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ARTICLE IN PRESSG ModelVAC-15966; No. of Pages 7

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Contents lists available at ScienceDirect

Vaccine

j our na l ho me page: www.elsev ier .com/ locate /vacc ine

valuation of adaptive immune responses and heterologousrotection induced by inactivated bluetongue virus vaccines

mmanuel Brearda,∗,1, Guillaume Belbisb,1, Cyril Viarougea, Kyriaki Nomikouc,ndy Haegemand, Kris De Clercqd, Pascal Hudelete, Claude Hamers f, Francis Moreaug,homas Liling, Benoit Durandh, Peter Mertensc, Damien Vitoura,orinne Sailleaua, Stéphan Zientaraa

ANSES, UMR 1161 Virologie ANSES-INRA-ENVA, 23 avenue du Général de Gaulle, 94704 Maisons-Alfort, FranceUniversité Paris-Est, Ecole Nationale Vétérinaire d’Alfort, Unité de Pathologie du Bétail, 7 avenue du Général de Gaulle, 94704 Maisons-Alfort, FranceVector-Borne Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey GU24 0NF, United KingdomCODA–CERVA, Department of Virology, Ukkel, BelgiumMERIAL S.A.S., 254 Rue Marcel Mérieux, 69007 Lyon, FranceMERIAL S.A.S., P.I. Plaine de l’Ain, Allée des Cyprès, 01150 Saint-Vulbas, FranceUniversité Paris-Est, Ecole Nationale Vétérinaire d’Alfort, Centre de recherche biomédicale, 7 avenue du Général de Gaulle, 94704 Maisons-Alfort, FranceANSES, unité Epidémiologie, 23 avenue du Général de Gaulle, 94704 Maisons-Alfort, France

r t i c l e i n f o

rticle history:eceived 22 October 2013eceived in revised form0 November 2014ccepted 28 November 2014vailable online xxx

eywords:luetongue virus disease

a b s t r a c t

Eradication of bluetongue virus is possible, as has been shown in several European countries. Newserotypes have emerged, however, for which there are no specific commercial vaccines. This studyaddressed whether heterologous vaccines would help protect against 2 serotypes. Thirty-seven sheepwere randomly allocated to 7 groups of 5 or 6 animals. Four groups were vaccinated with commercialvaccines against BTV strains 2, 4, and 9. A fifth positive control group was given a vaccine against BTV-8.The other 2 groups were unvaccinated controls. Sheep were then challenged by subcutaneous injection ofeither BTV-16 (2 groups) or BTV-8 (5 groups). Taken together, 24/25 sheep from the 4 experimental groupsdeveloped detectable antibodies against the vaccinated viruses. Furthermore, sheep that received het-

nactivated vaccinesxperimental infectioneterologous challengesheep

erologous vaccines showed significantly reduced viraemia and clinical scores for BTV-16 when comparedto unvaccinated controls. Reductions in clinical signs and viraemia among heterologously vaccinatedsheep were not as common after challenge with BTV-8. This study shows that heterologous protectioncan occur, but that it is difficult to predict if partial or complete protection will be achieved followinginactivated-BTV vaccination.

© 2014 Elsevier Ltd. All rights reserved.

. Introduction

Bluetongue (BT) is an infectious, OIE-listed arboviral disease thatnfects ruminants [1]. The disease is caused by viruses belonging toluetongue virus species [2]. There are 26 bluetongue virus (BTV)erotypes, which raise type specific neutralising antibodies (NA)

Please cite this article in press as: Breard E, et al. Evaluation of adaptinactivated bluetongue virus vaccines. Vaccine (2014), http://dx.doi.o

uring infection of their hosts [3]. BTV is transmitted primarily byiting midges (Culicoides spp.) but can be transmitted verticallyr via an oral route [4–6]. Since the 20th century, 10 serotypes

∗ Corresponding author. Tel.: +33 695063201; fax: +33 143689762.E-mail address: emmanuel.breard@anses.fr (E. Breard).

1 Both authors contributed equally.

ttp://dx.doi.org/10.1016/j.vaccine.2014.11.053264-410X/© 2014 Elsevier Ltd. All rights reserved.

have been detected in Europe, some of them on several differentoccasions [7].

