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Vaccine 27 (2009) 5603–5611

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

n silico prediction and ex vivo evaluation of potential T-cell epitopes inlycoproteins 4 and 5 and nucleocapsid protein of genotype-I (European) oforcine reproductive and respiratory syndrome virus

van Díaz a,∗, Joan Pujols a,b, Llilianne Ganges a,d, Mariona Gimeno a,c, Laila Darwich a,c,ariano Domingo a,c, Enric Mateu a,c

Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, SpainInstitut de Recerca i Tecnologia Agroalimentàries (IRTA), Barcelona, SpainDepartament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, SpainInstituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain

r t i c l e i n f o

rticle history:eceived 25 April 2009eceived in revised form 3 July 2009

a b s t r a c t

T-cell epitopes of porcine reproductive and respiratory syndrome virus (PRRSV) glycoproteins 4 (GP4),5 (GP5) and nucleocapsid (N) were predicted using bioinformatics and later tested by IFN-� ELISPOT inpigs immunized with either a modified live vaccine (MLV) or DNA (open reading frames 4, 5 or 7). For

ccepted 10 July 2009vailable online 29 July 2009

eywords:orcine reproductive and respiratoryyndrome virus-cell epitopes

MLV-vaccinated pigs, immunodominant epitopes were found in N but T-epitopes were also found in GP4and GP5. For DNA-immunized pigs, some peptides were differently recognized. Using a large set of PRRSVsequences it was shown that N contains a conserved epitope and that for GP5, the genotype-I counterpartsof previously reported epitopes of genotype-II strains were also immunogenic.

© 2009 Elsevier Ltd. All rights reserved.

ioinformatics

. Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV)as firstly isolated in 1991 by Dutch researchers [1] and thereafter

lassified as a member of the genus Arteriviridae [2]. At present,wo genotypes of PRRSV are known: genotype-I (European) andenotype-II (American). Antigenic and genetic diversity appears toe high within each genotype, particularly within European strains3–8].

PRRSV genome is composed of nine open reading frames (ORFs).RFs 1a and 1b account for approximately 75% of the viral genomend encode the non-structural proteins (nsp). ORFs 2a, 2b and–7 encode the PRRSV structural proteins of which resulting pro-eins 2a, 3, 4 and 5 are glycosylated (namely GP2a, GP3, GP4 andP5, respectively). ORF6 encodes the viral matrix protein (M) andRF7 encodes the nucleocapsid (N) [9–12]. Neutralizing antibod-

es are thought to be mainly induced by GP5 and GP4 [13]; inontrast, a very limited knowledge is available regarding T-cell epi-opes of PRRSV. Recently, two T-cell epitopes have been identifiedn GP5 of American-type strains based on their ability to induce

∗ Corresponding author. Tel.: +34 93 581 4565; fax: +34 93 581 4490.E-mail address: ivan.diaz@cresa.uab.cat (I. Díaz).

264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.07.029

IFN-� responses in cultures of peripheral blood mononuclear cells(PBMC) obtained from PRRSV-immunized and later challenged pigs[14].

Determination of T-cell epitopes is a difficult task. The system-atic approach based on the synthesis and testing of large sets ofoverlapping peptides is indicated when location of T-epitopes can,somehow, be estimated; however, when this knowledge lacks, thatis a very expensive and cumbersome way. In contrast, bioinformaticprediction is extremely cheap and may help to restrict the numberof peptides to be screened. Combination of T-cell epitope predictionand classical immunization experiments can be a useful strategythat permits to speed research in this area [15]. In the present study,bioinformatics were used to predict potential T-cell epitopes in GP4,GP5 and N proteins of PRRSV and were later tested on immunizedanimals.

2. Materials and methods

2.1. Peptide design and synthesis

Putative MHC-I epitopes were predicted after Lelystad virus(LV) sequences combining two different types of predictionalgorithms: binding matrices and artificial neural networks.Thus, in first step, peptides were predicted using MAPP

5 ne 27

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604 I. Díaz et al. / Vacci

ith the SYFPEITHY matrix (available at http://www.mpiib-erlin.mpg.de/MAPPP/binding.html) considering all nonamersith potential binding for any of the human or cattle alle-

es available, since swine genes software and haplotypes areot yet available. For each nonamer and allele, the programroduced a binding score that corresponded to the estimatef half-time of disassociation for a given peptide. That scoreas expressed as a percentage of the maximum score pos-

ible for a given allele. Finally, using the IEBD analysis resourcehttp://tools.immuneepitope.org/analyze/html/mhc binding.html)ased on an artificial neural network, IC50nM (concentration ofeptide that inhibits binding of a standard peptide by 50%) [16] wasalculated for binding peptides. It was considered that peptidescoring more than 35% of the maximum score in a given allelen SYFPEITHY and with IC50nM < 50 for at least one coincidingllele would be most likely T-epitopes of PRRSV. When overlappingeptides were identified, the best IC50nM score was selected. Withhese criteria the two best scoring peptides for each protein wereynthesized. For GP4, peptide no. 5 was originally predicted asLFAILLAI but a modification (substitution of the last I by a T) waseeded in order to permit adequate solubilization (Table 1).

