50ms in durationand 1 s apart at a voltage of 60V (ie 200Vcm) From thefirst injectionmouse serum samples were collected at 2-weekintervals and stored at minus80∘C

28 Antibody Assay The antibodies against human DKK1were evaluated using ELISA Briefly 96-well microtiter plateswere coated with 100120583L of recombinant human DKK1 pro-teins (200 ngmL RampD Systems) and incubated overnightat 4∘C After washing three times with PBST (PBS with005 Tween-20) the plates were blocked with 200 120583L of1 bovine serum albumin (BSA) in PBST for 2 h at 37∘CAfter washing 100120583L of diluted serum (1 200) was addedto each well Then the plates were incubated for 2 h at37∘C and washed five times with PBST HRP-conjugatedgoat anti-mouse IgG secondary antibodies (100 120583L) (1 10000ProteinTech Group) were added to each well followed byincubation for 1 h at 37∘C Next the plates were washed fivetimes with PBST After adding the substrate solution and stopsolution the absorbance was read at 450 nm by a microplatereader (Thermomax Technologies) The end-point titer ofantibodywas determined in the samewayThe serum sampleswere serially diluted from 1 200 to 1 12800

29 Preparation and Evaluation of Collagen-Induced ArthritisMouse Model DBA1 mice (119899 = 6) were immunized withplasmid pCMV-DP three times at 2-week intervals asdescribed above Control mice (119899 = 6) were immunized withan equal amount of empty vector pCMV One week after thefinal immunization all mice were injected intradermally atthe base of the tail with 100 120583g of bovine C II emulsifiedwith complete Freudrsquos adjuvant (CFA) containing 4mgmLof heat-killedMycobacterium tuberculosis On day 21 the ani-mals were given a booster injection with 100120583g of bovine C IIdissolved in incomplete Freudrsquos adjuvant (IFA) [22] Animalswere observed and recorded every 3 days after disease onsetClinical scores were assigned to evaluate the disease severityas follows 0 no signs of arthritis 1 swelling andor rednessof one paw or one digit 2 two joints involved 3 three ormore joints involved and 4 severe arthritis of the entire pawsand digits Each limb was graded independently resulting ina maximal clinical score of 16 per affected animal [23]

210 Microcomputer Tomography (CT) Scanning To deter-mine the protective effects of the DNA vaccine 40 daysafter the challenge with bovine C II a micro-CT-200 system(AlokA latheta Laboratory Japan) was employed to detect thebone mineral density (BMD) and the degree of bone erosionafter the mice were anaesthetized with chloral hydrate (10)X-ray images were analyzed by reconstructed 3D quantitativeanalyses using the software VGstudio MAX 20

211 Histopathology Murine hind paws were removed post-mortem stored in 10 neutral formalin decalcified in 20


Alpha regions-Garnier-Robson Beta regions-Garnier-Robson Turn regions-Garnier-Robson Hydrophilicity plot-Kyte-Doolittle Alpha amphipathic regions-Eisenberg

Beta amphipathic regions-Eisenberg Flexible regions-Karplus-Schulz Antigenic index-Jameson-Wolf Surface probability plot-Emini

20 40 60 80 100 120 140 160 180 200 220 240 260ABT0

F0

16

minus45

minus17

lowast

lowast

(a)

00

02

04

06

08

Affi

nity

(OD

450

)

KLH

P32

ndash42

P43

ndash65

P66

ndash109

P110

ndash133

P134

ndash144

P145

ndash152

P153

ndash181

P182

ndash189

P190

ndash216

P217

ndash227

P228

ndash253

P254

ndash266

(b)

00

05

10

15

20

KLH

Imm

unog

enic

ity (O

D450)

P32

ndash42

P43

ndash65

P66

ndash109

P110

ndash133

P134

ndash144

P145

ndash152

P153

ndash181

P182

ndash189

P190

ndash216

P217

ndash227

P228

ndash253

P254

ndash266

(c)

Control P145ndash152P66ndash109P43ndash65

(d)

Figure 1 Designation of the DNA vaccine(a) B cell epitope scanning of human DKK1 was performed with the software DNASTAR 71 (b)The affinity of epitopes was measured by indirect ELISA (c) The separated epitopes were immunized BALBc mice and the immunogenicityof epitopes was measured by sandwich ELISA (d) The separated epitopes were injected to BALBc mice for seven days TRAP staining wasperformed to identify the mature osteoclasts Magnification 200x data are expressed as the mean plusmn SEM

ethylenediaminetetraacetic acid (EDTA) for 6 weeks andthen dehydrated and embedded in paraffin Sections were cutalong the longitudinal axis and stained with toluidine blue(TB) as previously described [18]

212 Statistical Analysis Data were analyzed using the soft-ware SPSS (version 160) and presented as the mean plusmn SEMDifferences between two groups ofmicewere compared usingStudentrsquos 119905-test A 119875 value less than 005 was considered to bestatistically significant