After the emergence of BTV in Europe (1998), the first suc-cessful inactivated vaccine (IV) protected against BTV-2 [7–11].Subsequently, monovalent and bivalent IV were developed, usedin the field [9,10] and efficient for eradication of different homol-ogous BTV serotypes [12–14]. Since 2011, several countries thatwere previously infected with serotype 1, 2, 4 or 8 returned tofreedom-from-disease after mass vaccination campaigns [7,11].

Animals vaccinated with these IV develop protective immune

ive immune responses and heterologous protection induced byrg/10.1016/j.vaccine.2014.11.053

responses against the homologous serotype(s). The BTV outer-capsid proteins VP2 and VP5 (particularly VP2) are the BTV proteinsinducing NA [15]; VP2 is also the major protein involved in serotypedetermination [16,17]. Sheep inoculated with VP2 alone also

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roduced NA and were protected against challenge with homol-gous BTV serotype [18,19].

A vaccine that protects against multiple BTV types wouldrovide a valuable defence against incursions of new BTV serotypes.owever, such vaccines seem difficult to be create, despite evidence

hat cytotoxic T cells play a role in protection against BTV infection20–22]. Few studies report complete or even partial protectionfter immunization and heterologous challenge. However, a signif-cant level of cross-protection against a BTV challenge was obtainedfter immunization with a single BTV protein (VP7) without NAesponse against virus particles [23]. Studies show that inactivatedr virus-like particle vaccines can reduce the severity of heterol-gous BTV infections, even though the BTV serotypes used wereot closely related [24,25]. One study using a recombinant vac-ine against BTV-4 in IFNAR(−/−) knockout mice, showed effectiveross-protection against heterologous lethal challenges [26]. Lit-le is known about the mechanism(s) of protection after pan- or

ulti-serotype vaccinations and heterologous challenge.Earlier studies of sheep serially infected with BTV-3 and 4

emonstrated resistances to challenge with BTV-6 [21]. However,he level of protection induced in sheep against heterologouserotypes by simultaneous multivalent vaccination with IV has noteen reported. The nucleotype grouping for BTV segment 2 illus-rates the ‘relatedness’ of different strains [27]. Different serotypesithin a single BTV nucleotype are more likely to generate cross-

eactive responses to other viruses within the same nucleotype27].

The possibility that animals can be protected and to restrict themergence of new BTV serotypes in ‘free’ regions, using currentlyvailable IV in a polyvalent vaccination strategy, needs to be furtherssessed. The only IV registered within Europe are for serotypes 1,, 4, 8, and 9. No vaccine against BTV-16 is available in the marketven if a study demonstrated that two doses of BTV-16 IV protectheep [28]. Those would be the immediate solution in the case of anutbreak of an exotic serotype. In this study we evaluated the levelf heterologous protection offered by sequential vaccinations inheep using different BTV serotypes (-2, -4 or -9) against virulenturopean strains of BTV-8 and -16. These 3 inactivated vaccinesere available in 2006 when BTV-8 appeared in north of Europe. In

his study, we try to evaluate the potential protector effect (or not)f these 3 IV in a BTV-8 spread context (no IV against this serotypeas available) and also against a BTV-16 emergence.

. Materials and methods

.1. Experimental design

Thirty seven sheep were used for the study, randomly allo-ated to 7 groups of 5 or 6 and housed in group pens in biosafetyevel 3 animal facilities (Maisons-Alfort Veterinary National School,rance). All experimental protocols were reviewed by a state ethicsommission and have been approved by the competent authority.

On day (D) 0 or D21, 5 groups (G) of sheep received 1 mL each ofaccine (BTVPUR ALSAP® 2, 4, 9, Merial, France; see Table 1). Thenjections were performed subcutaneously, behind the left elbown D0 and the right elbow on D21. G4 was vaccinated only oncen D21, following the manufacturer’s recommendations for BTV-8accination in sheep (vaccine BTVPUR ALSAP® 8).

On D42, sheep were challenged, at two points subcutaneously inhe neck, with 3 mL of BTV-16 (G1 and G2) or 3 mL of BTV-8 strainG3 to G7).