For GP5, some of the predicted peptides were absolutely insol-ble in water and instead of nonamers, dodecamers with similarinding properties but more soluble were produced. In one casepeptide no. 3) a common variant found in genotype-I strains [17]ere synthesized as well.

able 1eptides used in this study and characteristics.

HC-I prediction

eptidea Amino acid sequence Position in LV SYF

P4-4† SYLYNADLL 93–101 A0P4-5 CLFAILLAT 175–183 A0P5-3 CALAALVCFVIR 117–128 A0P5-2 CAFAAFVCFVIR 117–128 A0-15 KPEKPHFPL 50–58 B07-6 FMLPVAHTV 105–113 A0

HC-II prediction

eptidea Amino acid sequence Positio

P4-9 FLLAGAQHI 7–15P4-10† FRPHGVSAA 53–6P4-11† MRWATTIAC 167–17

P5-14† LFLLPTGLS 20–2

P5-12† YQYIYNLTI 41–4

P5-13† FVIRAAKNC 125–13-7 IRHHLTQTE 64–7

-8 VRLIRVTST 113–1

eptides from Vashisht et al. [14] and Genotype-I counterparts

eptidea Amino acid sequence P

P5-EU-1 FAAFVCFVIRAAKNC 1P5-EU-1V LAALVCFVIRAAKNC 1P5-EU-2 RGRIHRWKSPIVVEK 1P5-US-1 LAALICFVIRLAKNC 1P5-US-2 KGRLYRWRSPVIVEK 1

a GP4 = glycoprotein 4; GP5 = glycoprotein 5; N = nucleocapsid.b MHC-I alleles to which the peptide was predicted to bind using the SYFPEITHY matrixc Alleles for which IC50nM was predicted to be less than 50.d MHC-II alleles to which the peptide was predicted to bind using Propred software; on

s well.† Peptides that were found not be recognized by PRRSV-immunized pigs in the first exp

(2009) 5603–5611

MHC-II prediction was done using Propred (available athttp://bic.uams.edu/mirror/propred). For each virus protein, non-amers were predicted setting the threshold of the software to the1% of the best scoring peptides for all the available human alleles.Of the resulting peptides, those with broader MHC-II allele scopewere selected (Table 1).

For GP5, additional peptides corresponding to the genotype-Icounterparts of T-epitopes identified by Vashisht et al. [14] wereproduced. These peptides corresponded to positions 119–133 (pep-tide GP5-EU-1) and 151–165 (GP5-EU-2) of the European GP5. Forthe first epitope, a variant was found in most of the examinedsequences of genotype-I strains from Spain in which two pheny-lalanines were substituted by two leucines (peptide GP5-EU-1V).The two genotype-II epitopic peptides described by Vashisht et al.[14] were synthesized as well (GP5-US-1 and GP5-US-2) (Table 1).

Peptides were purchased from Schafer-N (Copenhagen, Den-mark) at 95% purity except for peptide nos. 2, 3 and 5 that becauseof low solubility were only purified at 50%. An initial dilution ofpeptides based on actual peptide concentration (mg/ml) was donewith sterile double distilled water or methanol when solubility inwater was low.

2.2. Vaccine and virus strains

The commercial modified live vaccine (MLV) used for vaccina-tion (Porcilis PRRS, Intervet-Schering Plough) and the L-450 PRRSV

PEITHYb IC50nMc

201; H2Db; H2Kd, H2Ld A0201; A2403201 A0201; A0202, A203, A0206201; H2Db; H2Ld A0201; A0202; A0203; A0206; A3101201; H2Kk; H2Kd; H2Ld B1517; B150302; H2ld B0702

201; B0702; H2kd; H2kk; H2Ld A201; A0203; A202; A206, B1530

n in LV Propredd

DRB1: 0101; 0102; 0702; 0703; DRB5: 0101; 01051 DRB1: 0305; 0408; 0426; 0813

5 DRB1: 0401; 0402; 0408; 0410; 0426; 0101;1104; 1106; 1107; 1311

8 DRB1: 0401; 0402; 0408; 0410; 0423; 0426;0802; 0804; 0806; 1101; 1104; 1106; 1107; 1128;1305; 1307; 1311; DRB5: 0101; 0105

9 DRB1: 0402; 0404; 0408; 0701; 0703; 1128;1305; 1307; 1501; 1502; 1506; DRB5: 0101; 0105

3 DRB1: 0101; 0801; 0804; 0806; 0813; 08172 DRB1: 0401; 0402; 0405; 0410; 0421; 1301;

1304; 1327; 132821 DRB1: 0101; 0102; 0301; 0306; 0307; 0308;

0311; 1107

osition in LV or VR-2332

19–133 (LV)19–133 (LV)51–165 (LV)17–131 (VR)49–163 (VR)

.

ly nonamers were included, although 12-mers and 15-mers includes the sequence

eriment. They were not used in the second experiment.

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I. Díaz et al. / Vacci

train used for the stimulation of PBMC in ELISPOT assays and forNA constructions have been previously characterized [18,19]. Sim-

larity between the vaccine strain and strain L-450 was 93.5% forRF4, 94.0% for ORF5 and 96.3% for ORF7 [19].

