synthetic b- and t-cell epitope peptides of porcine reproductive and respiratory.pdf

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Vaccine 31 (2013) 18381847

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Vaccine

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Synthetic B- and T-cell epitope peptides ofporcine reproductive and respiratorysyndrome virus with Gp96 as adjuvant induced humoral and cell-mediatedimmunity

Caiwei Chen a,b,Jing Li a, Yuhai Bi a, Limin Yang a, Shanshan Meng a, Yuancheng Zhou a,XiaojuanJia a, Songdong Meng a, Lei Sun a, Wenjun Liu a,b,

a Key Laboratory of Pathogenic Microbiologyand Immunology, Institute ofMicrobiology, Chinese Academy of Sciences,Beijing 100101, Chinab University of Chinese Academy of Sciences,Beijing 100101, China

a r t i c l e i n f o

Article history:

Received 21 January 2012

Received in revised form 6 January 2013

Accepted 25 January 2013

Available online 7 February 2013

Keywords:

HP-PRRSV

Synthetic peptide vaccine

B-cell epitope

T-cell epitope

Humoral and cell-mediated immunity

Gp96

a b s t r a c t

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has recently caused

huge economic losses in the pig industry worldwide. Commercial vaccines, including inactivated vac-

cines and attenuated live vaccines, are available but fail to provide sustainable protection, especially

against genetically heterologous strains. Thus several approaches have been used to develop more effec-

tive PRRSV vaccines and/or immune modulators to accelerate and magnify immune responses to PRRSV

vaccines. Heat shock protein Gp96 is one such modulator that enhances both the innate and adaptive

immune responses. In the present study, two B-cell epitopes and seven T-cell epitopes from PRRSV and

a Pan DR T-helper cell epitope were synthesized and mixed with the N-terminal 22355 aa of Gp96

(Gp96N) as an adjuvant, and immune responses were evaluated. Our results show that Gp96N activated

PRRSV-specific humoral immune responses elicited by BCE-peptides and promoted the PRRSV-specific

cellular immunity induced by TCE-peptides. Moreover, higher levels ofIL-12 and TNF- and lower levels

ofIL-4 and IL-10 were observed in the serum ofGp96N-vaccinated piglets compared to piglets immunized

with no Gp96N, displaying a predominant Th1 type ofimmune response induced by Gp96N. Following

challenge with the virulent HP-PRRSV isolate JXwn06, piglets vaccinated with the mixture of peptidesand Gp96N presented with milder clinical symptoms, lower viremia, and less pathological lesions in their

lungs, however, this vaccine could not provide lasting and effective protection against HP-PRRSV infec-

tion. These data provide important bases for the development ofPRRSV epitope-based synthetic peptide

vaccines combined with Gp96N as attractive immunomodulators in swine.

2013 Elsevier Ltd. All rights reserved.

1. Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV)

has two major genotypes: the European type (type I; the Lelystad

strain as the prototype) and the North American type (type II;

the ATCC VR2332 strain as the prototype), both of which belong

to the Arteriviridae family, order Nidoviridales [1]. PRRSV, espe-

cially highly pathogenic PRRSV (HP-PRRSV), causes more than

two million cases of pig infection annually in China [2] and costs

the US pork industry approximately 664 million dollars in direct

losses per year [3]. Several approaches to develop PRRSV vaccines

have been evaluated, including live attenuated virus, inactivated

Corresponding author at: Center for Molecular Virology, Institute of Microbiol-

ogy, Chinese Academy of Sciences, Beijing 100101, China. Tel.: +86 10 64807497;

fax: +86 10 64807503.

E-mail address: [email protected](W. Liu).

virus, recombinant subunit vaccine systems (based upon plasmid

DNA, bacteria, baculovirus, adenovirus, fowlpox virus, and pseu-

dorabies virus carrying several PRRSV structural proteins) and

chimeric viruses [49]. However, commercially available vaccines

have limited effect against heterologous virus challenges with

potential to revert to virulence [410].