Please cite this article in press as: Breard E, et al. Evaluation of adapinactivated bluetongue virus vaccines. Vaccine (2014), http://dx.doi.o

.2. Vaccines

Vaccines used (Table 1) are inactivated, monovalent or bivalentreparations, in aqueous solution, adjuvanted with saponins and

PRESSxx (2014) xxx–xxx

aluminium hydroxide (i.e., BTVPUR ALSAP® 2, 4, 9 and 8 commer-cialized by Merial).

2.3. Challenge viruses

The BTV-16 challenge virus GRE2008/11 was obtained from theOrbivirus Reference Collection at The Pirbright Institute (UK). It wasisolated in 2008 from blood of an infected Greek sheep and thenpassaged twice in KC cells (derived from C. sonorensis) [29]. Thechallenge virus was tested by RT-qPCR targeting Seg-9 (LaboratoireService International, Lissieu, France) with Cq values ranging from6.86 to 9.18.

The BTV-8 challenge virus UKG2007/77 was isolated in 2007from blood of a BTV-8 infected cow in UK and passaged three timesin KC cells [30]. The challenge virus gave Cq values ranging from6.69 to 9.65 by RT-qPCR (Laboratoire Service International, Lissieu,France).

These challenge viruses were not cell-culture-adapted to mam-malian culture cells and the determination of their respectiveinfectious virus titre was not possible.

2.4. Serological analyses

On each day of sampling (D0, D5, D21, D28, D42 and D56),sheep were blood sampled and serum collected. All serum sam-ples were analyzed using VP7 specific cELISA assays (cELISA,ID-Screen Blue Tongue Competition Kit, ID VET, France). Resultsare expressed as an inhibition-percentage (IP), as follows:IP = (ODsample/ODnegative reference) × 100. IP < 35 was considered pos-itive.

Sera from D0, D21, D42 or D56 were also titrated for specificBTV-2, -4, -8, -9 or -16 NA by serum-neutralization tests (SNT) asdescribed previously [31].

2.5. Virological analyses

Viral RNA from EDTA blood samples was extracted using theNucleoSpin RNA Virus kit (Macherey-Nagel, Germany) accordingto the manufacturer’s instructions and analyzed at the CODA-CERVA Institute (Belgium) by a “pan-BTV” multiplex RT-qPCR assay[32]. The in-house BTV-8 RT-qPCR was carried out as previouslydescribed [33]. The analytical specificities were determined for thein-house BTV-2, 4, 9 and 16 PCRs in a similar fashion (specific datafor these PCRs are available on demand). Serotyping of the firstand last (day of euthanasia) positive pan-BTV results for each ani-mal was performed using in-house BTV-2, 4, 8, 9 and 16 serotypeRT-qPCR (depending on BTV serotypes used for vaccination andchallenge).

2.6. Body temperature and clinical signs

Rectal temperatures and clinical examination of all animalswere recorded at D42 and then daily until D56. Clinical scoringwas performed based upon typical BTV clinical signs as alreadydescribed [31]. The score for each clinical sign is specified in Table 3.

2.7. Statistical analysis

tive immune responses and heterologous protection induced byrg/10.1016/j.vaccine.2014.11.053

Data were analyzed using repeated measures ANOVA, the daypost-challenge being treated as a fixed effect and the subject as arandom effect. Significance threshold was set to 0.05. Data analysiswas performed using R version 3.1.1 [34].

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Table 1Vaccination and challenge protocols used in this study.

Groups Number ofsheep/group

BTV vaccines BTV challengeD42

Protection level

D0 D21

G1 5 BTV-9 BTV-2 and BTV-4 BTV-16 ?G2 6 No vaccination No vaccination BTV-16 NoneG3 5 BTV-9 BTV-2 and BTV-4 BTV-8 ?G4 5 No vaccination BTV-8 BTV-8 CompleteG5 5 BTV-2 and BTV-4 BTV-2 and BTV-4 BTV-8 ?