.3. Construction of the plasmid vectors expressing ORF4, ORF5nd ORF7 and expression in mammalian cells

ORF4 was amplified from strain L-450 by means of RT-PCR usinghe protocol described by Meulenberg et al. [10] including a restric-ion site for enzymes BamHI and HindIII (forward and reverse,espectively). For ORF5, primers were designed to include restric-ion sites for HindIII and XbaI (forward and reverse) and the Kozadequence in the forward oligonucleotide. For ORF7, amplificationas done according to van Nieuwstadt et al. [20] with restriction

ites BamHI (Table 2).The amplified fragments were cloned in the eukaryotic vector

cDNA3.1+ (Invitrogen) using the cytomegalovirus promoter. Eachlasmid was transfected in BHK-21 cells using Lipofectamine PlusInvitrogen) [21]. Verification of the expression of the viral pro-eins coded by the plasmids was carried out by immunoperoxidase

onolayer assay [1] using either monoclonal antibodies for GP5 andprotein (Ingenasa, Madrid) or for GP4 a polyclonal sera obtained

rom experimentally infected animals. All DNA preparations usedn the following experiments were endotoxin-free as determinedsing a commercial kit (Qiagen).

.4. Testing of synthesized peptides

An initial screening of peptides was done using 10 healthy 4-eek-old Landrace piglets that were split in two groups of five

nimals (A and B). Animals in group A were vaccinated with 2 mlf the commercial vaccine described above and the other five wereept as controls. At days 42 and 63 post-vaccination, blood samplesere collected in heparinized blood-collecting tubes and processed

o analyze the frequencies of IFN-�-secreting cells (IFN-�-SC) byLISPOT after overnight stimulation of PBMC with a given pep-ide or the whole virus. ELISPOT assay was selected since it is aensitive test to identify T-cell epitopes [14,15,22,23]. Moreover,ome authors have described a correlation of cell-mediated immu-ity measured by ELISPOT and protection against PRRSV [19,24,25].

n this first experiment peptides nos. 2–15 were tested. Pep-ides inducing non-significant responses were not further testedTable 1).

A second experiment was carried out to further confirm thatome of the selected peptides were T-epitopes of PRRSV. Thir-een healthy 3-week-old Landrace pigs PRRSV-free (determinedy serology and RT-PCR in serum) were selected. Animals wereransported to an experimental farm and they were randomly dis-ributed in five groups, namely: MLV (n = 5; animals A–E), ORF4n = 2, pigs F and G), ORF5 (n = 2; pigs H and I), ORF7 (n = 2; pigsand K) and C (n = 2, pigs L and M). Pigs in group MLV were vacci-ated intramuscularly with 2 ml of the commercial PRRSV vaccinetated above. Animals in groups ORF4, ORF5 and ORF7 were injectedith 400 �g (in 1.2 ml of sterile PBS) of the corresponding plasmidNA constructions containing ORF4, ORF5 or ORF7. In this case, pigs

eceived a second injection of plasmid DNA at day +17 and a thirdose at day +35 for a total of three inoculations. In all cases, two-hirds of the total DNA amount was injected intramuscularly andne-third of the DNA was injected subcutaneously in the ear [21].roup C animals were kept as controls receiving 1.2 ml of unaltered

lasmid pcDNA3.1+. In this second experiment, peptides that wereot recognized by pigs in the first experiment were discarded andot tested further. During the development of this study, Vashishtt al. [14] published a description of T-epitopes in GP5 of PRRSVenotype-II strains. According to their data, peptides reported by

(2009) 5603–5611 5605

those authors as well as their genotype-I counterparts were alsotested (Table 1). All the sequences of the genotype-I peptides usedin our second experiment were conserved between MLV, LV and L-450 strains, except for GP5-2/GP5-3 and GP5-EU-1/GP5-EU-1V (seeSection 2.1).

2.5. Specific IFN-�-secreting cells by ELISPOT

Heparinized blood samples were collected at days 42 and 63 inthe first experiment and at day 56 post-vaccination with the MLV(14 days after the third DNA injection) in the second experiment.PBMC were separated from whole blood by density-gradient cen-trifugation with Histopaque 1.077 (Sigma) and used in an IFN-�ELISPOT using commercial mABs (Porcine IFN-� P2G10 and biotinP2C11, BD Biosciences Pharmingen) as previously reported [26].PBMC were incubated at 5 × 105 cells/well and overnight stimu-lated with either wild-type virus (strain L-450) at m.o.i. 0.01 orindividual peptides. All tests were done in duplicate. In the ini-tial experiments, peptides were tested in different concentrationsranging from 5 to 40 �g/ml in order to determine the optimal con-centrations. Ten micrograms per milliliter resulted to be the optimalconcentration and, accordingly, this concentration was used fromthen onwards. Unstimulated cells and phytohaemagglutinin (PHA)-stimulated controls (10 �g/ml) were also included. To calculate theadjusted antigen-specific frequencies of IFN-�-SC, average countsof spots in unstimulated wells were subtracted from average countsin antigen-stimulated wells. Results were expressed as the numberof peptide-specific and PRRSV-specific IFN-�-SC per 106 PBMC. Inorder to further assure the specificity of the tests, adjusted countsof less than 10 spots/million were considered to be non-significant.