3 Results

31 Construction of the DNA Vaccine According to theanalysis of the potential B cell epitopes in human DKK1 bythe epitope prediction software DNASTAR 71 a panel oftwelve peptides fragments was synthesized (Figure 1(a)) Inaddition to select the high affinity and immunogenicity ofsynthesis peptides the titers of peptides were determinedand the peptides of grey-blue columns were candidates todesign DNA vaccine (Figures 1(b)-1(c)) Furthermore todecrease the pathological functions of DKK1 in peptides


CMV promoter

f1 ori

Amp resistant gene ColE1

SV40 ori

PolyA signal

pCMV6-XL5

4482bp

PADRE

AAY AAY AAY AAY AAY

203(R rarr G) (R rarr G)(Hrarr G)(K rarr G)

204 211 236

MCS

EcoR I Xbal I

SP1ndash31aa PADRE 110ndash144aa 153ndash181aa 182ndash216aa 228ndash253aa

(a)

MMALG AAGAT RVFVA MVAAA LGGHP LLGVS ATLNS VLNSN AIKNL PPPLG GAAGH PGSAV SAAPG ILYPG GNKYQ

TIDNY QPYPC AEDEE CGTDE YCASP TRGGD AGVQI CLACR KRRKR CMRHA MCCPG NYCKN GICVS SDQNH FRGEI

EETIT ESFGN DHSTL DGYSR RTTLS SKMYH TKGQE GSVCL RSSDC ASGLC WSKIC EGQVC TKHRR

KGSHG LEIFQ EGLSC RIQKD HHQAS NSSRL HTCQR H

CARHF KPVLK

RCYCG

(b)

MMALG AAGAT RVFVA MVAAA LGGHP LLGVS AAKFV AAWTL KAAAA AYICL ACRKR RKRCM RHAMC CPGNY CKNGI

CVSSD QNAAY TE FGNDH STLDG YSRRT TLSSK MYHTA AYKGQ EGSVC LRSSD CASGL FWSKI

KEAAY SHGLE YCGEG LSCRI QKDHH QAAYA KFVAA WTLKA AAIFQGC

CCAGG CGPVLSTI

(c)

Figure 2 Construction of the DNA vaccine (a)Themaps of recombinant DKK1DNA vaccine (b)The amino acid sequence of humanDKK1(c)The recombinant amino acid sequence of DKK1 DNA vaccine Red line 110ndash144aa green line 153ndash181aa orange line 182ndash216aa blue line228ndash253aa black line signal peptide box PADRE

the osteoclast-forming assay was performed Comparedwith controls no significant osteoclastogenesis was observedin peptides-treatment group (Figure 1(d)) The syntheticnucleotide sequence with muted four amino acids encodingthe DNA vaccine was cloned in the eukaryotic expressionvector pCMV6-XL5 (Figure 2)

32 Expression of the Multiepitope Chimera Gene In Vitro andIn Vivo The expression of the multiepitope DNA vaccine invitro was evaluated in COS7 cells ELISA showed that themultiepitopeDNA vaccine recombinant protein of DKK1wassecreted abundantly and was recognized by its polyclonalantibodies (Figure 3(a)) In addition Western blotting alsoshowed that COS7 cells transfected with plasmid pCMV-DP highly expressed the recombinant protein (Figure 3(b))To assess the expression of the target protein in vivo theinjected muscles of mice were further stained with an anti-DKK1 antibodyThe expression of recombinant protein in theDKK1 DNA vaccine was much higher in the muscles at theinjected site than in the control group (Figure 3(c)) Due to

these observations we concluded that the multiepitope genewas expressed in eukaryotes both in vitro and in vivo

33 The DNA Vaccine Induced a Specific Antibody A specificanti-human DKK1 antibody was identified in BALBc miceimmunized with plasmid pCMV-DP The serum IgG titerbegan to increase as early as 4 weeks after the primaryimmunization and reached its peak at 6 weeks (Figure 3(d))The end-point titer of the specific antibody was also deter-mined to evaluate its neutralizing effects in vitro (Figure 3(e))Although the end-point titer of anti-DKK1 was only 1 1600the specific antibody existed persistently in the serum up to 6months after immunization

34 Induction of Arthritis in DBA1 Mice DBA1 mice wereinjected with bovine C II twice at 3-week intervals afterimmunization The signs of arthritis appeared around day 24after injection with bovine C II and continued to developat later time points The disease incidence was 83 inprevaccinated mice and 100 in the positive control mice


pCMV-DP0

1

2

3

pCMV

OD

450

lowastlowastlowast

(a)

pCMV-DPpCMV

(b)

pCMV-DPpCMV

(c)

0 2 4 6 8 10 1200

02

04

06

08

10

pCMV-DPpCMV

Weeks after immunization

OD

450

lowast

(d)

pCMV-DP

00

05

10

15

PCMV

Serum dilution ratio

OD

450

1 200

1 400

1 800

1 1600

1 3200

1 6400

1 12800

(e)