It has been shown that synthetic peptides possess the capa-

bility to elicit critical epitope-specific immune responses without

adverse effects from the vaccines mentioned above. Advancements

in molecular biology, molecular immunology, and bioinformatics

have enhanced the discovery of antigenic epitopes of PRRSV. To

date, aa 3745 of the major envelope glycoprotein Gp5 is the main

target for viral neutralizing antibodies (NAs) in both the geno-

types of PRRSV [11]. In addition, type I PRRSVs strongly induce NAs

against aa 5768 of Gp4 [12,13], but whether this B-cell epitope

(BCE) from type II PRRSV strains can induce NA remains unclear.

Recently, two T-cell epitopes (TCEs) in Gp4 and three TCEs in the

nucleoprotein (N) of type I PRRSV strains, as well as two TCEs

0264-410X/$ seefrontmatter 2013 Elsevier Ltd. All rights reserved.

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C. Chen et al. / Vaccine31 (2013) 18381847 1839

Table 1

Characteristics of theB- and T-cell epitopes previously reported andused in thepresent study.

Epitope ID Reported information Synthetic peptide sequences in this study

aAA position and sequences Reference

Gp4-59B 59 SAAQEKISF67 (b I) [12,13] 59 SSPTIRKIS

Gp5-37B 37 SHL/FQLIYNL45 (cII) [11] 37 SHIQLIYNL

Gp4-7T 9FLLAGAQHI17 (II) [15] 7FLLVGFKCF

Gp4-170T 170 CLFAILLAT178 (II) 170 CLFAILLAI

Gp5-117T 117 LAALICFVIRLAKNC131 (II) [14,15] 117 LAALICFVIRLAKNC

Gp5-149T 149 KGRLYRWRSPVII/VEK163 (II) 149 KGRLYRWRSPVIVEK

N-49T 49 KPEKPHFPL57 (II) [15] 49 NPEKPHFPL

N-63T 63 VRHHLTQTE71 (II) 63 VRHHFTPSE

N-104T 104 FMLPVAHTVRLIRVTST120 (II) 104 FSLPTQHTVRLIRATASdPADRE AKFVAAWTLKAAA [27] AKFVAAWTLKAAA

The differentaa of theepitope used in the present study are marked by underlining.a AA, amino acid.b I, type I PRRSV.c II, type IIPRRSVd PADRE,Pan DR T-helper cell epitope.

in Gp5 of both types of strains were identified based on their

ability to induce IFN- secretion in cultures of peripheral blood

mononuclear cells (PBMCs) obtained from PRRSV-immunized pigs

[14,15]. Regardless, it is still unknown if these two BCE and seven

TCE peptides can induce PRRSV-specific and protective immuneresponses in pigs.

Due to the delayed and weak cell-mediated immunity and NA

production of the current PRRSV vaccines [1618], many immune

modulators have been used to provoke stronger immune responses

to experimental vaccines in pigs, including CD40L, GM-CSF, HSP70,

and CpG [68,19]. The heat shock protein (HSP) Gp96, as well as

its N-terminal fragments (aa22355,Gp96N), are able to bind pep-

tides and act as potent immuno-adjuvants to enhance both innate

and adaptiveimmune responses in mice models [2023]. However,

whether this fragment of porcine Gp96 can play the same role in

pigs remains to be determined.

In this study, bioinformatics were used to align the above BCEs

and TCEs among the type II PRRSVs, andconsensus sequences were

obtained. Humoral and cellular immune responses and the protec-tive efficacy of BCE + TCE-peptides with the Gp96N adjuvant were

investigated in piglets.

2. Materials and methods

2.1. Cell lines, viruses, and antibodies

MARC-145cells weremaintained in Dulbeccos modified Eagles

medium (DMEM, GIBCO) supplemented with 10% heat-inactivated

fetal bovine serum (FBS, GIBCO) and were used to propagate and

titer HP-PRRSV isolate JXwn06 (a kind gift of Prof. Hanchun Yang of

China Agricultural University, GenBank accession No. EF641008.1).

HRP-conjugated and TRITC-conjugated anti-swine IgG were pur-

chased from Santa Cruz.

