research article the peptide vaccine combined with prior...

Research ArticleThe Peptide Vaccine Combined withPrior Immunization of a Conventional Diphtheria-TetanusToxoid Vaccine Induced Amyloid 120573 Binding Antibodies onCynomolgus Monkeys and Guinea Pigs

Akira Yano1 Kaori Ito2 Yoshikatsu Miwa3 Yoshito Kanazawa4

Akiko Chiba5 Yutaka Iigo6 Yoshinori Kashimoto6 Akira Kanda7

Shinji Murata7 and Mitsuhiro Makino2

1 Iwate Biotechnology Research Center 22-174-4 Narita Kitakami Iwate 024-0003 Japan2Venture Science Laboratories RampD Division Daiichi-Sankyo Co Ltd 1-2-58 Hiromachi Shinagawa-ku Tokyo 140-8710 Japan3Quality Assurance Division Hayashibara Co Ltd 675-1 Fujisaki Naka-ku Okayama 702-8006 Japan4RampD Planning Department RampD Division Daiichi-Sankyo Co Ltd 1-2-58 Hiromachi Shinagawa-ku Tokyo 140-8710 Japan5Development Oversight Function RampDDivision Daiichi-Sankyo Co Ltd 1-2-58 Hiromachi Shinagawa-ku Tokyo 140-8710 Japan6Biological Research Department Drug Discovery and Biomedical Technology Unit Daiichi-Sankyo RampD Novare CoLtd 1-16-13 Kitakasai Edogawa-ku Tokyo 134-8630 Japan7Biological Research Laboratories RampD Division Daiichi-Sankyo Co Ltd 1-2-58 Hiromachi Shinagawa-ku Tokyo 140-8710 Japan

Correspondence should be addressed to Akira Yano akirayibrcorjp

Received 2 May 2015 Accepted 21 July 2015

Academic Editor Pedro A Reche

Copyright copy 2015 Akira Yano et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The reduction of brain amyloid beta (A120573) peptides by anti-A120573 antibodies is one of the possible therapies for Alzheimerrsquos diseaseWepreviously reported that the A120573 peptide vaccine including the T-cell epitope of diphtheria-tetanus combined toxoid (DT) inducedanti-A120573 antibodies and the prior immunization with conventional DT vaccine enhanced the immunogenicity of the peptideCynomolgus monkeys were given the peptide vaccine subcutaneously in combination with the prior DT vaccination Vaccinationwith a similar regimen was also performed on guinea pigs The peptide vaccine induced anti-A120573 antibodies in cynomolgusmonkeys and guinea pigs without chemical adjuvants and excessive immune responses were not observedThose antibodies couldpreferentially recognize A120573

40 and A120573

42compared to A120573 fibrils The levels of serum anti-A120573 antibodies and plasma A120573 peptides

increased in both animals and decreased the brain A12057340level of guinea pigs The peptide vaccine could induce a similar binding

profile of anti-A120573 antibodies in cynomolgus monkeys and guinea pigs The peptide vaccination could be expected to reduce thebrain A120573 peptides and their toxic effects via clearance of A120573 peptides by generated antibodies

1 Introduction

Alzheimerrsquos disease (AD) is a neurodegenerative diseasepathologically characterized by the deposition of the amyloidbeta (A120573) fragments derived from amyloid precursor protein(APP) in senile plaques and the accumulation of neurofib-rillary tangles composed of tau protein [1 2] Increasingevidence suggests that accumulation of A120573 plays a centralrole in the onset and progression of AD and therapeutic

interventions have been directed toward the reduction ofA120573 production using inhibitors of the 120573- and 120574-secretaseenzymes or enhancement of A120573 clearance by immunotherapy[3ndash5]

Regarding A120573 immunotherapy both active immuniza-tion against A120573 and passive immunization with monoclonalA120573 antibodies were reported to attenuate amyloid plaqueformation in the brains of APP transgenic mice [6ndash8] Thesetreatments also diminished the amyloid-associated pathology

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2015 Article ID 786501 9 pageshttpdxdoiorg1011552015786501

2 Journal of Immunology Research

[9ndash11] and improved learning deficits [12 13] In the clinicaltrials of the AN1792 vaccine the aggregated A120573

1ndash42 peptidewith aQS-21 adjuvant the long-term follow-up study analysisindicated that A120573

1ndash42 immunization resulted in clearance ofamyloid plaques in patients with AD however this clearancedid not lead to the prevention of progressive neurodegen-eration [14] In recent clinical trials passive immunizationwith the anti-A120573 antibody bapineuzumab and solanezumaband intravenous immunoglobulin treatment failed to show asignificant clinical benefit in patients with mild to moderateAD [15] Although the clinical results were disappointingthere is a consensus in the field that A120573 immunotherapyby earlier intervention targeting patients with early ADor mild cognitive impairment or presymptomatic subjectscould be an effective therapeutic and prophylactic treatmentAnti-amyloid combination therapies were also expected aspractical approach for AD by the results that inhibition of120574-secretase or 120573-secretase with anti-A120573 antibodies was moreeffective than either alone in animal models [16 17]

Based on the clinical results of the study of AN1792which was halted due to the development of meningoen-cephalitis potentially related to a proinflammatory T-cell-mediated immune response [18ndash20] next-generation vaccinestrategies for AD treatment will remain promising if thevaccine induces autoantibodies (anti-A120573 antibodies) withoutexcessive inflammatory responses

We have previously reported an A120573 peptide vaccineconstructed of two parts a T-cell epitope peptide on the N-terminal side and a B-cell epitope peptide connected by adilysine linker (KK) to the C-terminal side of the peptide [21]In order to enhance the immunogenicity of the peptide a cell-attachment motif (RGD) was added to the N-terminal sideof the peptide [21] and a multiagretope-type T-cell epitopewas used for induction of antibodies to a wide range ofMHC-II type individuals [22] Although the A120573

1ndash42 peptidethe antigen of AN1792 is estimated to contain many T-cellepitopes including cytotoxic epitopes the N-terminal regionof A120573 was thought to be an effective and safer target [23ndash25] Our vaccine contained only the A120573

1ndash13 as a target B-cellepitope peptide which is estimated to contain few cytotoxicT-cell epitopes by in silico analysis [22] Because the A120573

1ndash13was as weak as the B-cell epitope the utilization of theadditional T-cell epitope peptide recognizable by preexistingmemoryT-cells in the host was necessary for induction of theantibody to A120573

1ndash13 [26] We used the multiagretope-type T-cell epitope peptide fromdiphtheria toxin (DiTox