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G7 6 No vaccination

. Results

.1. Serology

All sheep were seronegative before vaccination or challenge

Please cite this article in press as: Breard E, et al. Evaluation of adaptinactivated bluetongue virus vaccines. Vaccine (2014), http://dx.doi.o

or groups control (Table 2). Twenty-one days after the first vac-ination, IP were reduced in all vaccinated groups, indicatingevelopment of anti-VP7 antibodies. On D42, animals vaccinated

able 2LISA, SNT and clinical score results after vaccination (at D42; against BTV-2, 4, 8 or -9) aP: inhibition percentage; +a: positive by BTV-2 and 4 SNT; +b: positive by BTV-2 SNT; +c:itre of each serum was defined as the highest dilution (log 10) allowing neutralization of

SNT titre results by SNT against ELISA r

BTV-2 BTV-4 BTV-9 BTV-8 BTV-16 IP

Lot No. sheep D42 D42 D42 D42 D56 D56 D0

G1 137 1.2 0.9 – 1.8 114

391 0.9 – – 1.5 109

250 0.9 0.9 – 0.9 120

568 1.2 0.9 – 1.5 106

109 0.9 0.9 – 2.1 124

G2 493 0.9 115

578 0.6 114

403 1.5 120

306 2.1 110

533 1.2 117

400 1.8 121

G3 1124 0.9 – – – 1.5 107

1152 0.6 – – – Dead 124

1331 – – – – 2.1 120

1379 0.9 0.9 – – >2.4 123

1451 0.6 0.6 – – Dead 108

G4 791 0.6 1.5 107

1138 0.6 >2.4 107

1407 1.2 2.1 118

1455 0.6 1.2 108

1466 – 1.2 107

G5 205 2.1 2.1 – – 120

1243 1.2 1.5 – >2.4 107

1253 1.5 1.5 – >2.4 112

1384 1.5 0.6 – >2.4 113

1406 2.1 2.1 – >2.4 92

G6 1127 – – 1.8 103

1268 – – 2.1 103

1321 – – 2.1 93

1383 0.9 – 0.9 97

1435 – – >2.4 129

G7 936 – Dead 114

1082 – >2.4 120

1151 – >2.4 77

1205 – >2.4 117

1351 – >2.4 97

1144 – 0.9 104

TV-9 BTV-8 ?o vaccination BTV-8 None

twice (G1, G3, G5 and G6) showed a marked boost in VP7 anti-body levels and all became ELISA positive (IP < 35). Exception wasthe sheep 1451 (G3), which remained ‘doubtful’ by ELISA, with anIP = 62 at D56. In the G4 (vaccinated once), 4 ELISA negative sheephave an IP ranged from 58 to 76, suggesting a low seroconversion.

ive immune responses and heterologous protection induced byrg/10.1016/j.vaccine.2014.11.053

The challenge at D42 induced a seroconversion in all unvaccinatedanimals and an increase of antibodies levels against VP7 in vacci-nated sheep.

nd challenge (at D56; against BTV-8 (G1 and G2) or -16 (G3 to G7)) for each sheep. positive by BTV-8 SNT; +d: positive by BTV-9 SNT; +e: positive by BTV-16 SNT. NA

the 100 TCID50. (Titre < 0.6 is considered as negative).

esults

Results at Day42

Results at Day56

D21 D28 D42 D56 ELISA SNT ELISA SNT Clinicalscore/sheep

62 5 2 2 + +a + +e 2057 5 10 6 + +b + +e 2163 8 5 4 + +a + +e 178 24 20 4 + +a + +e 1365 4 5 4 + +a + +e 14

115 109 109 7 – NT + +e 29113 109 108 6 – NT + +e 51112 122 119 6 – NT + +e 34106 108 105 15 – NT + +e 29110 118 118 19 – NT + +e 47114 114 107 7 – NT + +e 62

69 8 11 4 + +b + +f 2196 23 24 Dead + +b 4475 25 24 4 + – + +f 4697 43 13 4 + +a + +f 35102 68 62 Dead – +a 39

115 60 71 7 – +c + +f 1112 47 65 5 – +c + +f 4114 19 5 4 + +c + +f 5119 43 76 8 – +c + +f 13123 72 58 12 – – + +f 3

4 4 4 Dead + +a 1536 5 4 4 + +a + +f 3310 8 5 4 + +a + +f 5733 34 16 4 + +a + +f 255 4 4 4 + +a + +f 26