Figure 3 Expression and immunogenicity of the DNA vaccine (andashc) Expression of the multiepitope protein in vitro and in vivo weredetermined in cell culture supernatants by ELISA cell lysates byWestern blotting and themuscles of mice by immunohistochemical analysis(d-e) The DNA vaccine induced a specific IgG antibody against human DKK1 The titer and the end-point titer of the specific antibody weretested by ELISA Bars indicate 100 120583m Data are expressed as the mean plusmn SEM lowast119875 lt 005 lowastlowastlowast119875 lt 0001


on day 39 Paw erythema and swelling also became moresevere with time in control mice than the DKK1 vaccinegroup (Figures 4(a) and 4(b)) In addition the infiltrationof inflammatory cells was reduced in the vaccine-immunizedmice compared with the controls (Figure 4(c))

35 The DNA Vaccine Attenuated Bone Erosion in CIA MiceWe further evaluated the amelioration of arthritic bone andthe joint structure was well conserved in the vaccinated micecompared with the positive control mice as demonstratedby microcomputer tomography (CT) scanning (Figure 4(d))The total bone mineral density (BMD) of the mice in thevaccinated groups was significantly higher than that in thepositive control group (Figure 4(e)) The results of TB stain-ing showed that the degree of overall destruction of cartilagewas reduced markedly in the arthritic paws of vaccinatedmice compared with the positive controls (Figure 4(f)) Theresults of micro-CT scanning and TB staining demonstratedthat the DNA vaccine attenuated bone erosion in CIAmice

4 Discussion

DNA vaccines also termed nucleic acid vaccines or genevaccines were first described in the 1990s when Wolff foundthat DNA could be taken up and expressed by mouse skeletalmuscle cells in vivo [24] It is generally thought that the geneof interest in a eukaryotic expression vector is translated intoantigen protein using the cellular expression system of thehost Subsequently the antigen protein stimulates the hostto generate an immune response Compared with traditionalvaccines the greatest advantage of DNA vaccines is their abil-ity to elicit both humoral immunity and cellular immunitySimultaneously they are simple to prepare easy to deliverand relatively safe to apply Therefore DNA vaccination isa promising new technology for the prevention and therapyof diseases such as infectious diseases autoimmune diseasesand cancers

In this study we constructed a DNA vaccine targetinghuman DKK1 a major contributor to bone loss in RA Weexpected to use this vaccine to inhibit bone erosion in a RAmouse model through the blockade of the biological activityofDKK1We have adopted optimization strategies to improvethe antigen expression and the immunogenicity of the DNAvaccine which are discussed below

41 Construction Strategies for the DNA Vaccine To producespecific antibodies B cell epitopes of DKK1 were the firstchoice to construct the DNA vaccine To increase the titerof specific antibodies we combined the predictions of thesoftware DNASTAR 71 and the affinity and immunogenicityassays To reduce their potential bone resorption of naturalDKK1 the synthesis epitopes were injected to BALBc andno significant osteoclastogenesis was observed These datademonstrated that these separated epitopes were safe as avaccine utilized in vivo To increase the immunogenicity ofthe DNA vaccine as expressed in the host the key bindingsites of DKK1 (R203 H204 K211 and R236) to LRP56

and Kremen were mutated At the same time the mutatedprotein expressed in vivo enhanced the immunogenicity ofthe vaccine [20] To facilitate the epitope processing the fourselected epitopes were separated from one another with AAYspacers [25] AAYs expressed inside the selected epitopeswere easily recognized by proteasomes and the full-lengthprotein was more easily processed and presented in vivoDKK1 is expressed naturally in the body To further increasethe immunogenicity of the recombinant DNA vaccine thepan DR T helper epitope (PADRE) was added to enhanceantibody responses [26] Finally the signal peptide forhuman DKK1 was added at the N-terminus of the DNAvaccine to facilitate the extracellular secretion of the proteinantigen

42 Immunization Strategies The DNA vaccine deliverymethod is one of themost important factors that affect immu-nization efficiency The methods for DNA vaccine deliverycurrently used include intramuscular intraperitonealintravenous intradermal subcutaneous injection andothers The delivery method for the plasmid directly affectsthe uptake of the gene and further affects the expression ofthe target protein Intramuscular injection is the earliest andsimplest method for DNA delivery However the majority ofthe administered plasmid DNA was blocked by perimysia ofthe skeletal muscles where most of the DNA was degradedby DNase I in the plasma Less than 1 of the DNA actuallyentered the nucleus [27] Recently electroporation has beenused for DNA vaccine delivery and has proven to be a betterDNA delivery method In this study intramuscular injectionof plasmidDNA followed by electroporation (DNA+EP)wasemployed The DC electric current disrupts cell membranesfor a short time allowing the plasmidDNA to cross themem-branes into the nucleus [28] In addition the electric currentcauses cell destruction and a local inflammatory reaction Alarge number of inflammatory cells including macrophagesand other antigen-presenting cells infiltrated the injectedmuscles which enhanced the efficiency of antigenpresentation In our study the highly specific IgG antibodiesin serumwere induced as early as 4weeks which also demon-strated the efficiency of the electroporation of the DNAvaccine