382ndash401)Thediphtheria toxin was used as a conventional vaccine antigensuch as diphtheria and tetanus (DT) vaccines designed toinduce the toxin-neutralizing antibodies byTh2 type humoralimmunities and the major memory T-cells responding toDT epitopes were estimated to induce Th2 type immuneresponses Our peptide vaccine the RGD-DiTox

382ndash401-KK-A1205731ndash13 peptide could induce the anti-A120573 antibodies to

C57BL6 by boosting the T-cell reaction preimmunized byDT vaccination without chemical adjuvants [26] This resultprovided motivation to investigate whether our peptidevaccine will also be effective to other species

In this study we investigated the immunogenicity of thepeptide with vaccination to cynomolgusmonkeys and guinea

pigs and studied the effects of antibodies by monitoring theA120573 peptides

2 Methods

21 Peptides ARGD-DiTox382ndash401-KK-A1205731ndash13 peptide (RGD-

AYNFVESIINLFQVVHNSYN-KK-DAEFRHDSGYEVH thenumbers following DiTox indicated the position of theamino acids on the precursor protein of diphtheria toxinincluding the 32-amino acids signal peptide) synthesizedand verified by MALDI-TOFMS as over the 95 puritywas obtained from Operon Biotechnologies KK (TokyoJapan) The single-letter universally accepted notation foramino acids is used throughout this text Human A120573 peptidefragments used in this study were purchased from AnaSpecInc (CA USA)

22 Animals The vaccination studies on male cynomolgusmonkeys (3 to 4 years of age at the start of the study) wereperformed at Mitsubishi Chemical Medience Corporation(Shibaura Tokyo Japan)Male guinea pigs (SlcHartley) werepurchased from Japan SLC Inc (Hamamatsu Japan) andimmunization began at 5 weeks of age All experimentalprocedures were performed in accordance with the in-house guideline of the Institutional Animal Care and UseCommittee of Daiichi Sankyo Co Ltd

23 Immunization Cynomolgus monkeys were primed with05mL of absorbed diphtheria-tetanus combined toxoid (DTvaccine The Kitasato Institute Tokyo Japan) three weeksbefore peptide immunization The A120573 peptide vaccine wassubcutaneously administrated with 05 or 25mg05mLhead eight times every two weeks

Guinea pigs were primed subcutaneously with 50 120583Lhead of DT vaccine before the peptide immunization Threeweeks after the DT vaccination guinea pigs were immu-nized subcutaneously with 200 120583g200120583Lhead of RGD-DiTox

382ndash401-KK-A1205731ndash13 peptide solution or 200120583L of vehicle(distilled water) for the control group Four identical boosterdoses were given at 3-week intervals

24 Sample Collection Peripheral blood was collected every2 weeks from the cynomolgus monkeys then plasma andserum were prepared and stored at minus20∘C for the followingexperiments

Approximately 100 120583L of blood was collected from theguinea pigs by tail bleeding one week after each peptideadministration Plasma samples were prepared by centrifu-gation and stored individually with a complete proteaseinhibitor cocktail (Roche Diagnostics KK Tokyo Japan)One week after the last booster dose cerebrospinal fluid(CSF) was obtained then the animals were bled and plasmasamples were prepared CSF and plasma samples were pre-pared by centrifugation and then stored at minus20∘CThe brainswere removed frozen on dry ice and stored at minus80∘C for anELISA assay

25 ELISA for Anti-A120573 Antibodies Plates were coated withA1205731ndash42 dissolved in distilled water and then washed with

Journal of Immunology Research 3

wash buffer (005 Tween 20 in phosphate buffered salinePBS) Next the plates were blocked with 1 Block Ace(Bio-Rad Laboratories Inc Hercules CA USA) in PBS atroom temperature and then washed with wash buffer Plasmasamples were diluted 100- to 1000-fold The autologous2H8 mouse monoclonal anti-A120573 antibody (Thermo FisherScientific KK Yokohama Japan) was used to generate acalibration curve for antibody titers Each sample was appliedto a well and incubated at 4∘C overnight After washing theplate the wells were incubated with horseradish peroxidase-(HRP-) conjugated anti-mouse IgG and anti-guinea pigantibody (Sigma-Aldrich Japan Inc Tokyo Japan) at 4∘C for2 h Next they were incubated with 221015840-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt (ABTS)substrate (Bio-Rad Laboratories Inc) at room temperaturein the dark After sufficient color development had occurred2M phosphate buffer was added to stop the reaction Theabsorbance of each well at 405 nm was measured with aspectrophotometer and antibody titers were then calculated

ELISAs for antibody epitope-mapping were performedusing the following A120573 peptides RGD-DiTox

382ndash401-KK-A1205731ndash13 A1205731ndash13 A1205731ndash40 A1205731ndash42 and A120573

1ndash42 fibrils (fA1205731ndash42)as an immobilized antigen and ELISAs were performedin the same way as described above The preparation offA1205731ndash42 is described as follows LyophilizedA1205731ndash42 in PBSwas

incubated at 37∘C for three days and then the resulting A120573solution was centrifuged at 4∘C for 10min at 10000timesg thenthe precipitated fraction was suspended in distilled water andused in this study

26 ELISA for Brain and Plasma The brains of guinea pigswere thawed on ice and then homogenized in 5 volumes (vw)of 42 formic acid solution including protease inhibitorsusing a homogenizer and a sonicator and were incubatedovernight at 37∘C The homogenates were centrifuged at37000 rpm for 60min at 4∘C (OptimaTM L-100XP rotor504Ti Beckman Coulter Inc Tokyo Japan) and the super-natants were neutralized with 11 volumes (vv) of 1M Trissolution and then centrifuged at 10000 rpm for 10min at 4∘C(himac CT13R Hitachi Koki Co Ltd Tokyo Japan) Thesupernatants were collected as a brain A120573 fraction

Levels of A12057340

and A12057342

in brain and plasma sampleswere measured using a HumanRat 120573 Amyloid (40) ELISAKit Wako II (Wako Pure Chemical Industries Ltd TokyoJapan) HumanRat 120573 Amyloid (42) ELISA Kit Wako High-Sensitive (Wako Pure Chemical Industries Ltd) andHumanAmyloid120573Oligomers (82E1-specific)AssayKit (IBLCo LtdGunma Japan) and were used according to the manufac-turerrsquos instructions

27 Detection of Cytokines from Peripheral Blood of Cynomol-gus Monkeys Plasma of cynomolgus monkeys at days 22 to36 64 to 78 and 106 to 120was pooled andused for the ELISAIL-2 IL-4 IL-10 and TNF120572 were measured by a MonkeyELISA Kit (Invitrogen) according to the manufacturerrsquosinstruction