47 11 16 2 + – + +f 1434 10 6 4 + – + +f 4154 46 36 4 Dbt – + +f 4021 5 4 4 + +d + +f 531 4 5 4 + – + +f 50

110 106 112 Dead – NT 36112 107 112 6 – NT + +f 3838 39 63 4 – NT + +f 65112 112 105 8 – NT + +f 30123 108 106 12 – NT + +f 50108 98 103 14 – NT + +f 4

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In the majority of vaccinated sheep, NA (titres ranging from 0.6o 2.1) against the BTV-2, -4, and -8 at D42 were detected (Table 2).owever, NA against BTV-9 was found at D42 in only 1 (no. 1383

n G6) out the 15 sheep vaccinated (once or twice) with the BTV-9accine. All challenged sheep still alive at D56 had significant levelsf NA against BTV-16 or -8 (titres ranging from 0.9 to up than 2.4).

Taken together, VP7 and/or neutralizing antibodies wereetected in 24/25 animals at D42. Only sheep no. 1466 (G4) wasLISA and NA negative. All challenged sheep, still alive at D56, hadeveloped NA against the BTV-8 or -16 challenge strains (Table 2).

.2. Detection of BTV RNA in post-challenge sheep by RT-qPCR

No BTV RNA was detected in the 37 animals prior to challengesFig. 1A and B). RT-qPCR serotyping results confirmed the presencef the challenge virus by consistently identifying the serotype cor-esponding to the challenge inoculum, but not the vaccine (dataot shown).

All BTV-16 challenged animals were viraemic at the first samp-ing post infection (D47). However, the intensity of the BTV-16iraemia in G1 was reduced (tenfold) compared to unvaccinated2, showing a significant effect (p < 0.0001) of the heterologousaccination (against BTV-9, 2 and 4) (Fig. 1A).

The average level of viraemia in the vaccinated G3 and G5 chal-enged with BTV-8 was similar to that of the unvaccinated G7. Inontrast, G6 had a reduced level of viraemia when compared with7 (p = 0.07) (Fig. 1B). This was due to 2 animals (no.1127 and 1383)

hat developed no viraemia. The three other animals of G6 had airaemia that was similar to that of G7. The BTV-8 vaccination (G4)locked viraemia in 4/5 animals, post BTV-8 challenge. Only onenimal in G4 (no. 1138) developed a low viraemia (Cq values ran-ing from 31.7 (D47) to 37.4 (D56)). In the unvaccinated G7, 5/6heep had a strong viraemia after BTV-8 challenge, although oneheep (no. 1144) showed no viraemia until D56 with a Cq value of4.5 from the blood sample.

.3. Clinical signs and body temperature

The clinical signs most frequently observed during BTV-16nfection (G2) were: congestion of skin, conjunctivitis, respiratoryifficulties (slight nasal discharge), fever (40–41 ◦C) and locomo-

Please cite this article in press as: Breard E, et al. Evaluation of adapinactivated bluetongue virus vaccines. Vaccine (2014), http://dx.doi.o

ion impairment (stiffness) (Table 3). In heterologous vaccinated1, conjunctivitis, slight nasal discharge and fever (40–41 ◦C) werelso observed but with a low frequency, while congestion of the skinnd locomotion impairment were only observed in one animal. The

Days

fter challenge with BTV-16 (A) or BTV-8 (B).

total clinical score per animal at D56 was reduced (p = 0.02) in G1(17), compared with the unvaccinated G2 (41.8).

In the BTV-8 challenged groups, two sheep in G3, one in G5 andin G7 were euthanized for ethical reasons between D51 and 53, dueto the severity of disease. They showed severe oedema, depressedbehaviour and several days of fever (>40 ◦C). All sheep in G4 onlyshowed conjunctivitis, with individual clinical scores significantlylower (p < 0.0001) than the other unvaccinated or heterologous vac-cinated groups (Tables 2 and 3). The individual clinical score in G7was the highest (37.2) but was not statistically different from indi-vidual scores in the heterologous-vaccinated sheep that were alsochallenged with BTV-8.