In order to ascertain whether or not a given peptide was rec-ognized by immunized pigs, results of the IFN-� ELISPOT weretabulated as reported previously [14] and the following values werecalculated: (1) the maximum response, namely, the frequency ofIFN-�-SC from the highest responder pig within the tested group;(2) the total response, namely the sum of all of the specific IFN-�-SCobserved in each group; (3) the average response, that is the sumof all of the specific IFN-�-SC in each group divided by the num-ber of pigs in that group; (4) the percentage of peptide-responsivepigs, namely the proportion of pigs in each group for which thepeptide-specific IFN-�-SC response was equal or higher than 10;(5) the average response of peptide-responsive pigs, namely, theaverage in each group of the peptide-specific IFN-�-SC response ofall pigs exhibiting a response to the peptide equal or higher than 10.With the purpose of having a more precise picture of the results,two additional values were calculated: (6) OverELISPOT (averageand standard deviation), namely, the average resulting from theindividual ratios obtained when the frequency of peptide-specificresponses (IFN-�-SC) were divided by the frequencies observedusing the whole virus (L-450) as stimulus; (7) individual identi-fication (Id) of non-responding pigs.

2.6. Conservation of identified GP4, GP5 and N epitopes inEuropean and American-type PRRSV strains

In order to determine if the reactive peptides were more or lessconserved, nucleotide sequences of PRRSV strains form differentcountries were retrieved from Genbank. The corresponding aminoacid sequences were predicted with Bio-Edit and alignment of theresulting proteins was done using ClustalX. In this analysis wereincluded: (a) 16 sequences for ORF4 (12 belonging to genotype-I and

4 belonging to genotype-II); (b) 82 sequences for ORF5 (64 belong-ing to genotype-I and including representatives of sub-genotypes 1,2, 3 and 4, and 18 genotype-II sequences) and (c) 79 sequences forORF7 (60 of genotype-I; sub-genotypes 1–4 and 19 of genotype-II).Table 3 shows the characteristics of the abovementioned strains.

5606 I. Díaz et al. / Vaccine 27 (2009) 5603–5611

Table 2Primers used for amplification in PCR of ORFs 4, 5 and 7 of L-450 PRRSV strain for plasmid constructions.

ORF Primer sequence Expected size ofthe PCR product

4 F: 5′-GGCAATTGGATCCATTTGGA-3′ 619 pbR: 5′-AGAAGCAAGCTTGCGGAGTC-3′

5 F: 5′-GCAAGCTTGCCACCGCCATTCTCTTGGCAATATGAGATGTTCT-3′ 623 pbTCTAG

CGG-3A-3′

3

3

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sea

TS

G

O

O

O

R: 5′-ATTCTAGAATCGTTGCAAAAATCG

7 F: 5′-GATTGGATCCTCGTCAAGTATGGCR: 5′-CTAAGGATCCTGTCAAATTAACTTGC

. Results

.1. Screening of peptides using IFN-�-SC by ELISPOT

In the first study, 14 peptides were tested (nos. 2–15); 6 of themere predicted to bind MHC-I and 8 were predicted to bind MHC-

I. The initial screening of those peptides by ELISPOT showed thatof them (8/14; 57.1%) – peptides 2, 3, 5, 6, 7, 8, 9, 15 – were rec-

gnized by at least 1/5 immunized pigs at both 42 and 63 daysost-vaccination with OverELISPOT values ranging from 3% to 18%.hat group of peptides included five potential MHC-I epitopes (5/6;

3.3%) and three (3/8; 37.5%) potential MHC-II epitopes.

In the second experiment, reactive peptides selected in the firsttudy were retested as well as GP5 peptides reported by Vashishtt al. [14] and their genotype-I counterparts. As shown in Table 4And B, at 56 days post-vaccination all MLV-vaccinated pigs strongly

able 3equences (GenBank accession numbers) used to study the impact of genetic diversity of

enotype Sub-genotype Country and

RF4I (European) 1 China: EU07

DQ864705;II (American) NA China: AY03

RF5I (European) Sub-genotype 1 Belgium: AY

AY035902; AAY035923; AM96262 (LeDQ324680;DQ345736;EF429114; EUnited StateDQ324681 (

Sub-genotype 2 Belarus: DQAF378802; A

Sub-genotype 3 Belarus: DQDQ324687

Sub genotype 4 Belarus: DQII (American) NA Canada: Z82

U03040; U3DQ475065;(Ingelvac AT

RF7I (European) Sub-genotype 1 Belgium: AY

AY035954; AAY035967; AAY035976; AAF438360; DX92942; AYStates: AY36DQ324712 (

Sub-genotype 2 Belarus: DQSub-genotype 3 Belarus: DQ

DQ324699 (Sub genotype 4 Belarus: DQ

II (American) NA Canada: U64EF441819; EAF043960; A

GCCTCCCA-3′

′ 398 pb

responded against peptides 5 and 9 derived from PRRSV GP4 (aver-age frequencies 34 and 24.8 IFN-�-SC/106 PBMC, respectively;OverELISPOT: 0.46 and 0.35, respectively). Interestingly, animalsvaccinated with plasmid DNA containing GP4 only recognized pep-tide no. 5. For GP5 peptides, MLV-vaccinated animals recognized(3/5 pigs) mainly peptide no. 3 (average of responsive pigs: 26IFN-�-SC/106 PBMC). All other GP5 peptides (no. 2, EU-1, EU-1Vand EU-2) derived from genotype-I sequences were recognizedalthough at a much lower extent (not more than 3/5 animalswith frequencies ≤24 IFN-�-SC/106 PBMC and OverELISPOT valuesbetween 0.05 and 0.19). In this case, the American-type peptides