28 A1205731ndash42 Toxicity Assay The soluble A120573

1ndash42 peptide wasadded to the 7th day after passage of rat adrenal medulla

derived pheochromocytoma PC12 (Dainippon SumitomoPharma Co Ltd Tokyo Japan) cell line at 0 to 2 120583Mand the viability of the cells was evaluated by AlamarBlue(Pierce Thermo Fisher Scientific Inc Waltham MA USA)according to themanufacturerrsquos instructionsThe anti-A120573

1ndash42monoclonal antibody (033 120583M of 6E10 abcam CambridgeUSA) was used as a control for the protection of PC12cells from cytotoxic A120573

1ndash42 peptide The antibody (033 120583Mof mouse IgG Sigma-Aldrich St Louis MO USA) wasused as a negative control The serum and antibodies werepreincubated with A120573

1ndash42 peptides at 4∘C for 2 h then added

to the cells and cultured for 24 h

29 Statistical Analysis Data were expressed as mean plusmnstandard error (SE) Data from the passive avoidance testand the brain plasma and CSF A120573 levels were analyzedby one-way analysis of variance (ANOVA) Data from thesplenic T-cell proliferation assay were analyzed by the one-way layout andmultiple comparisonmethod ofDunnett SASSystem Release 82 (SAS Institute Inc) was used to performall analyses and 119875 values of less than 005 were considered tobe statistically significant

3 Results

31 Immunization ofA120573Peptide inCynomolgusMonkeys Thetime courses of plasma antibody concentration against theA120573 peptides by subcutaneous administration of the RGD-DiTox

382ndash401-KK-A1205731ndash13 peptide (05 and 25mghead) orvehicle (distilled water) in cynomolgus monkeys are shownin Figure 1(a) Compared to the vehicle group the 25mgpeptide administrated group showed significantly higherantibody concentrations after the initial peptide adminis-tration (8 days after the first administration) To recognizethe upper limit of the antibody concentration induced bythe peptide booster immunizations were continued over100 days after the first immunization The marked elevationfollowing the third immunization was not observed andfinal serum antibody concentrations were reached about 8times higher than that of the vehicle group The plasma con-centrations of A120573

40were not significantly different between

the groups but the concentration of A12057342

peptide in the25mghead vaccination group was significantly higher thanthe vehicle group (Figure 1(b))The result of epitope-mappingis indicated in Figure 1(c) The peptide induced antibodiesrecognized not only the A120573 vaccine peptide (KK-A120573

1ndash13) butalso the full-lengthA120573

1ndash40 andA1205731ndash42 peptideThe antibodieswere less reactive to A120573 fibrils (fA120573

1ndash42) than A1205731ndash42 peptide

The antibody reactivities against the A12057313ndash16 and A120573

1ndash10peptide were relatively weaker than that against the A120573

1ndash13peptide suggesting that the C-terminal side of the A120573

1ndash13peptide was the main epitope of the antibody generated bythis peptide vaccination

The cytokine responses to the peptide immunizationwereinvestigated by ELISA Plasma of the cynomolgusmonkeys atdays 22 to 36 64 to 78 and 106 to 120 was pooled and usedfor ELISA IL-2 IL-4 IL-10 and TNF120572 were measured butall cytokines remained under the detection limits (Table 1


lowastlowastlowastlowast

lowastlowast

lowastlowast

Seru

m an

ti-A120573

abs (

ngm

L)

Days after the initial peptide vaccination

600

500

400

300

200

100

0

minus6 1 8 15 22 29 36435057647178 85 9299

106

113

120

VehicleKK-A1205731ndash13 (05mghead)KK-A1205731ndash13 (25mghead)

lowastlowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

(a)

Plasma A12057340 (AUC) Plasma A12057342 (AUC)

Fold

incr

ease

Fold

incr

ease

Vehicle 05mg 25mg Vehicle 05mg 25mgKK-A1205731ndash13 KK-A1205731ndash13

14

1

06

12

1

08

lowastP lt 005 versus vehicle (Dunnett)

lowast

(b)

A1205731

ndash40

A1205731

ndash42

fA1205731

ndash42

A1205731

ndash13

A1205734

ndash13

A1205731

ndash10

A12057313

ndash16

KK-A1205731

ndash13

Antigen

Abso

rban

ce (O

D405

) 25

2

15

1

05

0

VehicleKK-A120573 (05mghead)KK-A120573 (25mghead)

(c)

Figure 1 Induction of the anti-A120573 antibodies andA120573 peptides in the peripheral blood by immunization of the peptide vaccine to cynomolgusmonkeys The serum anti-A120573 antibody levels of the cynomolgus monkeys treated with vehicle or RGD-DiTox

20-KK-A120573

1ndash13 (a) The plasmaA12057340and A120573

42peptide (b) level (nM) of cynomolgus monkeys Diphtheria and tetanus toxoids (DT 05mLhead sc) were administered

to cynomolgus monkeys Three weeks after the DT treatment vehicle or the peptide (05 or 25mghead sc) was administered at intervalsof 2 weeks (arrows total 9 times) Blood sampling was performed every week after the initial treatment Results are represented as mean plusmnSE (119899 = 5) lowast119875 lt 005 and lowastlowast119875 lt 001 as compared with the vehicle control group (the Dunnett test) Epitope-mapping of plasma anti-A120573 antibodies immunized with vehicle or RGD-DiTox

382ndash401-KK-A1205731ndash13 peptide (c) Epitope-mapping of antibodies was performed usingeach peptide-precoated ELISA with plasma collected at two weeks after the final treatment Results are represented as mean plusmn SE (119899 = 5)KK-A120573

1ndash13 RGD-DiTox20-KK-A1205731ndash13 peptide


15

125

1

075

05

025

0

A1205731

ndash40

A1205731

ndash42

fA1205731

ndash42

A1205731

ndash13

A1205734

ndash13

A1205731

ndash10

A12057313

ndash16

KK-A1205731

ndash13

Antigen

Abso

rban

ce (O

D405

)

(a)

Plas

maA

120573(p

mol

L)

Plasma anti-A120573 antibody (120583gmL) (times1000)

200

150

100

50

00 1 2 3 4 5

r = 06383141

r = 05459

A1205731ndash40

A1205731ndash42

P = 0000001

P = 000006

(b)

Vehicle KK-A1205731ndash13

A12057340

leve

l in

brai

ns (p

mol

g)

25

20

15

10

05

00

lowast

(c)


A12057342

leve

l in

brai

ns (p

mol

g)

25

20

15

10

05

00

(d)

Figure 2 Induction of the anti-A120573 antibodies in the plasma and A120573 peptides in the brain by immunization of the peptide vaccine toguinea pigs Epitope-mapping of plasma anti-A120573 antibodies immunized with RGD-DiTox