2.2. Synthetic peptides and purification of Gp95N

According to the aa sequences of each BCE and TCE shown in

Table 1, Gp4-59B (SSPTIRKIS, purity: 98.4%), Gp5-37B (SHIQLIYNL,

purity: 98.6%), Gp4-7T (FLLVGFKCF, purity: 97.8%), Gp4-170T

(CLFAILLAI, purity: 97.4%), Gp5-117T (LAALICFVIRLAKNC, purity:

87.5%), Gp5-149T (KGRLYRWRSPVIVEK, purity: 85.9%), N-49T

(NPEKPHFPL, purity: 96.7%), N-63T (VRHHFTPSE, purity: 97.2%), N-

104T (FSLPTQHTVRLIRATAS, purity: 81.2%), and a Pan DR T-helpercell epitope (PADRE) peptide (AKFVAAWTLKAAA, purity: 82.0%)

were all synthesized and purified at Scilight Biotech, Beijing, China.

Gp4-59B and Gp5-37B were coupled to the carrier protein keyhole

limpet hemocyanin (KLH) or bovine serum albumin (BSA) by the

same company.

Recombinant Gp96N was expressed in Escherichiacoli and puri-

fied using nickel-nitrilotriacetic acid (Ni-NTA) chromatography

(GE). Endotoxins in the protein preparation were removed using

a bacterial Endotoxin Kit (Hycult Biotech).

2.3. Pig immunization and HP-PRRSV challenge

Sixteen 4-week-old piglets were obtained from the BeijingCenter for SPF Swine Breeding & Management. All piglets were

negative for anti-PRRSV antibodies as demonstrated by commer-

cial ELISA kit (Herdcheck, IDEXX). The animals were also negative

for porcine circovirus type 2 (PCV2), Classical Swine Fever Virus

(CSFV), Pseudorabies virus (PRV), and PRRSV by PCR and/or RT-

PCR. Then, piglets were randomly separated into four groups (four

piglets per group) and housed at Ceva-China Biotech, Beijing.

Piglets were intramuscularly injected three times at 2-week inter-

vals according to Table 2. Sera and PBMCs separated from the

blood at 0, 14, 28, and 42 days post injection (dpi) were ana-

lyzed by ELISAs, lymphocyte proliferation assays, and cytokine

assays.

Then, the vaccinated piglets were intranasally challenged with

2105 TCID50

HP-PRRSV JXwn06 at 42dpi and monitored daily

until they died. Rectal temperatures were measured daily, and the

severity of the clinical symptoms was evaluated based on a point

scale established by Lee et al. (2005) that independently consid-

ers attitude (04 points), respiratory rate (02 points), respiratory

distress (03 points), and cough (01 point) [24]. Sequential blood

samples were collected from all animals at 0, 3, 4, 5 and 7 days

Table 2

Immunizationregimensof peptides and Gp95N in piglets.

Group ID PBS Gp BT BT-Gp

Antigens PBS Gp96N aBCEsand TCEs peptides bBT peptides with Gp96N

Dose 2 ml per pig 0.5 mg/2 ml per pig (0.2 mg ofeachpeptidewithor without 0.5mg Gp96N)/2 ml perpig

a Themixture of two BCE peptides and eight TCE peptides shown in Table 1.b

The samepeptidesas the BT group mixed with Gp96N as an adjuvant.


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1840 C. Chen et al. / Vaccine31 (2013) 18381847

post-challenge (dpc). At 4 dpc, one piglet from each group was

euthanized for pathological examination.

All the animal research wasapprovedby the Beijing Association

for Science and Technology, with approval ID SYXK (Beijing) 2010-

0006 and SYXK(Beijing) 2006-0008, and complied with the Beijing

Laboratory Animal Welfare and Ethical Guidelines of the Beijing

Administration Committee of Laboratory Animals.