5 Conclusions

DKK1 a negative regulator of the canonical Wnt signalingpathway plays an important role in RA bone destructionInhibition of its biological functions can attenuate bonedestruction in RA [9] Our results showed that the recom-binant human DKK1 multiepitope DNA vaccine inducedspecific antibodies that effectively neutralized the biologicalactivities of DKK1 and attenuated bone destruction in CIAmice This study may provide a potential therapy for RA andother bone loss diseases

Conflict of Interests

The authors declare no conflict of interests


21 24 27 30 33 36 390

20

40

60

80

100

Days after immunization

Inci

denc

e (

)

pCMV-DPpCMV

(a)

21 24 27 30 33 36 39Days after immunization

pCMV-DP

0

2

4

6

8

10

Clin

ical

scor

e

pCMV

(b)

pCMV pCMV-DP

(c)

pCMV

pCMV-DP

(d)

650

700

750

800

pCMV pCMV-DP

BMD

(mg

cm3 )

lowast

(e)

pCMV-DPpCMV

(f)

Figure 4 The DNA vaccine attenuated bone erosion in CIA mice Mice were injected intradermally with bovine type II collagen to inducearthritis (119899 = 6group) (a-b)The incidence of arthritis and clinical score were recorded until day 39 (cndashf) Representative joints on the day ofsacrifice were analyzed with HE staining Micro-CT images BMD analysis and TB staining images Bars indicate 500 120583m and 200 120583m Dataare expressed as the mean plusmn SEM lowast119875 lt 005


Authorsrsquo Contribution

Xiaoqing Zhang Huihui Yuan and Wenming Zhao con-ceived and designed the experiments Xiaoqing Zhang andSibo Liu performed the experiments Yuxuan Du and Yun-peng Dou analyzed the data Shentao Li and ZhanguoLi contributed reagentsmaterialsanalysis tools XiaoqingZhang andHuihui Yuanwrote the paper XiaoqingZhang andSibo Liu contributed equally to this work

Acknowledgments

This study was supported by the State Key Development Pro-gram for Basic Research of China (Grant no 2010CB529106)and the National Nature Science Foundations of China(Grant no 31400767)

References

[1] D L Scott ldquoBiologics-based therapy for the treatment ofrheumatoid arthritisrdquo Clinical Pharmacology and Therapeuticsvol 91 no 1 pp 30ndash43 2012

[2] J M Davis and E L Matteson ldquoMy treatment approach torheumatoid arthritisrdquoMayo Clinic Proceedings vol 87 no 7 pp659ndash673 2012

[3] I Colmegna B R Ohata and H A Menard ldquoCurrent under-standing of rheumatoid arthritis therapyrdquo Clinical Pharmacol-ogy andTherapeutics vol 91 no 4 pp 607ndash620 2012

[4] P J Mease ldquoImproving the routine management of rheumatoidarthritis the value of tight controlrdquo Journal of Rheumatologyvol 37 no 8 pp 1570ndash1578 2010

[5] A K Brown P G Conaghan Z Karim et al ldquoAn explanationfor the apparent dissociation between clinical remission andcontinued structural deterioration in rheumatoid arthritisrdquoArthritis and Rheumatism vol 58 no 10 pp 2958ndash2967 2008

[6] E T HMolenaar A E Voskuyl H J Dinant P D Bezemer MBoers and B A C Dijkmans ldquoProgression of radiologic dam-age in patients with rheumatoid arthritis in clinical remissionrdquoArthritis and Rheumatism vol 50 no 1 pp 36ndash42 2004

[7] Y Kawano and R Kypta ldquoSecreted antagonists of the wntsignalling pathwayrdquo Journal of Cell Science vol 116 no 13 pp2627ndash2634 2003

[8] E Bourhis C Tam Y Franke et al ldquoReconstitution of aFrizzled8sdotWnt3asdotLRP6 signaling complex reveals multiple Wntand Dkk1 binding sites on LRP6rdquo The Journal of BiologicalChemistry vol 285 no 12 pp 9172ndash9179 2010

[9] D Diarra M Stolina K Polzer et al ldquoDickkopf-1 is a masterregulator of joint remodelingrdquo Nature Medicine vol 13 no 2pp 156ndash163 2007

[10] D Daoussis and A P Andonopoulos ldquoThe emerging role ofdickkopf -1 in bone biology is it the main switch controllingbone and joint remodelingrdquo Seminars in Arthritis and Rheuma-tism vol 41 no 2 pp 170ndash177 2011

[11] A Bafico G Liu A Yaniv A Gazit and S A Aaronson ldquoNovelmechanism ofWnt signalling inhibitionmediated byDickkopf-1 interaction with LRP6Arrowrdquo Nature Cell Biology vol 3 no7 pp 683ndash686 2001