382ndash401-KK-A1205731ndash13 peptide (a) Epitope-mappingof antibodies was performed using each peptide-precoated ELISA with plasma collected at two weeks after the final treatment Results arerepresented as mean plusmn SE 119899 = 5 The plasma anti-A120573 antibody levels of the guinea pigs treated with RGD-DiTox

20-KK-A120573

1ndash13 peptides andthe correlation of anti-A120573 antibody concentrations to A120573

40and A120573

42peptide doses in the plasma (b) The A120573 peptide levels in the brain

of guinea pigs (c and d) Diphtheria and tetanus toxoids (DT 200120583Lhead sc) were administered to guinea pigs Three weeks after theDT treatment vehicle or the peptide (200 120583ghead) was administered at intervals of 3 weeks (arrows total 6 times) Blood sampling wasperformed every week after the initial treatment Results are represented as mean plusmn SE (119899 = 5) lowast119875 lt 005 and lowastlowast119875 lt 001 as compared to thevehicle control group (the Dunnett test)

Table 1 Cytokines in the peripheral blood of cynomolgusmonkeys

Th1 type Th2 typeTNF120572 IL-2 IL-4 IL-10

Control nd nd nd nd05mghead-KK-A120573 peptide nd nd nd nd25mghead-KK-A120573 peptide nd nd nd ndDetection limits TNF 2 pgmL Il-2 2 pgmL Il-4 3 pgmL and Il-1010 pgmL

TNF120572 lt 2 pgmL Il-2 lt 2 pgmL Il-4 lt 3 pgmL and Il-10lt 10 pgmL)

32 Immunization of A120573 Peptide in Guinea Pigs TheA120573 pep-tide RGD-DiTox

382ndash401-KK-A1205731ndash13 peptide (200120583ghead)

or vehicle (distilled water) was subcutaneously administratedin guinea pigs Compared to the vehicle group the peptideadministrated group showed 6 to 8 times higher antibodytiters throughout the experiment (data not shown) Theepitope-mapping performed with the serum from the finalbleed indicated that the peptide vaccination induced anti-bodies in guinea pigs in the same patterns of epitopes as incynomolgusmonkeys except that the antibodieswere reactiveto fA120573 as like as A120573

1ndash42 peptide (Figure 2(a))The concentration of the plasma A120573

1ndash40 peptide tendedto increase due to the peptide immunization and showed acorrelation with the level of anti-A120573 antibodies in the serum(Figure 2(b)) The concentration of the plasma A120573

42peptide

was ten times lower than A12057340peptide and their orders were

unchanged by increasing anti-A120573 antibody concentration


A1205731ndash42 (mM)

Prot

ectio

n (

)

140

120

100

80

60

40

20

00 01 02 05 1 2

ControlmAb (6E10)Ab (IgG)

(a)


Prot

ectio

n (

)

140

120

100

80

60

40

20

00 01 02 05 1 2

ControlSerum (low KK-A120573)Serum (high KK-A120573)

(b)

Figure 3 Effects of antiserum from cynomolgus monkeys vaccinated with RGD-DiTox20-KK-A120573

1ndash13 peptides or vehicle on A120573-inducedcytotoxicity of PC12 cells The effects of the anti-A120573 monoclonal antibodies (positive control black bar) and nonspecific immunoglobulin(gray bar) on the cytotoxicity of A120573 peptides (base control of cytotoxicity white bar) are indicated (a) The effects of the serum of A120573

1ndash42peptide-immunized monkey (05mghead low dose black bar and 25mghead high dose gray bar) on the cytotoxic A120573

42peptides (buffer

control white bar) are indicated

A12057340peptide levels in brains were significantly decreased

by the peptide immunization group (Figure 2(c)) but thelevel of A120573

42peptide did not change by immunization

(Figure 2(d))

33 A1205731ndash42 Toxicity Assay with a Nerve Cell Culture To

evaluate the protective effect of the peptide induced anti-A120573 antibodies A120573

1ndash42 toxicity assay with a PC12 cell culturewas performedThe anti-A120573 serum (25 final concentration)in cynomolgus monkeys collected one week after the finalpeptide immunization was investigated Only the anti-A120573antibody could protect PC12 cells from the toxic A120573 pep-tide (Figure 3(a)) The anti-A120573 serum of high-dose peptide-immunized monkeys protected PC12 cells from the damageof A120573

1ndash42 peptide (Figure 3(b))

4 Discussion

The RGD-DiTox382ndash401-KK-A1205731ndash13 peptide with preimmu-

nized DT vaccine induced anti-A120573 antibodies in monkeysand guinea pigs without any chemical adjuvants The vaccinepeptide induced about 6 to 8 times higher anti-A120573 antibodiesthan vehicle-treated animals following third immunizationThose results clearly indicated the vaccine peptide wasimmunogenic to both kinds of animals The multiagretope-type T-cell epitope peptide on the N-terminal side of our

peptide worked as T-cell epitope of DT vaccine to the animalswith many types of MHC [22] and the results of guinea pigsand cynomolgus monkeys showed the T-cell epitopes of eachanimal were included in the peptide

The T-cell epitope sequence was derived from con-ventional DT vaccine for induction of antibodies to A120573peptide Davtyan et al [27] reported the same concept ofAD peptide vaccine Lu AF20513 that used memory Th cellsgenerated by tetanus toxoid vaccine for induction of A120573antibodies Lu AF20513 was immunized with strong adju-vants CFAIFA Quil-A or Alhydrogel andmore than severalhundred times higher titers of antibodies were inducedIt supported the availability of our peptide vaccine if thearrangement of the epitopes and the amino acid sequenceof T-cell epitope were different from our peptide Ourpeptide would also induce more strong immune responsesimmunized with strong chemical adjuvants (supplementalexperiments see Supplementary Material available onlineat httpdxdoiorg1011552015786501) In this study wetried to avoid the induction of the strong immune responsesfor reduction of the risk by unexpected immune responsesnamely cytotoxic responses reported in the clinical trialof AN1792 [14] The excessive entry of antibodies in thebrain also might be a risk of encephalitis Following the 9administrations of the peptide significant levels of cytokineswere not induced in cynomolgus monkeys (Table 1)


The result of the A1205731ndash42 toxicity assay using PC12 cells

presented the effectiveness of the anti-A120573 serum induced bythe weak immune responses by our peptide vaccination Inaddition our peptide vaccination induced antibodies work-ing for clearance of A120573 peptides from the brain of the guineapigs (Figure 2) [7] The epitope-mapping of antibodies ofboth animals indicated the antibodies commonly recognizethe C-terminal side of the A120573