2.4. Immunofluorescence assay (IFA)

IFA was performed according to our previous report with slight

modifications [25]. Briefly, MARC-145 cells were infected with

PRRSV JXwn06 at a multiplicity of infection (MOI) of 1. Twenty-

four hours later, the cells were washed with PBS, fixed in 4%

paraformaldehyde, permeabilized with PBST (0.1% Triton X-100 in

PBS), and then incubated for 1h at 37C with 4% BSA in PBS. T he

cells were incubated with pig antisera (1:100) at 42dpi from the

fourgroups for 1 h at37 C. After washing five times with PBST, the

cells were incubated for 1h at 37 C with TRITC-conjugated anti-

swine IgG (1:100). After washing five times with PBST, the cells

were stained for nuclei by DAPI and observed using a Leica confocal

microscope.

2.5. Purification of PRRS virions

MARC-145 cells were infected with PRRSV JXwn06 isolate for

72h. The infected cells with cell cultures were frozen and thawed

three times and then centrifuged at 3000gat 4 C for half an hour.

The clarified culture supernatant (containing virus) was then con-

centrated by centrifugation (Beckman Coulter SW41 Ti rotor) at

100,000gat 4 C for 2 h. The precipitate (virions) was resuspended

in PBS. Subsequently, the virus was purified by ultracentrifuga-

tion through a 30% sucrose cushion using a SW41 Ti rotor at

100,000g for 2h, and the purified virions were resuspended in

PBS and heat-inactivated at 56C for 30 m in. At the same time,

the uninfected cells were purified with the same methods and

used as negative control (cell control). The concentration of the

purified virions (or cell control) was determined by the Bradford

assay using BSA as a standard, and the virions were stored at

80 C.

2.6. Monitoring antibodies by ELISA

BCE peptide-specific and PRRSV-specific antibody responses

weredetermined using an indirectELISA (iELISA)with BSA-coupled

BCE peptides or inactivated purified-PRRS virions (as described

above) as coating antigens. The 96-well ELISA plates were coated

overnight at 4 C with 1g/well coating antigens diluted in 100l

of 50mmol/l sodium carbonate buffer (pH 9.6). The plates were

washed three times withPBST (0.05% Tween-20in PBS)and blockedfor 1 h at 37 C with blocking buffer (3% BSA in PBS). Then, the

serum samples from piglets (including the negative sera collected

at 0 dpi) were diluted 1:100 in blocking buffer, added to each well

(100l/well) in duplicate, and incubated for 1h at 37 C. After

six washes, 100l of HRP-conjugated goat anti-swine IgG, diluted

1:5000 in blocking buffer, was added to each well and incubated at

37 C for 1h. After six washes, 100l of substrate solution (0.4M

tetramethylbenzidine and 1 mM H2O2 in 100mM acetate buffer,

pH 5.6) was added and incubated for 20min at room tempera-

ture in the dark, and the reaction was stopped by the addition

of 50l 2 M H2SO4 to each well. The absorbance was read at

450 nm (OD450) using an ELISA reader (Melgan). The OD450value

above 0.1 was determined as positive while under 0.1 as nega-

tive.

2.7. Lymphocyte proliferative response assay

Lymphocyte proliferation assays were performed using PBMCs

from the immunized piglets. The PBMCs were plated in 96-

well flat-bottom plates at 100l RMPI 1640 medium (containing

5% FBS, 100U /ml of penicillin and 100g /ml of streptomycin,

2105 cells/well) in triplicate. Subsequently, the inactivated

purified-PRRS virions (20g/ml, MARC-145 cells derived anti-

gens as a negative control) or each of the seven TCE peptide

(20g/ml, PBS as a negative control) was added and mixed.

Con A (5g/ml, Sigma) was used as a positive control. The

proliferative activity was measured according to a previously

described method with slight modifications [6]. Briefly, after a

72h incubation, 5mg/ml 3-(4,5-dimethylthylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS,

Sigma) was added to each well, and the plates were incubated for

anadditional 5 h, followed byadding 150l/well DMSOfor another

10min.At theendof theincubation,the plateswerereadat 570nm.

The stimulation index (SI) was calculated as the ratio of the aver-

age OD valueof the wellscontaining antigen-stimulatedcells to the

average OD value of negative control wells.