The kinetics of clinical signs (Fig. 2A) show a significantreduction in clinical scores and partial protection against BTV-16challenge in G1 when compared with G2, which can be attributed toheterologous-vaccinations against BTV-9, 2 and 4. Only the sheepin G4 (vaccinated against BTV-8) showed a protection against chal-lenge (Fig. 2B). The other vaccinated groups showed similar clinicalpatterns to G7. The mean of the individual clinical scores in G6 wasslightly lower (30) than in G7 (37.2), although this is due to the 2animals without viraemia which had low individual clinical scores(Table 2).

Interestingly, the duration and intensity of fever reported for allanimals challenged with BTV-16 (Fig. 3A) and BTV-8 (Fig. 3B) weremostly similar, except for G4.

4. Discussion

After the different vaccination protocols used and before chal-lenge infection, the serological data (ELISA and SNT) demonstratedthat 24/25 sheep developed an adaptive immune response againstIV detectable by ELISA or/and SNT. Sheep no. 1466 (in G4; ELISA andNA negative at D42) was also protected against the homologouschallenge. Following challenge, 33 sheep developed both neutral-izing and VP7 antibodies. Four sheep died due to the severity of theclinical signs induced after the BTV-8 challenge.

Antibodies levels of the sheep vaccinated once showed a lowanti-VP7 antibody response, as previously observed after primovaccination of ruminants with IV [28,30]. However, it has beenshown (experimentally and in the field) that vaccinated sheep, withlow or without NA, can be effectively protected against homolo-gous challenges after a single IV dose, [7–11,14]. The sheep in G4

tive immune responses and heterologous protection induced byrg/10.1016/j.vaccine.2014.11.053

vaccinated once with the BTV-8 IV confirmed these observations,and although one animal showed a low viraemia after challenge, nofever or clinical signs were observed (although some conjunctiviswere attributable to the fact that these animals were usually kept

Please cite this article in press as: Breard E, et al. Evaluation of adaptive immune responses and heterologous protection induced byinactivated bluetongue virus vaccines. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.11.053

ARTICLE IN PRESSG ModelJVAC-15966; No. of Pages 7

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Table 3Mean of clinical score per sign and per sheep for each group after BTV-16 (G1 and G2) or BTV-8 (G3 to G7) challenges.

Score/sign Mean of score/sign/sheep/group

G1 G2 G3 G4 G5 G6 G7

Behaviuor Apathetic 1 0.7 0.2 0.2Depressed 2 0.8 0.4Prostated 3

Oedema Submandibular 1 0.2Face 1 0.2 0.2Nose 1 0.6 0.8

Congestion Skin 4 1.6 12.7 15.2 0.8 15.2 10.4 13.3

Locomotion Lameness 1 1.0 0.8 0.8 0.4 1.3Stiffness 2 0.4 2.7

Respiratory Slight nasal discharge 1 3.4 5.8 4.2 1 3.6 5.8 4.8Important nasal discharge 2 0.7 2.8 0.8 0.4 2.3Coughing 1 0.2 0.2 1 0.4 0.5

Digestive Diarrhea 1 0.4

Other Conjonctivite 1 6.6 12.3 5.6 2.8 5.4 6.8 6.7Ulcers 1 1.2 0.2 0.4 0.2 0.6 1.0Hypersalivation 1 0.3 0.6 0.7

Fever 40 to 41 1 2.8 4.2 2.8 0.4 3.6 1.6 2.041 to 42 2 0.4 1.0 2.8 1.2 2.8 2.7>42 4 0.7

Mean of score/sheep/group 17 41.8 37 5.2 31.2 30 37.2

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ARTICLEVAC-15966; No. of Pages 7

E. Breard et al. / Va

n free range and that they were during this study in rooms in level containment conditions).

The BTV-8 and 16 strains used as virulent inocula were fieldtrains with low passages in KC cells. These strains efficientlynduced severe clinical signs in the unvaccinated groups, con-rming that low passages BTV strains grown in Culicoides cellultures are more virulent than a mammalian cell-culture derivedTV-8 strain [30].