1ndash13 full-length A1205731ndash40 and

A1205731ndash42 peptides (Figures 1(b) and 2(a)) A1205731ndash42 fibril was also

recognized by antiserum of guinea pigs but less reactive toantiserum of cynomolgus monkeys (Figures 2(a) and 1(c))The antisera of our peptide would be more suitable fornerve cell protection from A120573

1ndash42 peptides than dissolutionof the A120573

1ndash42 fibrils This might be a major differencebetween AN1792 and our peptide Lu AF20513 also usedthe A120573

1ndash12 peptide as B-cell epitope and the epitope of theinduced antibodies was thought to be common between LuAF20513 and our peptide The antibodies induced by ourpeptide significantly increased the plasma A120573

1ndash40 peptidesand reduced the A120573

1ndash40 peptides in the brains (Figures 1and 2) It would be considered that the anti-A120573 antibodiestransport the A120573 peptides from the brain to the blood stream[28 29] In contrast plasma and brain A120573

1ndash42 peptide wasalways lower than A120573

1ndash40 peptides and was not changedin the guinea pigs It was expected that the basal level ofA1205731ndash42 peptides in the wild type guinea pigs was three times

lower than A1205731ndash40 peptide and most of anti-A120573 antibodies

would preferably react to A1205731ndash40 peptide compared to A120573

1ndash42peptide Then it might be difficult to show the effects of theanti-A120573

1ndash42 antibodies in Figure 2(d)We also investigated the effect of the peptide vaccine by

immunization of Tg2576 mice expressing the Swedish muta-tion of APP (APPK670N M671L) (supplements) Unfortu-nately the antibodies were not induced without chemicaladjuvants and Freundrsquos incomplete adjuvant (FIA) was usedthree weeks after the DT vaccination Tg2576 mice expressedexcessive APPs in all tissues and strong adjuvants were nec-essary for induction of antibodies to A120573 peptides (Figure S1)The peptide vaccine could induce anti-A120573 antibodies to APP-TgmiceThe binding patterns of antibodies showed the sameepitopes to guinea pigs and cynomolgus monkeys (FigureS1A) The results of the Tg2576 indicated that the vaccineinduced antibodies increased A120573 peptides in plasma whichis consistent with the results inmonkeys and guinea pigs andCSF (Figures S1B C D and E) The brain A120573 deposition andA120573 oligomer of APP-Tg mice reduced compared to vehicle-treated animals (Figures S1F G H and I) Those resultssuggested that antibodies transported the A120573 peptides fromthe brain to the blood stream through the cerebrospinal fluid

We investigated memory changes in a passive avoidancetest after the last booster vaccination Compared to non-Tgmice the learning impairment had already been initiated inTg2576 mice at the initial immunization (data not shown)Six months after the initial immunization the learningimpairment inmice treated with A120573 peptide was significantlyless than that in mice treated with vehicle suggesting thatA120573 peptide vaccination slowed the progression of learningimpairment Biochemical analysis indicated a significantreduction of the levels of insoluble A120573

1ndash40 A1205731ndash42 and A120573

oligomers in the brain of KK-A120573 peptide vaccinated miceA significant reduction of A120573

1ndash42 immunostaining in theparietal cortex and hippocampus was also observed (datanot shown) suggesting that the vaccination with the RGD-DiTox

382ndash401-KK-A1205731ndash13 peptide could have a potential toreduce andor to inhibit the formation of senile plaques in thebrains of Tg2576miceThese results are similar to other find-ings of vaccinations [30 31] and suggest that the vaccinationwith the RGD-DiTox

382ndash401-KK-A1205731ndash13 peptide might slowthe progression of learning impairment mediated throughthe reduction of senile plaques soluble A120573 oligomers andorinhibition of senile plaque formation in the brain Thosepharmaceutical effects observed in Tg2576 mice would beexpected in wild type guinea pigs and cynomolgus monkeys

5 Conclusions

RGD-DiTox382ndash401-KK-A1205731ndash13 peptide vaccination in com-

bination with a prior vaccination of the conventionaldiphtheria-tetanus combined toxoid vaccine induced anti-A12057340 and 42 antibodies in cynomolgus monkeys and guineapigs It promoted A120573 clearance in the brains of guinea pigsThe peptide vaccination did not show any excessive immuneresponses in any tested animals We propose that this peptidewould be a possible therapeutic or prophylactic candidate forAD

Abbreviations

A120573 Amyloid betaDT Diphtheria-tetanus combined toxoidAPP Amyloid precursor proteinAD Alzheimerrsquos diseaseCSF Cerebrospinal fluidPBS Phosphate buffered salineHRP Horseradish peroxidaseBBB Blood-brain barrier

Conflict of Interests

The authors declared that they have no competing interests

Authorsrsquo Contribution

Akira Yano participated in the design of the study andin writing the paper Kaori Ito Yoshikatsu Miwa YoshitoKanazawa and Mitsuhiro Makino participated in the designof the study and Kaori Ito Mitsuhiro Makino and AkikoChiba performed the experiments Yutaka Iigo and YoshinoriKashimoto performed the data analysis Akira Kanda andShinji Murata supported the animal experiments Akira Yanoand Kaori Ito made equal contribution

Acknowledgments

The authors would like to thank Chikako Nishimoto andHaruka Endo for their assistance in conducting the studyThis work was supported by Daiichi-Sankyo Co Ltd


References

[1] J Hardy and D J Selkoe ldquoThe amyloid hypothesis of Alz-heimerrsquos disease progress and problems on the road to thera-peuticsrdquo Science vol 297 no 5580 pp 353ndash356 2002

[2] A Nikolaev T McLaughlin D D M OrsquoLeary and M Tessier-Lavigne ldquoAPP binds DR6 to trigger axon pruning and neurondeath via distinct caspasesrdquo Nature vol 457 no 7232 pp 981ndash989 2009

[3] M S Rafii and P S Aisen ldquoRecent developments in Alzheimerrsquosdisease therapeuticsrdquo BMCMedicine vol 7 article 7 2009

[4] DMHoltzman J CMorris andAMGoate ldquoAlzheimerrsquos dis-ease the challenge of the second centuryrdquo Science TranslationalMedicine vol 3 article 77sr1 2011

[5] M Citron ldquoAlzheimerrsquos disease strategies for disease modifica-tionrdquo Nature Reviews Drug Discovery vol 9 no 5 pp 387ndash3982010

[6] D Schenk R Barbour W Dunn et al ldquoImmunization withamyloid-120573 attenuates Alzheimer disease-like pathology in thePDAPP mouserdquo Nature vol 400 no 6740 pp 173ndash177 1999