2.8. Cytokine assay

The levels of IL-4, IL-6, IL-10, IL-12, IFN-, and TNF- in the

sera and in the supernatant of the activated PBMCs were quan-

titatively determined using commercially available cytokine ELISA

Kits (R&D). PBMCs separated from the vaccinated piglets at 42dpi

were plated in 96-well flat-bottom plates at 100l RMPI 1640

medium (containing 5% FBS and antibiotics as described in Section

2.7, 2105 cells/well) in triplicate (fresh medium without cellswas

used as a blank control) and stimulated with purified inactivated-

PRRS virions (or MARC-145 cells derived antigens as a negative

control). After culturing for 72h, the supernatant from each well

was collected and immediately quantified by ELISA. Cytokines in

the sera at 0, 14, 28, and 42 days post primary immunization

were also examined according to the kits procedure. The cytokine

concentration of each sample was expressed as the mean of thetriplicates, while the cytokine quality of each group at the indi-

cated time point was calculated as the mean standard deviation

(SD) of four piglets.

2.9. Pathological examination

To estimate the severity of pathological lesions, the histologi-

cal pathology of lungs from the pigs at 4 dpc was determined as

described [26].

2.10. Statistical analysis

Statistical analyses and graphical presentation were performed

using GraphPad Prism 5.0software. All of the data are expressed asthe mean of four pigsSD. Differences in cell-mediated immune

responses, cytokine levels and viral titers at each sampling point

wereanalyzedusing the Wilconxon test.The clinicalsigns recorded

of pigs for each group were analyzed using the Fishers exact test.

P-values


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1001 sequences of Gp5, and 226 sequences of N were analyzed by

DNAStar andWeblogosoftware. As shown in Fig.1, eightof the epi-

topes were highly conserved, while Gp4-59B was more divergent.

Gp5-37B, Gp4-7T, and Gp4-170T displayed poor antigenic charac-

teristics using the Jameson-Wolf Antigenic analysis, while strong

antigenicity have been showed in Gp5-59B, Gp5-117T, Gp5-149T,

N-49T, N-63T, and N-104T. Thus, the consensus sequences of these

epitopes, together with PADRE, were chosen and synthesized with

KLH or BSA conjugated to Gp4-59B and Gp5-37B.

3.2. Humoral immune responses induced by BCE-peptides

First, IFA was used to determine whether the sera from the

BCE peptide-vaccinated piglets were PRRSV-specific. As shown in

Fig. 2A, JXwn06 PRRSV could be detected with pig sera against BCE

peptides (groups BT and BT-Gp). No staining was detected in the

infected cells with the sera from the pigs receiving PBS or Gp96N.

These results demonstrated that the two BCE peptides could stim-

ulate PRRSV-specific antibodies.

Next, the BSA-conjugated BCE peptides or the inactivated

purified-PRRS virions were used as coating antigens to monitor the

IgG responses by ELISA. A very low level of anti-BEC-peptides and

anti-PRRSV antibodies was detected in piglets from group BT after

three immunizations, while a low level of IgG was detected after

the second immunization and increased notably by the third vac-

cination in group BT-GP (Fig. 2B and C). Thus, Gp96N increased the

levels of BCE-specific (Fig. 2B) andPRRSV-specific(Fig.2C) IgGs. We

also determined thelevel ofNAs inthe sera from immunizedpiglets

by sera-virus neutralization assays. Unfortunately, no NA could be

detected in the sera of the immunized piglets.

3.3. TCE-induced cell-mediated immunity by TCE peptides

First, inactivated purified PRRS virions were used as a stimu-

latorto perform the lymphocyte proliferation assays. As shown in

Fig.3A, comparedto the PBSgroup ateachindicated time point,sig-

nificant PRRSV-specific lymphocyte proliferative responses were

exhibited in the PBMCs from the peptide-vaccinated piglets only

at 42dpi (P


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Fig. 4. Th1-type cytokine response bias by Gp96N in the peptides-vaccinated piglets. (A) PBMCs were collected and stimulated as mentioned in Fig. 3A. Six cytokinesweretested by quantitative ELISA from the supernatants of the PBMC cultures immediately after 72h of stimulation. (B) Cytokinesin the serum from the pigs of the PBS, Gp, BT,

and BT-Gp groups were detected by quantitative ELISA.Data areshownas themean SDof fourpigsfromeach group. *P