Although BTV-16 did not kill the challenged sheep, the meanlinical score (41.8) for the unvaccinated sheep (G2), was similaro that observed in the unvaccinated G7 challenged with BTV-

(37.2). The sheep that received heterologous vaccinations (G1)learly showed reduced viraemia and clinical scores caused by BTV-6 (Fig. 1A and Tables 2 and 3) when compared with G2, and specificlinical signs were either not observed (congestion of skin and stiff-ess) or were highly reduced (nasal discharge or conjunctivis). Evenhough the ‘curve’ for body temperature (Fig. 3A) in G1 was con-istently below the G2 curve, there was no difference in terms ofntensity or length of pyrexia. Taken together, these data suggesthat IV against serotype 9, 2 and 4 induced a partial protectiongainst challenge with BTV-16, as seen in the clinical and virologicalata and particularly by a reduction in some of the clinical signs.

Group 3, also vaccinated against serotypes 9, 2 and 4, washallenged with BTV-8. The viraemia and the clinical scores wereimilar to those observed in the unvaccinated G7. Moreover, 2/5heep from G3 died after BTV-8 challenge. The partial protectionbserved in G1 (against BTV-16) is clearly not seen in G3 (againstTV-8), although the vaccination protocols were identical. This

ndicates that even partial protection against heterologous chal-enge is highly dependent on the challenge serotype. Furthermore,his partial protection is not due to a cross protection against phy-ogenetically related serotypes [25] as serotypes 8 and 16 have aery weak interrelationship with serotypes 2, 4 and 9 [27,35].

Two of the 5 sheep in G6 (no. 1127 and 1383), vaccinated twicegainst BTV-9, showed no viraemia after challenge with BTV-8.owever, NA against the BTV-8 challenge were detected in bothnimals, demonstrating that they were properly challenged at D42.he average clinical score and viraemia were slightly (not statisti-ally significant) reduced in this group when compared with G7, ashe response against BTV-8 was heterologous in G6. In this group,he heterologous vaccination seemed to induce a complete protec-ion for 2 of the 5 sheep, while the other three did not show evenartial protection. Individual protection therefore seemed to be anall or nothing” effect.

One of the six unvaccinated sheep in G7 showed no viraemiantil day 14 after the BTV-8 challenge. This animal developed noever and no clinical signs (clinical score = 5) but seroconverted (byLISA and SNT) with similar kinetics for antibody development tohe other five animals in the group, demonstrating that the inocu-um had been correctly administrated at D42. To our knowledge,o pre-existing condition or remarkable events had affected thisxperimental result.

Taken together, our results suggest 3 different scenarios afterulti-type vaccinations and heterologous challenge. The first: par-

ial protection observed in vaccinated animals challenged with virulent heterologous BTV serotype, with a reduction of theiraemia and a decrease or disappearance of some clinical signs.n this case, a protective effect was not observed on body tem-erature, showing that fever is not a predictable clinical sign of

protective effect. During BTV spread, one of the requirementsor virus transmission is a sufficiently high viraemia in the hostor infection of feeding insect vectors [36,37]. The reduction of the

Please cite this article in press as: Breard E, et al. Evaluation of adapinactivated bluetongue virus vaccines. Vaccine (2014), http://dx.doi.o

iraemia induced by heterologous IV in sheep is an important factoro help control an outbreak and spread. The second: no protectionas observed (G3 and G5 challenged against BTV-8) showing that

he same heterologous vaccination protocol can generate different

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levels of protection against different challenge serotypes (G1 andG3). The third scenario is illustrated by the heterogeneous responseagainst BTV-8 in sheep vaccinated twice against BTV-9 with anindividual “all or nothing” protection effect.

This study shows that it is difficult to predict if par-tial or complete heterologous protection will be achieved postinactivated-BTV vaccination. This may reflect different levels ofcell-mediated immunity that may be generated by IV against het-erologous and phylogenetically unrelated BTV serotypes as wellas unpredictable natural resistance against BTV disease in sheep[17,19,38–41].

Acknowledgements

We thank for their financial support the European Orbivac GrantAgreement Project (no.: 245266) coordinated by Prof. Polly Roy(London School of Hygiene and Tropical Medicine).

®: BTVPUR ALSAP is a registered trademark of Merial in theEuropean Union and elsewhere.

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