[7] F Bard C Cannon R Barbour et al ldquoPeripherally adminis-tered antibodies against amyloid 120573-peptide enter the centralnervous system and reduce pathology in a mouse model ofAlzheimer diseaserdquo Nature Medicine vol 6 no 8 pp 916ndash9192000

[8] DMWilcockA Rojiani A Rosenthal et al ldquoPassive immuno-therapy against A120573 in aged APP-transgenic mice reversescognitive deficits and depletes parenchymal amyloid depositsin spite of increased vascular amyloid and microhemorrhagerdquoJournal of Neuroinflammation vol 1 article 24 2004

[9] J A Lombardo E A Stern M E McLellan et al ldquoAmyloid-120573 antibody treatment leads to rapid normalization of plaque-induced neuritic alterationsrdquo The Journal of Neuroscience vol23 no 34 pp 10879ndash10883 2003

[10] S Oddo L Billings J P Kesslak D H Cribbs and F MLaFerla ldquoA120573 immunotherapy leads to clearance of early but notlate hyperphosphorylated tau aggregates via the proteasomerdquoNeuron vol 43 no 3 pp 321ndash332 2004

[11] R P Brendza B J Bacskai J R Cirrito et al ldquoAnti-A120573 antibodytreatment promotes the rapid recovery of amyloid-associatedneuritic dystrophy in PDAPP transgenic micerdquo The Journal ofClinical Investigation vol 115 no 2 pp 428ndash433 2005

[12] C Janus J Pearson J McLaurin et al ldquoA120573 peptide immuniza-tion reduces behavioural impairment and plaques in a modelof Alzheimerrsquos diseaserdquo Nature vol 408 no 6815 pp 979ndash9822000

[13] D Morgan D M Diamond P E Gottschall et al ldquoA 120573 peptidevaccination prevents memory loss in an animal model ofAlzheimerrsquos diseaserdquo Nature vol 408 no 6815 pp 982ndash9852000

[14] C Holmes D Boche D Wilkinson et al ldquoLong-term effectsof A12057342 immunisation in Alzheimerrsquos disease follow-up of arandomised placebo-controlled phase I trialrdquo The Lancet vol372 no 9634 pp 216ndash223 2008

[15] M Sarazin G Dorothee L C de Souza and P AucouturierldquoImmunotherapy in Alzheimerrsquos disease do we have all thepieces of the puzzlerdquo Biological Psychiatry vol 74 no 5 pp329ndash332 2013

[16] E Giacobini and G Gold ldquoAlzheimer disease therapymdashmovingfrom amyloid-120573 to taurdquoNature Reviews Neurology vol 9 no 12pp 677ndash686 2013

[17] H Jacobsen L Ozmen A Caruso et al ldquoCombined treatmentwith a BACE inhibitor and anti-A120573 antibody gantenerumabenhances amyloid reduction in APPLondon micerdquoThe Journalof Neuroscience vol 34 no 35 pp 11621ndash11630 2014

[18] J-M Orgogozo S Gilman J-F Dartigues et al ldquoSubacutemeningoencephalitis in a subset of patients with AD after A12057342immunizationrdquo Neurology vol 61 no 1 pp 46ndash54 2003

[19] J A R Nicolll DWilkinson CHolmes P Steart HMarkhamandROWeller ldquoNeuropathology of humanAlzheimer diseaseafter immunization with amyloid-120573 peptide a case reportrdquoNature Medicine vol 9 no 4 pp 448ndash452 2003

[20] I Ferrer M Boada Rovira M L Sanchez Guerra M J Reyand F Costa-Jussa ldquoNeuropathology and pathogenesis ofencephalitis following amyloid-120573 immunization in Alzheimerrsquosdiseaserdquo Brain Pathology vol 14 no 1 pp 11ndash20 2004

[21] A Yano A Onozuka K Matin S Imai N Hanada and TNisizawa ldquoRGD motif enhances immunogenicity and adju-vanicity of peptide antigens following intranasal immuniza-tionrdquo Vaccine vol 22 no 2 pp 237ndash243 2003

[22] A Yano A Onozuka Y Asahi-Ozaki et al ldquoAn ingeniousdesign for peptide vaccinesrdquoVaccine vol 23 no 17-18 pp 2322ndash2326 2005

[23] D S Gelinas K DaSilva D Fenili P St George-Hyslopand J McLaurin ldquoImmunotherapy for Alzheimerrsquos diseaserdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 2 pp 14657ndash14662 2004

[24] F Bard R Barbour CCannon et al ldquoEpitope and isotype speci-ficities of antibodies to120573-amyloid peptide for protection againstAlzheimerrsquos disease-like neuropathologyrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 4 pp 2023ndash2028 2003

[25] D H Cribbs A Ghochikyan V Vasilevko et al ldquoAdjuvant-dependent modulation of T

ℎ1 and T

ℎ2 responses to immuniza-

tion with 120573-amyloidrdquo International Immunology vol 15 no 4pp 505ndash514 2003

[26] A Yano Y Miwa Y Kanazawa et al ldquoA novel method forenhancement of peptide vaccination utilizing T-cell epitopesfrom conventional vaccinesrdquo Vaccine vol 31 no 11 pp 1510ndash1515 2013

[27] H Davtyan A Ghochikyan I Petrushina et al ldquoImmuno-genicity efficacy safety and mechanism of action of epitopevaccine (Lu AF20513) for Alzheimerrsquos disease prelude to aclinical trialrdquo Journal of Neuroscience vol 33 no 11 pp 4923ndash4934 2013

[28] R B DeMattos K R Bales D J Cummins J-C Dodart S MPaul and D M Holtzman ldquoPeripheral anti-A120573 antibody altersCNS and plasma A120573 clearance and decreases brain A120573 burdenin a mouse model of Alzheimerrsquos diseaserdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 98 no 15 pp 8850ndash8855 2001

[29] F LemialeW-PKong LMAkyurek et al ldquoEnhancedmucosalimmunoglobulin A response of intranasal adenoviral vectorhuman immunodeficiency virus vaccine and localization in thecentral nervous systemrdquo Journal of Virology vol 77 no 18 pp10078ndash10087 2003


[30] F Goni F Prelli Y Ji et al ldquoImmunomodulation targetingabnormal protein conformation reduces pathology in a mousemodel of alzheimerrsquos diseaserdquo PLoS ONE vol 5 no 10 ArticleID e13391 2010

[31] S Rasool R Albay III H Martinez-Coria et al ldquoVaccinationwith a non-human random sequence amyloid oligomer mimicresults in improved cognitive function and reduced plaquedeposition and micro hemorrhage in Tg2576 micerdquo MolecularNeurodegeneration vol 7 no 1 article 37 2012