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Fig. 5. Detection of clinical sign and viremia of the pigs after challenge with HP-

PRRSV isolate JXwn06. (A) Rectal temperature of the pigs, with values expressed

as the mean SD. (B) Clinical sign scores of pigs per group are expressed as the

mean SD of allclinicalsignsrecordeddailypost challenge, includingattitude(03

points),respiratoryrate (02points),respiratorydistress (03points),and coughing

(01points). (C)The viremiaof thepigs isexpressed asthe viraltitersin seradetected

at 0 (before challenge), 3, 4, 5, and 7 dpc. The data are shown as the mean SD for

four orthreepigsper group.*P


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Fig. 6. Pathological examination.At 4 dpc, the lungs of the pigs were examined by HE staining in the PBS (A), Gp (B), BT (C), and BT-Gp (D) groups, as well as a healthy lung

from an uninfected pig (E).Pulmonaryalveoli were marked with hollow pentagon; solidtriangle exhibited thatfoci inflammatorycells infiltrated into the pulmonary alveoli,

and inflammatory cells infiltrated into the lumens as hollow triangle or near the vessel of the bronchus as solid arrow; hollow arrow represented the blood congestion in

lung. Scale bar: 200m.

is related to the cell-mediated immunity against PRRSV, was not

influenced by peptides, Gp96N, or peptides+ Gp96N (Fig. 4B). The

Th2 type cytokine IL-4 was down-regulated by peptides but not

Gp96N, and the combination of peptides+ Gp96N suppressed the

production of IL-4after the second immunization(Fig.4B). Further-

more, Gp96N inhibited the production of IL-10. It should be noted

that IL-10 is a potent immunosuppressive cytokine that can signif-

icantly inhibit production of several pro-inflammatory cytokinesand down-modulate APC activities, resulting in inhibition of innate

and adaptive immunity, particularly the cell-mediated immune

responses [46,47]. Concerning the inflammatory cytokines, Gp96N

or peptides did not alter the production of IL-6 (Fig. 4B). The other

innate anti-viral cytokine,TNF-, was significantlyup-regulatedby

Gp96N, but impaired by peptides because Fig.4A showed that pep-

tides might contribute to the down-regulated TNF- in group BT.

In addition, the TNF- level did not greatly increase until the third

immunization (at 42dpi in the BT-Gp group, Fig. 4B-iv). There may

be other unknown reasons that the TCE-peptides would delay the

up-regulation of TNF- by Gp96N. Taken together, Gp96N was an

effective Th1-biased adjuvant to improve the innate and adaptive

immune responses induced by the epitope-based vaccine, whichis

consistent with our previous report [25].

To determine the protective immune response induced by the

epitope-based peptides, the pigs were challenged with a high

dose of virulent HP-PRRSV isolate JXwn06. Temperature, clinical

symptoms, and viremia were monitored. Our results illustrate that

BCE+ TCE peptides with Gp96N as an adjuvant provided greater

protection than BCE +TCE peptides alone during the first 5 days

post HP-PRRSV infection. However, PRRSV infection was not fully

prevented in that all of the pigs in the BT-Gp group died at 7 dpcwitha hightiterof livePRRSVin their sera(Fig.5C) andserious clin-

ical symptoms (Fig. 5A and B, Fig. 6). One reason for this could be

that HP-PRRSV JXwn06 is a highly pathogenic isolate and causes

severe disease that leads to 100% mortality in 6-week-old pigs

within 513 days and 80% mortality in 12-week-old pigs [48]. In

this study, the piglets were 10 weeks old when challenged, which

mightbe related thedeath of the piglets. Additionally, the high dose

of HP-PRRSV JXwn06 challenge is another probable cause for the

death of the infected piglets. The viral dosage was 2105 TCID50in the present study, which is two times greater than the previous

report [48]. Based on the high level of mortality with 105 TCID50of HP-PRRSV infect, twice more dosage of virulent challenge virus

may cause much more serious mortality. This is the probable rea-

son for the 100% mortality at 7 dpc in the present study. Thus,


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