[12] S-Y Wang Y-Y Liu H Ye et al ldquoCirculating dickkopf-1 iscorrelated with bone erosion and inflammation in rheumatoidarthritisrdquo Journal of Rheumatology vol 38 no 5 pp 821ndash8272011

[13] L Li F Saade and N Petrovsky ldquoThe future of human DNAvaccinesrdquo Journal of Biotechnology vol 162 no 2 pp 171ndash1822012

[14] F Saade and N Petrovsky ldquoTechnologies for enhanced efficacyof DNA vaccinesrdquo Expert Review of Vaccines vol 11 no 2 pp189ndash209 2012

[15] D M Klinman S Klaschik D Tross H Shirota and FSteinhagen ldquoFDA guidance on prophylactic DNA vaccinesanalysis and recommendationsrdquo Vaccine vol 28 no 16 pp2801ndash2805 2010

[16] M Mooney S McWeeney G Canderan and R-P Sekaly ldquoAsystems framework for vaccine designrdquo Current Opinion inImmunology vol 25 no 5 pp 551ndash555 2013

[17] M A Kutzler andD BWeiner ldquoDNA vaccines ready for primetimerdquoNature ReviewsGenetics vol 9 no 10 pp 776ndash788 2008

[18] H Yuan H Qian S Liu et al ldquoTherapeutic role of a vaccinetargeting RANKL and TNF-120572 on collagen-induced arthritisrdquoBiomaterials vol 33 no 32 pp 8177ndash8185 2012

[19] A-R Kim H S Kim J M Lee et al ldquoArctigenin sup-presses receptor activator of nuclear factor 120581b ligand (RANKL)-mediated osteoclast differentiation in bone marrow-derivedmacrophagesrdquo European Journal of Pharmacology vol 682 no1ndash3 pp 29ndash36 2012

[20] K Wang Y Zhang X Li et al ldquoCharacterization of thekremen-binding site on Dkk1 and elucidation of the role ofkremen inDkk-mediatedwnt antagonismrdquo Journal of BiologicalChemistry vol 283 no 34 pp 23371ndash23375 2008

[21] B Peng Y Zhao H Lu W Pang and Y Xu ldquoIn vivo plasmidDNA electroporation resulted in transfection of satellite cellsand lasting transgene expression in regenerated muscle fibersrdquoBiochemical and Biophysical Research Communications vol 338no 3 pp 1490ndash1498 2005

[22] D D Brand K A Latham and E F Rosloniec ldquoCollagen-induced arthritisrdquo Nature Protocols vol 2 no 5 pp 1269ndash12752007

[23] S Cuzzocrea E Ayroldi R di Paola et al ldquoRole ofglucocorticoid-induced TNF receptor family gene (gitr) incollagen-induced arthritisrdquo The FASEB Journal vol 19 no 10pp 1253ndash1265 2005

[24] J A Wolff R W Malone P Williams G Acsadi A Jani andP L Felgner ldquoDirect gene transfer into mouse muscle in vivordquoScience vol 247 no 4949 I pp 1465ndash1468 1990

[25] X Wang W Xu D Tong et al ldquoA chimeric multi-epitope DNAvaccine elicited specific antibody response against severe acuterespiratory syndrome-associated coronavirus which attenuatedthe virulence of SARS-CoV in vitrordquo Immunology Letters vol119 no 1-2 pp 71ndash77 2008

[26] M-F Del Guercio J Alexander R T Kubo et al ldquoPotentimmunogenic short linear peptide constructs composed ofB cell epitopes and Pan DR T helper epitopes (PADRE) forantibody responses in vivordquo Vaccine vol 15 no 4 pp 441ndash4481997

[27] MA LiuWMcClements J BUlmer J Shiver and J DonnellyldquoImmunization of non-human primates with DNA vaccinesrdquoVaccine vol 15 no 8 pp 909ndash912 1997

[28] M Golzio J Teissie and M-P Rols ldquoDirect visualizationat the single-cell level of electrically mediated gene deliveryrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 99 no 3 pp 1292ndash1297 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


protein 56 (LRP56) on the cell surface This stabilizes120573-catenin which is eventually translocated into the nucleusand activates the TCFLEF-mediated transcription of targetgenes that elicit a variety of effects including the inductionof osteoblast differentiation and proliferation [8] Dickkopf-1 (DKK1) a soluble and natural inhibitor of the canonicalWnt signaling pathway may play an active role in inhibitingosteogenesis by binding the ligands ofWnt proteins [9 10] Inaddition to LPR56 DKK1 was also found to bind to Kremenanother cell surface coreceptor forming a ternary complexthat is rapidly endocytosed resulting in the depletion ofcell surface LRP56 [11] Studies have demonstrated that thelevels of DKK1 in serum were significantly higher in RApatients and were correlated with the severity of the disease[12] In a mouse model of RA treatment with an anti-DKK1 antibody has attenuated bone erosion [9] ThereforeDKK1 may be a promising therapeutic target for RA boneloss