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


[9ndash11] and improved learning deficits [12 13] In the clinicaltrials of the AN1792 vaccine the aggregated A120573

1ndash42 peptidewith aQS-21 adjuvant the long-term follow-up study analysisindicated that A120573

1ndash42 immunization resulted in clearance ofamyloid plaques in patients with AD however this clearancedid not lead to the prevention of progressive neurodegen-eration [14] In recent clinical trials passive immunizationwith the anti-A120573 antibody bapineuzumab and solanezumaband intravenous immunoglobulin treatment failed to show asignificant clinical benefit in patients with mild to moderateAD [15] Although the clinical results were disappointingthere is a consensus in the field that A120573 immunotherapyby earlier intervention targeting patients with early ADor mild cognitive impairment or presymptomatic subjectscould be an effective therapeutic and prophylactic treatmentAnti-amyloid combination therapies were also expected aspractical approach for AD by the results that inhibition of120574-secretase or 120573-secretase with anti-A120573 antibodies was moreeffective than either alone in animal models [16 17]

Based on the clinical results of the study of AN1792which was halted due to the development of meningoen-cephalitis potentially related to a proinflammatory T-cell-mediated immune response [18ndash20] next-generation vaccinestrategies for AD treatment will remain promising if thevaccine induces autoantibodies (anti-A120573 antibodies) withoutexcessive inflammatory responses

We have previously reported an A120573 peptide vaccineconstructed of two parts a T-cell epitope peptide on the N-terminal side and a B-cell epitope peptide connected by adilysine linker (KK) to the C-terminal side of the peptide [21]In order to enhance the immunogenicity of the peptide a cell-attachment motif (RGD) was added to the N-terminal sideof the peptide [21] and a multiagretope-type T-cell epitopewas used for induction of antibodies to a wide range ofMHC-II type individuals [22] Although the A120573

1ndash42 peptidethe antigen of AN1792 is estimated to contain many T-cellepitopes including cytotoxic epitopes the N-terminal regionof A120573 was thought to be an effective and safer target [23ndash25] Our vaccine contained only the A120573

1ndash13 as a target B-cellepitope peptide which is estimated to contain few cytotoxicT-cell epitopes by in silico analysis [22] Because the A120573

1ndash13was as weak as the B-cell epitope the utilization of theadditional T-cell epitope peptide recognizable by preexistingmemoryT-cells in the host was necessary for induction of theantibody to A120573

1ndash13 [26] We used the multiagretope-type T-cell epitope peptide fromdiphtheria toxin (DiTox

382ndash401)Thediphtheria toxin was used as a conventional vaccine antigensuch as diphtheria and tetanus (DT) vaccines designed toinduce the toxin-neutralizing antibodies byTh2 type humoralimmunities and the major memory T-cells responding toDT epitopes were estimated to induce Th2 type immuneresponses Our peptide vaccine the RGD-DiTox

382ndash401-KK-A1205731ndash13 peptide could induce the anti-A120573 antibodies to

C57BL6 by boosting the T-cell reaction preimmunized byDT vaccination without chemical adjuvants [26] This resultprovided motivation to investigate whether our peptidevaccine will also be effective to other species

In this study we investigated the immunogenicity of thepeptide with vaccination to cynomolgusmonkeys and guinea

pigs and studied the effects of antibodies by monitoring theA120573 peptides

2 Methods

21 Peptides ARGD-DiTox382ndash401-KK-A1205731ndash13 peptide (RGD-

AYNFVESIINLFQVVHNSYN-KK-DAEFRHDSGYEVH thenumbers following DiTox indicated the position of theamino acids on the precursor protein of diphtheria toxinincluding the 32-amino acids signal peptide) synthesizedand verified by MALDI-TOFMS as over the 95 puritywas obtained from Operon Biotechnologies KK (TokyoJapan) The single-letter universally accepted notation foramino acids is used throughout this text Human A120573 peptidefragments used in this study were purchased from AnaSpecInc (CA USA)

22 Animals The vaccination studies on male cynomolgusmonkeys (3 to 4 years of age at the start of the study) wereperformed at Mitsubishi Chemical Medience Corporation(Shibaura Tokyo Japan)Male guinea pigs (SlcHartley) werepurchased from Japan SLC Inc (Hamamatsu Japan) andimmunization began at 5 weeks of age All experimentalprocedures were performed in accordance with the in-house guideline of the Institutional Animal Care and UseCommittee of Daiichi Sankyo Co Ltd

23 Immunization Cynomolgus monkeys were primed with05mL of absorbed diphtheria-tetanus combined toxoid (DTvaccine The Kitasato Institute Tokyo Japan) three weeksbefore peptide immunization The A120573 peptide vaccine wassubcutaneously administrated with 05 or 25mg05mLhead eight times every two weeks

Guinea pigs were primed subcutaneously with 50 120583Lhead of DT vaccine before the peptide immunization Threeweeks after the DT vaccination guinea pigs were immu-nized subcutaneously with 200 120583g200120583Lhead of RGD-DiTox

382ndash401-KK-A1205731ndash13 peptide solution or 200120583L of vehicle(distilled water) for the control group Four identical boosterdoses were given at 3-week intervals

24 Sample Collection Peripheral blood was collected every2 weeks from the cynomolgus monkeys then plasma andserum were prepared and stored at minus20∘C for the followingexperiments

Approximately 100 120583L of blood was collected from theguinea pigs by tail bleeding one week after each peptideadministration Plasma samples were prepared by centrifu-gation and stored individually with a complete proteaseinhibitor cocktail (Roche Diagnostics KK Tokyo Japan)One week after the last booster dose cerebrospinal fluid(CSF) was obtained then the animals were bled and plasmasamples were prepared CSF and plasma samples were pre-pared by centrifugation and then stored at minus20∘CThe brainswere removed frozen on dry ice and stored at minus80∘C for anELISA assay

25 ELISA for Anti-A120573 Antibodies Plates were coated withA1205731ndash42 dissolved in distilled water and then washed with








Levels of A12057340

and A12057342











3 Results
















lowastlowast

lowastlowast

Seru

m an

ti-A120573

abs (

ngm

L)


600

500

400

300

200

100

0

minus6 1 8 15 22 29 36435057647178 85 9299

106

113

120


lowastlowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

(a)


Fold

incr

ease

Fold

incr

ease


14

1

06

12

1

08


lowast

(b)

A1205731

ndash40

A1205731

ndash42

fA1205731

ndash42

A1205731

ndash13

A1205734

ndash13

A1205731

ndash10

A12057313

ndash16

KK-A1205731

ndash13

Antigen

Abso

rban

ce (O

D405

) 25

2

15

1

05

0


(c)