were not recognized but in one animal. In ORF5 DNA-vaccinatedpigs only peptide EU-2 was recognized by one animal. For N pro-tein, significant IFN-�-SC responses were observed for peptides 6, 7,8 and 15 although only peptides 7 and 8 induced responses in mostpigs (5/5 and 4/5 pigs with average frequencies in responding pigs of

PRRSV upon the composition of the predicted T-epitopes.

accession number

6704; Netherlands: M96262; FJ004949; AY588319; Spain: DQ064785; Thailand:United States: AY366525; DQ489311; Vaccines: DQ064784 (Amervac); XXX Porcilis2626; EU880437; United States: U87392; AF325691

035900, AY035901; Czech Republic: AF253537; China: EU076704; Denmark:Y035905; AY035909; AY035912; France: AY035919; Germany: AY035921;Y035925; Italy: AY035927; AY035930; AY035931; AY035933; Netherlands:

lystad virus); AY588319; Poland: DQ324673; DQ324674; DQ324675; DQ324679;Spain: AF495509; AF495512; AF495516; AY035935; AY035936; DQ009647;DQ345743; DQ345746; DQ345750; DQ345755; EF429101; EF429106; EF429110;F429118; X92942; Thailand: DQ864705; United Kingdom: AY035939; AY035940;s: AY366525; DQ489311; Vaccines: DQ324668 (Amervac); DQ324678 (Porcilis);PYRSVAC-183)324671; DQ324672; DQ324683; DQ324689; Lithuania: AF378800; AF378801;F378803; DQ324667; DQ324682

324669 (sub-genotype 3/2); DQ324670 (sub-genotype 3/2); DQ324676; DQ324686;

324677995; China: EU480738; EU480743; EU480754; Japan: AB175691; United States:4297; U66392; U66393; U87392 (prototype strain VR-2332); AF176426;DQ475114; DQ475152; EU759705; EU759727; EU759859; Vaccine: AY656991P)

035945; AY035946; China: EF473138; Denmark: AY035947; AY035950;Y035957; AY035960; AY035962; France: AY035964; Z92527; Germany:Y035968; Z92538; Hungary: AF297099; Italy: AY035970; AY035973; AY035975;Y035977; Netherlands: M96262 (Lelystad virus); Z92533; Z92534; Poland:Q324703; DQ324704; DQ324705; DQ324711; DQ324715; DQ324716; Spain:

03579; AY035980; DQ009648; United Kingdom: L77920; L77924; L77926; United6525; AY749400; AY749406; Vaccines: DQ32698 (Amervac); DQ324710 (Porcilis);

PYRSVAC-183)324722; DQ324723; DQ324724; DQ324725; Lithuania: AF438362; AF438363324717; DQ324718; DQ3247219; DQ324720; DQ324728; DQ324731; DQ324734;sub-genotype 3/2); DQ324700 (sub-genotype 3/2)324708; DQ324709931; China: EU880439; EU880442; EU880443; Korea: EF441812; EF441815;

F441822; EF441852; EF441861; EF441866; United States: AF043952; AF043954;F043970; EU756843; EU756845; EU757731; U87392

I.Díaz

etal./Vaccine

27 (2009) 5603–56115607

Table 4Peptide-specific IFN-�-secreting cells from (A) modified-live vaccine and (B) DNA-vaccinated pigs.

Values Peptide

GP4-5 GP4-9 GP5-2 GP5-3 EU-1 EU-1V EU-2 US-1 US-2 N-6 N-7 N-8 N-15

(A) Vaccinated with a modified-live vaccineMaximum responsea 80 36 18 46 22 32 28 0 14 13 82 74 17Total responseb 170 124 20 82 40 48 61 0 14 24 280 166 23Average responsec 34 24.8 4 16.4 8 9.6 12.2 0 2.8 4.8 56 33.2 4.6% of responsive pigsd 100% 100% 20% 60% 40% 40% 60% 0% 20% 20% 100% 80% 20%Avg. responsive pigse 34 24.8 18 26 21.3 24 19.7 0 14 13 56 41.5 17OverELISPOTf,g 0.46 ± 0.3 0.35 ± 0.22 0.05 ± 0.1 0.21 ± 0.24 0.1 ± 0.1 0.11 ± 0.16 0.19 ± 0.17 0 0.05 ± 0.12 0.06± 0.06 0.84 ± 0.51 0.41 ± 0.34 0.008 ± 0.0Identification of non-responding pigsh – – A, C, D, E B, D C, D, E C, D, E A, E A–E A, B, C, E A, C, D, E – C A, B, D, E