Over the last 20 years great progress has been made indeveloping DNA vaccines [13] DNA vaccines have manyadvantages over conventional chemical agents biologicalagents and protein vaccines in the treatment of diseasesFirst plasmid preparation is rapid and cost effective anddoes not suffer from problems such as improper proteinfolding Plasmid DNA is highly stable and flexible allowingfor the modification of plasmid sequences [14] In additionthe antigen presenting cells (APCs) process and present theepitopes from antigens on MHC I and II molecules therebyinducing both humoral immunity and cellular immunityActive immunotherapy is feasible to reverse the disorders ofautoimmune diseases DNA vaccines have proven effective inanimal models including RA Crohnrsquos disease systemic ery-thematosus lupus (SLE) and infectious diseases [15] and havebeen extensively evaluated in humans The advancementsof bioinformation software and molecular immunology pro-moted the development of DNA vaccine [16] Currently 72Phase I 20 Phase II and 2 Phase III clinical trials have beenidentified [17]

In this study we selected a panel of twelve peptides usingthe software DNASTAR 71 and screened high affinity andimmunogenicity epitopes in vitro and in vivo assays Thenwe optimized four B cell epitopes and designed a novel DKK1multiepitope DNA vaccine determined its immunogenicityand evaluated its protective effects Our data demonstratedthat this DNA vaccine ameliorated bone erosion significantlyin mice with collagen-induced arthritis (CIA)

2 Materials and Methods

21 Cells and Mice COS7 cells were grown in Dulbeccorsquosmodified Eaglersquos medium (DMEM) supplemented with 5fetal bovine serum (FBS) in a humidified 5 CO

2incubator

at 37∘CSix-week-old female BALBc (H-2d) mice and 5-week-

old male DBA1 mice (a CIA-susceptible mouse strain H-2q) were both purchased from HFK Biotechnology Co LtdAll mice were maintained in a specific pathogen-free envi-ronment All animal experiments were performed according

to the guidelines of the Animal Care and Use Committee ofCapital Medical University

22 Construction and Preparation of the DNA Vaccine B cellepitopes in the amino acid sequence of human DKK1 wereanalyzed using the software DNASTAR 71 The separatedepitopes were synthesis from Invitrogen (Life TechnologiesCalifornia USA) Subsequently the indirect ELISA wasutilized to detect the affinity of separated epitopes Simply96-well plates were coated with peptides (1 120583gmL) overnightat 4∘C The DKK1 polyclonal antibodies (diluted 1 1000200120583L RampD Systems Minneapolis MN USA) were addedto wells and incubated overnight at 4∘C After washing theHRP conjugate goat anti-human DKK1 secondary antibody(100 120583L diluted 1 2000 Southern Biotech BirminghamAL USA) was added and the plates were incubated for1 h at 37∘C Then the peptides were immunized BALBcmice and the immunogenicity was measured as previouslydescribed [18] The reactions were stopped by the addition of50 120583L of Stop Solution (RampD Systems) and OD450 readingswere measured with an ELISA plate reader To reduce thebioactivity of DKK1 vaccine the peptides (100 120583ginjectionintraperitoneally) were applied for a week The tibias werecollected and an acid phosphatase kit (Sigma-Aldrich StLouis MO USA) was used to demonstrate osteoclasts [19]

Four fragments in the DKK1 sequence (110ndash144aa 153ndash181aa 182ndash216aa and 228ndash253aa) with high affinity andimmunogenicity were selected to construct the multiepitopeDNA vaccine The DNA sequences encoding four aminoacids (R203 H204 K211 and R236) in DKK1 were mutatedto glutamate to reduce the biological activity of DKK1 [20]AAY spacers between two adjacent epitope fragments wereused to link the four selected epitopes A Th2 cell epitopewas added at both sequence termini Finally the signalpeptide of human DKK1 was added at the N-terminus of thesequenceThe synthetic nucleotide sequence named DP wasincorporated into the expression vector pCMV6-XL5 usinga standard DNA recombination procedure resulting in therecombinant plasmid pCMV-DP Plasmids for immunizationwere extracted and purified from transformed Escherichiacoli strain DH5120572 using an endotoxin-free plasmid extractionkit (Roche Mannheim Germany) according to the manu-facturerrsquos instructions The purified plasmids were adjustedto a concentration of 1mgmL in sterile saline and stored atminus80∘C

23 Transfection of Plasmid pCMV-DP into COS7 CellsCOS7 cells were cultured in a 6-well tissue culture plateuntil the cells reached approximately 60 to 80 confluenceThe cells were transfected with the purified plasmid DNAusing TurboFect in vitro Transfection Reagent (FermentasThermoScientific Pittsburgh PA USA) according to themanufacturerrsquos instructions Briefly 4 120583L of plasmid DNA(1mgmL) was diluted in 390120583L of serum-free DMEMTurboFect reagents (6 120583L) were added to the diluted DNAAfter immediate mixing and incubation for 20min at roomtemperature 400 120583L of the mixture was distributed dropwiseinto culturedCOS7 cells in a 6-well plateThe transfected cells