20-KK-A120573







15

125

1

075

05

025

0

A1205731

ndash40

A1205731

ndash42

fA1205731

ndash42

A1205731

ndash13

A1205734

ndash13

A1205731

ndash10

A12057313

ndash16

KK-A1205731

ndash13

Antigen

Abso

rban

ce (O

D405

)

(a)

Plas

maA

120573(p

mol

L)


200

150

100

50

00 1 2 3 4 5

r = 06383141

r = 05459

A1205731ndash40

A1205731ndash42

P = 0000001

P = 000006

(b)


A12057340

leve

l in

brai

ns (p

mol

g)

25

20

15

10

05

00

lowast

(c)


A12057342

leve

l in

brai

ns (p

mol

g)

25

20

15

10

05

00

(d)



20-KK-A120573


40and A120573












42peptide





Prot

ectio

n (

)

140

120

100

80

60

40

20

00 01 02 05 1 2


(a)


Prot

ectio

n (

)

140

120

100

80

60

40

20

00 01 02 05 1 2


(b)




42peptides (buffer





(Figure 2(d))





4 Discussion





























5 Conclusions



Abbreviations






Acknowledgments



References


























ℎ1 and T















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Levels of A12057340

and A12057342











3 Results
















lowastlowast

lowastlowast

Seru

m an

ti-A120573

abs (

ngm

L)


600

500

400

300

200

100

0

minus6 1 8 15 22 29 36435057647178 85 9299

106

113

120


lowastlowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

(a)


Fold

incr

ease

Fold

incr

ease


14

1

06

12

1

08


lowast

(b)

A1205731

ndash40

A1205731

ndash42

fA1205731

ndash42

A1205731

ndash13

A1205734

ndash13

A1205731

ndash10

A12057313

ndash16

KK-A1205731

ndash13

Antigen

Abso

rban

ce (O

D405

) 25

2

15

1

05

0


(c)


20-KK-A120573







15

125

1

075

05

025

0

A1205731

ndash40

A1205731

ndash42

fA1205731

ndash42

A1205731

ndash13

A1205734

ndash13

A1205731

ndash10

A12057313

ndash16

KK-A1205731

ndash13

Antigen

Abso

rban

ce (O

D405

)

(a)

Plas

maA

120573(p

mol

L)


200

150

100

50

00 1 2 3 4 5

r = 06383141

r = 05459

A1205731ndash40

A1205731ndash42

P = 0000001

P = 000006

(b)


A12057340

leve

l in

brai

ns (p

mol

g)

25

20

15

10

05

00

lowast

(c)


A12057342

leve

l in

brai

ns (p

mol

g)

25

20

15

10

05

00

(d)



20-KK-A120573


40and A120573












42peptide





Prot

ectio

n (

)

140

120

100

80

60

40

20

00 01 02 05 1 2


(a)


Prot

ectio

n (

)

140

120

100

80

60

40

20

00 01 02 05 1 2


(b)




42peptides (buffer





(Figure 2(d))





4 Discussion





























5 Conclusions



Abbreviations






Acknowledgments



References


























ℎ1 and T















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















lowastlowast

lowastlowast

Seru

m an

ti-A120573

abs (

ngm

L)


600

500

400

300

200

100

0

minus6 1 8 15 22 29 36435057647178 85 9299

106

113

120


lowastlowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

(a)


Fold

incr

ease

Fold

incr

ease


14

1

06

12

1

08


lowast

(b)

A1205731

ndash40

A1205731

ndash42

fA1205731

ndash42

A1205731

ndash13

A1205734

ndash13

A1205731

ndash10

A12057313

ndash16

KK-A1205731

ndash13

Antigen

Abso

rban

ce (O

D405

) 25

2

15

1

05

0


(c)


20-KK-A120573







15

125

1

075

05

025

0

A1205731

ndash40

A1205731

ndash42

fA1205731

ndash42

A1205731

ndash13

A1205734

ndash13

A1205731

ndash10

A12057313

ndash16

KK-A1205731

ndash13

Antigen

Abso

rban

ce (O

D405

)

(a)

Plas

maA

120573(p

mol

L)


200

150

100

50

00 1 2 3 4 5

r = 06383141

r = 05459

A1205731ndash40

A1205731ndash42

P = 0000001

P = 000006

(b)


A12057340

leve

l in

brai

ns (p

mol

g)

25

20

15

10

05

00

lowast

(c)


A12057342

leve

l in

brai

ns (p

mol

g)

25

20

15

10

05

00

(d)



20-KK-A120573


40and A120573












42peptide





Prot

ectio

n (

)

140

120

100

80

60

40

20

00 01 02 05 1 2


(a)


Prot

ectio

n (

)

140

120

100

80

60

40

20

00 01 02 05 1 2


(b)




42peptides (buffer





(Figure 2(d))





4 Discussion





























5 Conclusions



Abbreviations






Acknowledgments



References


























ℎ1 and T















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















15

125

1

075

05

025

0

A1205731

ndash40

A1205731

ndash42

fA1205731

ndash42

A1205731

ndash13

A1205734

ndash13

A1205731

ndash10

A12057313

ndash16

KK-A1205731

ndash13

Antigen

Abso

rban

ce (O

D405

)

(a)

Plas

maA

120573(p

mol

L)


200

150

100

50

00 1 2 3 4 5

r = 06383141

r = 05459

A1205731ndash40

A1205731ndash42

P = 0000001

P = 000006

(b)


A12057340

leve

l in

brai

ns (p

mol

g)

25

20

15

10

05

00

lowast

(c)


A12057342

leve

l in

brai

ns (p

mol

g)

25

20

15

10

05

00

(d)



20-KK-A120573


40and A120573












42peptide





Prot

ectio

n (

)

140

120

100

80

60

40

20

00 01 02 05 1 2


(a)


Prot

ectio

n (

)

140

120

100

80

60

40

20

00 01 02 05 1 2


(b)




42peptides (buffer





(Figure 2(d))





4 Discussion





























5 Conclusions



Abbreviations






Acknowledgments



References


























ℎ1 and T















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Prot

ectio

n (

)

140

120

100

80

60

40

20

00 01 02 05 1 2


(a)


Prot

ectio

n (

)

140

120

100

80

60

40

20

00 01 02 05 1 2


(b)




42peptides (buffer





(Figure 2(d))





4 Discussion





























5 Conclusions



Abbreviations






Acknowledgments



References


























ℎ1 and T















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































5 Conclusions



Abbreviations






Acknowledgments



References


























ℎ1 and T















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References


























ℎ1 and T















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article the peptide vaccine combined with prior...

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