Values Peptide

ORF4 ORF5 ORF7

GP4-5 EU-2 N-6 N-7 N-8 N-15

(B) DNA vaccinationMaximum responsea 136 10 200 18 11 22Total responseb 166 13 201 32 11 22Average responsec 83 6.5 100.5 16 5.5 11% of responsive pigsd 100% 50% 50% 100% 50% 50%Avg. responsive pigse 83 10 200 16 11 22OverELISPOTf,g 34.5 ± 47.4 1 ± 0 1.78 ± 2.52 0.40 ± 0.07 0.09 ± 0.14 0.27 ± 0.39Identification of non-responding pigsh – H J – J J

Peptide-specific IFN-�-secreting cells (IFN-�-SC)/106 peripheral blood mononuclear cells belonging to pigs vaccinated with a modified-live vaccine (namely A–E) (Table A) and ORF4 (F–G), ORF5 (H–I) and ORF7 (J–K) vaccinatedpigs (Table B). In Table B, peptides not inducing detectable IFN-�-SC are not shown.

a The maximum response, namely, the frequency of IFN-�-SC from the highest responder pig within the tested group.b The total response, namely the sum of all of the specific IFN-�-SC observed in each group.c The average response, that is the sum of all of the specific IFN-�-SC in each group divided by the number of pigs in that group.d The percentage of peptide-responsive pigs, namely the proportion of pigs in each group for which the peptide-specific IFN-�-SC response was equal or higher than 10.e The average response of peptide-responsive pigs, namely, the average in each group of the peptide-specific IFN-�-SC response of all pigs exhibiting a response to the peptide equal or higher than 10.f OverELISPOT (average and standard deviation), namely, the average resulting from the individual ratios obtained when the frequency of peptide-specific responses (IFN-�-SC) were divided by the frequencies observed using

the whole virus (L-450) as stimulus.g Mean ± SD.h Individual identification (Id) of non-responding pigs.

5608 I. Díaz et al. / Vaccine 27 (2009) 5603–5611

Table 5Conservation of PRRSV GP5 T-cell epitopes.

Segment changes Genotype-I (European) Genotype-II (American) (n = 18)

Sub-genotype-1 (n = 48) Sub-genotype-2 (n = 10) Sub-genotype-3 (n=5) Sub-genotype-4 (n = 1)

C117 → F – – 2 – –C117 → S – 3 – – –F119 → L 7 7 1 – 17F119 → S 1 – – – –A120 → T 1 – – – –F122 → L 28 10 1 – 17V123 → L – – 2 – –V123 → A – – 2 – –V123 → I – – – – 12V123 → T – – – – 4V126 → G 1 – – – –V126 → A 2 2 1 – 2V126 → I – 2 – – 3I127 → T 1 – – – –I127 → V – 4 – – 1I127 → A – 1 1 – –A129 → V – 10 – – –A129 → L – – – – 12A129 → P – – – – 5A130 → V 1 1 1 1 2A130 → T – – – – 5K –

F NC) wg e rela

5ODacdtw

TC

C

K

I

F

V

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131 → R – –

or GP5 amino acid segment compromising positions 117–133 (CAFAAFVCFVIRAAKenotype-I or genotype-II PRRSV strains examined. Superscript numbers indicate th

6 and 41.5 and OverELISPOT values of 0.84 and 0.41, respectively).nly peptide 7 induced a significance response in the two ORF7NA-vaccinated pigs. Interestingly, among ORF7 DNA-vaccinatednimals, pig J did not respond to other N protein peptides. In all

ases, DNA-vaccinated animals responded only to the peptideserived from the protein encoded by the plasmid that was injectedo them. Unvaccinated control pigs were negative to all peptides asell as to the whole virus.

able 6onservation of PRRSV N protein T-cell epitopes.

hanges Epitope

Genotype-I (European)

Sub-genotype-1 (n = 43) Sub-genotype-2 (n = 6) Sub

PEKPHFPLK50 → R 3 1 6K50 → N – – –K50 → S – – –P51 → S 1 – –P51 → L – – –E52 → D 1 – –

RHHLTQTEI64 → V 4 6 9L68 → F 1 – –T69 → A – 2 4Q70 → G – – 1Q70 → S – – 2

MLPVAHTVA110 → V 1 – –A110 → N – – 6A110 → T – 1 1A110 → G 1 – –

RLIRVTSTT119 → R 1 – –T121 → A – 4 1S120T121 → AS – – –S120T121 → AP – – –V118T119S120T121 → ITAP – – –

pitope: peptide originally predicted as a T-cell epitope in N protein. Changes: variants oxamined. Superscript numbers indicate the relative position of a given amino acid in Llternatively, the counterpart amino acid sequence.

– 1

as considered. Segment changes: variants over the sequence found in the differenttive position of a given amino acid in Lelystad virus GP5.