2O2for 20min to











20 40 60 80 100 120 140 160 180 200 220 240 260ABT0

F0

16

minus45

minus17

lowast

lowast

(a)

00

02

04

06

08

Affi

nity

(OD

450

)

KLH

P32

ndash42

P43

ndash65

P66

ndash109

P110

ndash133

P134

ndash144

P145

ndash152

P153

ndash181

P182

ndash189

P190

ndash216

P217

ndash227

P228

ndash253

P254

ndash266

(b)

00

05

10

15

20

KLH

Imm

unog

enic

ity (O

D450)

P32

ndash42

P43

ndash65

P66

ndash109

P110

ndash133

P134

ndash144

P145

ndash152

P153

ndash181

P182

ndash189

P190

ndash216

P217

ndash227

P228

ndash253

P254

ndash266

(c)


(d)




3 Results



CMV promoter

f1 ori


SV40 ori

PolyA signal

pCMV6-XL5

4482bp

PADRE

AAY AAY AAY AAY AAY


204 211 236

MCS

EcoR I Xbal I


(a)





CARHF KPVLK

RCYCG

(b)




CCAGG CGPVLSTI

(c)








pCMV-DP0

1

2

3

pCMV

OD

450

lowastlowastlowast

(a)

pCMV-DPpCMV

(b)

pCMV-DPpCMV

(c)

0 2 4 6 8 10 1200

02

04

06

08

10

pCMV-DPpCMV


OD

450

lowast

(d)

pCMV-DP

00

05

10

15

PCMV


OD

450

1 200

1 400

1 800

1 1600

1 3200

1 6400

1 12800

(e)





4 Discussion






5 Conclusions





21 24 27 30 33 36 390

20

40

60

80

100


Inci

denc

e (

)

pCMV-DPpCMV

(a)


pCMV-DP

0

2

4

6

8

10

Clin

ical

scor

e

pCMV

(b)

pCMV pCMV-DP

(c)

pCMV

pCMV-DP

(d)

650

700

750

800

pCMV pCMV-DP

BMD

(mg

cm3 )

lowast

(e)

pCMV-DPpCMV

(f)





Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























2O2for 20min to











20 40 60 80 100 120 140 160 180 200 220 240 260ABT0

F0

16

minus45

minus17

lowast

lowast

(a)

00

02

04

06

08

Affi

nity

(OD

450

)

KLH

P32

ndash42

P43

ndash65

P66

ndash109

P110

ndash133

P134

ndash144

P145

ndash152

P153

ndash181

P182

ndash189

P190

ndash216

P217

ndash227

P228

ndash253

P254

ndash266

(b)

00

05

10

15

20

KLH

Imm

unog

enic

ity (O

D450)

P32

ndash42

P43

ndash65

P66

ndash109

P110

ndash133

P134

ndash144

P145

ndash152

P153

ndash181

P182

ndash189

P190

ndash216

P217

ndash227

P228

ndash253

P254

ndash266

(c)


(d)




3 Results



CMV promoter

f1 ori


SV40 ori

PolyA signal

pCMV6-XL5

4482bp

PADRE

AAY AAY AAY AAY AAY


204 211 236

MCS

EcoR I Xbal I


(a)





CARHF KPVLK

RCYCG

(b)




CCAGG CGPVLSTI

(c)








pCMV-DP0

1

2

3

pCMV

OD

450

lowastlowastlowast

(a)

pCMV-DPpCMV

(b)

pCMV-DPpCMV

(c)

0 2 4 6 8 10 1200

02

04

06

08

10

pCMV-DPpCMV


OD

450

lowast

(d)

pCMV-DP

00

05

10

15

PCMV


OD

450

1 200

1 400

1 800

1 1600

1 3200

1 6400

1 12800

(e)





4 Discussion






5 Conclusions





21 24 27 30 33 36 390

20

40

60

80

100


Inci

denc

e (

)

pCMV-DPpCMV

(a)


pCMV-DP

0

2

4

6

8

10

Clin

ical

scor

e

pCMV

(b)

pCMV pCMV-DP

(c)

pCMV

pCMV-DP

(d)

650

700

750

800

pCMV pCMV-DP

BMD

(mg

cm3 )

lowast

(e)

pCMV-DPpCMV

(f)





Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















20 40 60 80 100 120 140 160 180 200 220 240 260ABT0

F0

16

minus45

minus17

lowast

lowast

(a)

00

02

04

06

08

Affi

nity

(OD

450

)

KLH

P32

ndash42

P43

ndash65

P66

ndash109

P110

ndash133

P134

ndash144

P145

ndash152

P153

ndash181

P182

ndash189

P190

ndash216

P217

ndash227

P228

ndash253

P254

ndash266

(b)

00

05

10

15

20

KLH

Imm

unog

enic

ity (O

D450)

P32

ndash42

P43

ndash65

P66

ndash109

P110

ndash133

P134

ndash144

P145

ndash152

P153

ndash181

P182

ndash189

P190

ndash216

P217

ndash227

P228

ndash253

P254

ndash266

(c)


(d)




3 Results



CMV promoter

f1 ori


SV40 ori

PolyA signal

pCMV6-XL5

4482bp

PADRE

AAY AAY AAY AAY AAY


204 211 236

MCS

EcoR I Xbal I


(a)





CARHF KPVLK

RCYCG

(b)




CCAGG CGPVLSTI

(c)








pCMV-DP0

1

2

3

pCMV

OD

450

lowastlowastlowast

(a)

pCMV-DPpCMV

(b)

pCMV-DPpCMV

(c)

0 2 4 6 8 10 1200

02

04

06

08

10

pCMV-DPpCMV


OD

450

lowast

(d)

pCMV-DP

00

05

10

15

PCMV


OD

450

1 200

1 400

1 800

1 1600

1 3200

1 6400

1 12800

(e)





4 Discussion






5 Conclusions





21 24 27 30 33 36 390

20

40

60

80

100


Inci

denc

e (

)

pCMV-DPpCMV

(a)


pCMV-DP

0

2

4

6

8

10

Clin

ical

scor

e

pCMV

(b)

pCMV pCMV-DP

(c)

pCMV

pCMV-DP

(d)

650

700

750

800

pCMV pCMV-DP

BMD

(mg

cm3 )

lowast

(e)

pCMV-DPpCMV

(f)





Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CMV promoter

f1 ori


SV40 ori

PolyA signal

pCMV6-XL5

4482bp

PADRE

AAY AAY AAY AAY AAY


204 211 236

MCS

EcoR I Xbal I


(a)





CARHF KPVLK

RCYCG

(b)




CCAGG CGPVLSTI

(c)








pCMV-DP0

1

2

3

pCMV

OD

450

lowastlowastlowast

(a)

pCMV-DPpCMV

(b)

pCMV-DPpCMV

(c)

0 2 4 6 8 10 1200

02

04

06

08

10

pCMV-DPpCMV


OD

450

lowast

(d)

pCMV-DP

00

05

10

15

PCMV


OD

450

1 200

1 400

1 800

1 1600

1 3200

1 6400

1 12800

(e)





4 Discussion






5 Conclusions





21 24 27 30 33 36 390

20

40

60

80

100


Inci

denc

e (

)

pCMV-DPpCMV

(a)


pCMV-DP

0

2

4

6

8

10

Clin

ical

scor

e

pCMV

(b)

pCMV pCMV-DP

(c)

pCMV

pCMV-DP

(d)

650

700

750

800

pCMV pCMV-DP

BMD

(mg

cm3 )

lowast

(e)

pCMV-DPpCMV

(f)





Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















pCMV-DP0

1

2

3

pCMV

OD

450

lowastlowastlowast

(a)

pCMV-DPpCMV

(b)

pCMV-DPpCMV

(c)

0 2 4 6 8 10 1200

02

04

06

08

10

pCMV-DPpCMV


OD

450

lowast

(d)

pCMV-DP

00

05

10

15

PCMV


OD

450

1 200

1 400

1 800

1 1600

1 3200

1 6400

1 12800

(e)





4 Discussion






5 Conclusions





21 24 27 30 33 36 390

20

40

60

80

100


Inci

denc

e (

)

pCMV-DPpCMV

(a)


pCMV-DP

0

2

4

6

8

10

Clin

ical

scor

e

pCMV

(b)

pCMV pCMV-DP

(c)

pCMV

pCMV-DP

(d)

650

700

750

800

pCMV pCMV-DP

BMD

(mg

cm3 )

lowast

(e)

pCMV-DPpCMV

(f)





Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















4 Discussion






5 Conclusions





21 24 27 30 33 36 390

20

40

60

80

100


Inci

denc

e (

)

pCMV-DPpCMV

(a)


pCMV-DP

0

2

4

6

8

10

Clin

ical

scor

e

pCMV

(b)

pCMV pCMV-DP

(c)

pCMV

pCMV-DP

(d)

650

700

750

800

pCMV pCMV-DP

BMD

(mg

cm3 )

lowast

(e)

pCMV-DPpCMV

(f)





Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















21 24 27 30 33 36 390

20

40

60

80

100


Inci

denc

e (

)

pCMV-DPpCMV

(a)


pCMV-DP

0

2

4

6

8

10

Clin

ical

scor

e

pCMV

(b)

pCMV pCMV-DP

(c)

pCMV

pCMV-DP

(d)

650

700

750

800

pCMV pCMV-DP

BMD

(mg

cm3 )

lowast

(e)

pCMV-DPpCMV

(f)





Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Designation of a Novel DKK1 Multiepitope...

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Transcript of Research Article Designation of a Novel DKK1 Multiepitope...