3.2. Conservation of T-cell epitopes identified in European PRRSV

For GP4, two peptides, nos. 5 and 9, were recognized by immu-nized animals: CLFAILLAT (positions 175–183) and FLLAGAQHI

(positions 7–15). For the first peptide, the sequence was fullyconserved among genotype-I strains, and in genotype-II modifi-cations (peptide CLLPSLLAI) were only seen in a Chinese strainreported as corresponding to a high fever syndrome (Genbank

Genotype-II (American) (n = 19)

-genotype-3 (n = 9) Sub-genotype-4 (n = 2)

– –– 16– 2– –– 1– –

– VRHHFTP(S/G)E––––

– FSLPTAHTV–––

– –– –– 13– 4– 1

ver a given epitope found in the different genotype-I or genotype-II PRRSV strainselystad virus N protein. For genotype-II changes are shown in the last column or

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I. Díaz et al. / Vacci

ccession no. EU880437). For the second one, the following changesere observed in the set of analyzed sequences: H14 → Y in 4/12

enotype-I strains and I15 → L (4/12) or F (2/12). The genotype-IIounterpart of this peptide was FL(L/V)GTKC(F/L).

For GP5, changes in the segment of amino acids comprisedetween positions 117 and 133 relative to LV (that is, amino acidsresent in peptides nos. 2, 3, EU-1, EU-1V and EU-2) were commonnd thus, the only amino acids for which changes were never foundn any of the examined sequences were A118; A121; C124, F125, R128,

132 and C133. In contrast, F119 changed to L in 7/48 European strainsf sub-genotype 1, 7/10 in sub-genotype 2 and 1/5 in sub-genotype. F122 changed to L in 28/48 European strains in sub-genotypeand 10/10 in sub-genotype 2 and, finally A129 changed to V in

0/10 sub-genotype 2 European strains. In genotype-II strains, mostommon changes were F119 → L (17/18 strains); F122 → L (17/18);123 → I (12/18); A129 → L (12/18); A129 → P (5/18) and A130 → T

5/18). Table 5 shows these and other changes found in this epitopicegment.

In N protein, peptide KPEKPHFPL was conserved with only sixhanges in the total of sub-genotype 1, 2 and 4 strains. In contrast,strains of sub-genotype 3 carried an R instead of a K in position

0. For genotype-II strains, the most common change was K50 → Nut most other amino acids were equal to the genotype-I sequence.eptide FMLPVAHTV was almost conserved as well in genotype-strains of sub-genotypes 1, 2 and 4 with only three changes

A110 → V, T or G) but in sub-genotype 3 A110 changed to N in 6/9trains. The genotype-II counterpart of this peptide was FSLPTAHTVor which no changes were observed in the examined sequences.eptide IRHHLTQTE was again almost conserved among European-ype strains with only 4 strains with one change in sub-genotype 1,in sub-genotype 2 and 9 in sub-genotype 3. The genotype-II coun-

erpart of this peptide was VRHHFTP(S/G)E. Peptide VRLIRVTST wasgain almost conserved among European strains and thus only 6/60trains showed some change (Table 6).

. Discussion

Control of PRRSV has proven to be difficult. Several reasons canxplain this difficulty but one of the most important factors is thatvailable vaccines provide a limited protection against the infec-ion, particularly taking into account the high genetic diversity ofRRSV. Development of new and more efficacious vaccines againsthis virus faces the fact that our understanding of the host’s protec-ive immune responses against the virus or about what parts of theirus induce protective responses is very far from being complete.

Several papers emphasized the role of neutralizing antibodies13,27,28] as well as of the cell-mediated immunity (CMI) [15,17,18].n the first case, at least one neutralizing epitope has been identifiedn GP5 [29,30] and others have been suggested to exist in GP4 and

aybe in GP3 and M [10,31–34]. Regarding CMI, a recent paper14] reported the identification of two distinct regions in ORF5hat contain immunodominant T-cell epitopes based on their abil-ty to induce IFN-�-SC. The approach for identifying those regions

as based on the synthesis of overlapping pentadecamers (n = 96)erived from the ORF5 sequences, which represents approximately% of the whole genome, of four PRRSV strains. In the present study,different approach was taken by using bioinformatics as a mean

or the prediction of T-epitopes and focusing in in vivo/ex vivo exper-ments for screening and testing of reduced number of peptidesielding the higher predictive scores with different methods of

rediction. Obviously, a more classical approach using overlappingeptides from ORFs that encode for all the structural proteins wouldake a much more exhaustive determination of T-cell epitopes.

hus, some of the reasons lying behind bioinformatic approachere to save time, resources and speeding research by selecting

(2009) 5603–5611 5609

only those peptides with a higher probability of being immunolog-ically relevant. Combining the in silico T-cell epitope prediction anda classical in vitro/ex vivo confirmation, approach known as Fishingfor antigen using epitopes as bait [35], could accelerate the discoveryprocess more than 10-fold [36].

The approach of the present work permitted the preliminaryidentification of immunodominant T-epitopes in GP4, GP5 and Nproteins of genotype-I PRRSV: peptides 5 and 9 for GP4 and peptides7 and 8 for N protein, whereas, peptide 3 and the European-typecounterparts of the peptides reported by Vashisht et al. [14] wererelevant for GP5. Interestingly, in terms of proportion of respon-ders and of strength of the response, GP4 and N proteins might bemore immunodominant than GP5. As indicated by other authors,nucleocapsid proteins often induce strong CMI responses [37,38].However, the results obtained with GP5 are relevant by severalreasons. First of all the results confirm the epitopic nature ofEuropean-type counterparts of already reported American-typeepitopes [14]; secondly, the predicted peptide no. 3 was the oneinducing the stronger responses in GP5, and this confirm thevalidity of the prediction system used in comparison with the over-lapping peptide synthesis and testing used by Vashisht et al. [14]and, thirdly, it was evident that small changes between peptideGP5-EU-1 and GP5-US-1 were enough to produce lack of recog-nition of the American-type peptide by pigs vaccinated with anEuropean-type vaccine.

A second point of interest is the different nature of the responseobtained in animals vaccinated with a MLV vaccine or with a DNAvaccine. As can be seen in Table 4A and B, for example peptideno. 9 (GP4) did not elicit an IFN-� response in pigs vaccinatedwith an ORF4 plasmid while other peptides, for example no. 6(N protein) was better recognized by one DNA-immunized pig.One possible explanation for that lack of response is a failure inthe DNA vaccination protocol. This has to be discarded becauseother peptides derived from the same protein were perfectly rec-ognized by the DNA-immunized pigs. Therefore, these differencesseem to be more attributable to the way in which proteins areprocessed or presented to the immune system in the course of avaccination with a live virus or with DNA. This must be taken intoaccount and suggest that DNA-vaccination approaches for deter-mining potential viral epitopes should be interpreted cautiously forextrapolation to the events taking place in the course of a naturalinfection.

As Vashisht et al. [14] discussed in their paper, it cannot beruled out from our results that a temporal development in the T-cell responses occurred and affected GP4, GP5 and N responses.Therefore, the immunodominance between some T-cell epitopescould change in the course of time after an infection or vaccina-tion. However, it is important to note that at similar times after theinitial immunization, we found similar responses to similar GP5epitopes compared to the abovementioned authors. A second con-sideration that must be done is the fact that animals carrying adifferent MHC haplotype could have responded to other PRRSV pep-tides. This could have been the case of pig J (vaccinated with plasmidDNA containing ORF7) that only recognized peptide 7. Finally, theinfluence of the peptide lengths in the responses obtained has tobe also considered. The majority of peptides used in our study werenonamers. It is well known that short peptides (8–10 amino acids)can bind better to MHC-I than to MHC-II [39]. On the other hand, theassay used (IFN-� ELISPOT) predominantly measures Th1 and cyto-toxic T lymphocyte responses. Therefore, we cannot rule out that iflonger peptides or other assays with higher rate of success deter-

mining MHC-II epitopes had been used clear and stronger responseswould have obtained for MHC-II peptides.

In spite of our results could be the tip of an iceberg in thecell-immunity response against PRRSV, the proportion of actualepitopes identified in the present study and the repeatability among

5 ne 27

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xperiments done by us or others [14] suggests that, at least themmunodominant ones are probably common epitopes of PRRSV.

In a second part of our study, we examined the variability ofhe identified epitopes among genotype-I and genotype-II strains.t is difficult to draw conclusions about GP4 peptides because of thecarce number of genotype-I sequences; however, from the avail-ble data it seems that peptide no. 5 was almost fully conservedmong genotype-I and genotype-II strains except for a one Chi-ese strain associated to the recent very virulent PRRSV outbreaks.he potential relationship of this change with virulence should be

nvestigated. For GP5 and N protein changes were mostly seen inub-genotype 3 strains of Eastern Europe. Although we did notest animals immunized with those strains, the extent of changesffecting almost all of the identified epitopes suggest that cross-eactivity would probably be low. When looking at the genotype-IItrains, peptides 6 and 15 only have two amino acid differences withegards to the genotype-I sequences, suggesting potential cross-eaction. In fact, re-prediction using the genotype-II sequences forhose peptides produced the same results as the original predic-ion. In contrast, peptides 7 and 8 were substantially different inenotype-II strains suggesting a potential lack of cross-reactivity.evertheless, as seen for GP5 peptides, this does not preclude that

he genotype-II counterparts cannot be recognized by genotype-IImmunized pigs.

In spite the use of immunomic tools has been largely appliedn other species, few papers have been published regarding pigsr other domestic species [40–42]. To the best of our knowledge,his is the first time that an in silico prediction and in vitro/in vivoonfirmation has been used for evaluating CMI responses againstpig disease and in particular for PRRSV. Results of the present

tudy showed that the bioinformatic prediction can be used to fishmmunologically relevant epitopes to be further tested for its role inrotection. Furthermore, we showed that PRRSV N protein and GP4ontain T-cell epitopes, that N protein is probably immunodom-nant in cell-mediated responses over GP4 and particularly GP5nd that the genotype-I counterparts of the previously reportedenotype-II GP5 epitopes are also immunogenic. All these informa-ion is relevant for the design of newer and better vaccines. Also,hese results may be used to refine the precision of tools such ashe IFN-� ELISPOT for PRRSV immunology.

cknowledgements

We would like to thank Núria Navarro and Esmeralda Cano forheir excellent technical assistance. Our grateful thanks to Joan Tar-adas and Cristina Lorca for their help in the construction of DNAaccines. Thanks to Dr. Javier Dominguez and Dr. Belén Álvarez forelp and advice. Dr. Ivan Díaz has been financially supported byroject “Porcivir” CDS2006-00007 of the Spanish Ministry of Sci-nce and Innovation. This study has been funded by the Spanishinistry of Science and Innovation project AGL2005-07073-C02-

2/GAN.

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