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Vaccine journal homepage: www.elsevier.com/locate/vaccine
Evaluation of adaptive immune responses and heterologous protection induced by inactivated bluetongue virus vaccines
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Emmanuel Breard a,∗,1 , Guillaume Belbis b,1 , Cyril Viarouge a , Kyriaki Nomikou c , Andy Haegeman d , Kris De Clercq d , Pascal Hudelet e , Claude Hamers f , Francis Moreau g , Thomas Lilin g , Benoit Durand h , Peter Mertens c , Damien Vitour a , Corinne Sailleau a , Stéphan Zientara a a
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ANSES, UMR 1161 Virologie ANSES-INRA-ENVA, 23 avenue du Général de Gaulle, 94704 Maisons-Alfort, France Université Paris-Est, Ecole Nationale Vétérinaire d’Alfort, Unité de Pathologie du Bétail, 7 avenue du Général de Gaulle, 94704 Maisons-Alfort, France c Vector-Borne Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey GU24 0NF, United Kingdom d CODA–CERVA, Department of Virology, Ukkel, Belgium e MERIAL S.A.S., 254 Rue Marcel Mérieux, 69007 Lyon, France f MERIAL S.A.S., P.I. Plaine de l’Ain, Allée des Cyprès, 01150 Saint-Vulbas, France g Université Paris-Est, Ecole Nationale Vétérinaire d’Alfort, Centre de recherche biomédicale, 7 avenue du Général de Gaulle, 94704 Maisons-Alfort, France h ANSES, unité Epidémiologie, 23 avenue du Général de Gaulle, 94704 Maisons-Alfort, France b
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Article history: Received 22 October 2013 Received in revised form 20 November 2014 Accepted 28 November 2014 Available online xxx
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Keywords: Bluetongue virus disease Inactivated vaccines Experimental infection Heterologous challenges Sheep
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1. Introduction
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Eradication of bluetongue virus is possible, as has been shown in several European countries. New serotypes have emerged, however, for which there are no specific commercial vaccines. This study addressed whether heterologous vaccines would help protect against 2 serotypes. Thirty-seven sheep were randomly allocated to 7 groups of 5 or 6 animals. Four groups were vaccinated with commercial vaccines 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 of either BTV-16 (2 groups) or BTV-8 (5 groups). Taken together, 24/25 sheep from the 4 experimental groups developed detectable antibodies against the vaccinated viruses. Furthermore, sheep that received heterologous vaccines showed significantly reduced viraemia and clinical scores for BTV-16 when compared to unvaccinated controls. Reductions in clinical signs and viraemia among heterologously vaccinated sheep were not as common after challenge with BTV-8. This study shows that heterologous protection can occur, but that it is difficult to predict if partial or complete protection will be achieved following inactivated-BTV vaccination. © 2014 Published by Elsevier Ltd.
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Bluetongue (BT) is an infectious, OIE-listed arboviral disease that infects ruminants [1]. The disease is caused by viruses belonging to Bluetongue virus species [2]. There are 26 bluetongue virus (BTV) serotypes, which raise type specific neutralising antibodies (NA) during infection of their hosts [3]. BTV is transmitted primarily by biting midges (Culicoides spp.) but can be transmitted vertically or via an oral route [4–6]. Since the 20th century, 10 serotypes
∗ Corresponding author. Tel.: +33 695063201; fax: +33 143689762. E-mail address: [email protected] (E. Breard). 1 Both authors contributed equally.
have been detected in Europe, some of them on several different occasions [7]. After the emergence of BTV in Europe (1998), the first successful inactivated vaccine (IV) protected against BTV-2 [7–11]. Subsequently, monovalent and bivalent IV were developed, used in the field [9,10] and efficient for eradication of different homologous BTV serotypes [12–14]. Since 2011, several countries that were previously infected with serotype 1, 2, 4 or 8 returned to freedom-from-disease after mass vaccination campaigns [7,11]. Animals vaccinated with these IV develop protective immune responses against the homologous serotype(s). The BTV outercapsid proteins VP2 and VP5 (particularly VP2) are the BTV proteins inducing NA [15]; VP2 is also the major protein involved in serotype determination [16,17]. Sheep inoculated with VP2 alone also
http://dx.doi.org/10.1016/j.vaccine.2014.11.053 0264-410X/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: Breard E, et al. Evaluation of adaptive immune responses and heterologous protection induced by inactivated bluetongue virus vaccines. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.11.053
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produced NA and were protected against challenge with homologous BTV serotype [18,19]. A vaccine that protects against multiple BTV types would provide a valuable defence against incursions of new BTV serotypes. However, such vaccines seem difficult to be create, despite evidence that cytotoxic T cells play a role in protection against BTV infection [20–22]. Few studies report complete or even partial protection after immunization and heterologous challenge. However, a significant level of cross-protection against a BTV challenge was obtained after immunization with a single BTV protein (VP7) without NA response against virus particles [23]. Studies show that inactivated or virus-like particle vaccines can reduce the severity of heterologous BTV infections, even though the BTV serotypes used were not closely related [24,25]. One study using a recombinant vaccine against BTV-4 in IFNAR(−/−) knockout mice, showed effective cross-protection against heterologous lethal challenges [26]. Little is known about the mechanism(s) of protection after pan- or multi-serotype vaccinations and heterologous challenge. Earlier studies of sheep serially infected with BTV-3 and 4 demonstrated resistances to challenge with BTV-6 [21]. However, the level of protection induced in sheep against heterologous serotypes by simultaneous multivalent vaccination with IV has not been reported. The nucleotype grouping for BTV segment 2 illustrates the ‘relatedness’ of different strains [27]. Different serotypes within a single BTV nucleotype are more likely to generate crossreactive responses to other viruses within the same nucleotype [27]. The possibility that animals can be protected and to restrict the emergence of new BTV serotypes in ‘free’ regions, using currently available IV in a polyvalent vaccination strategy, needs to be further assessed. The only IV registered within Europe are for serotypes 1, 2, 4, 8, and 9. No vaccine against BTV-16 is available in the market even if a study demonstrated that two doses of BTV-16 IV protect sheep [28]. Those would be the immediate solution in the case of an outbreak of an exotic serotype. In this study we evaluated the level of heterologous protection offered by sequential vaccinations in sheep using different BTV serotypes (-2, -4 or -9) against virulent European strains of BTV-8 and -16. These 3 inactivated vaccines were available in 2006 when BTV-8 appeared in north of Europe. In this study, we try to evaluate the potential protector effect (or not) of these 3 IV in a BTV-8 spread context (no IV against this serotype was available) and also against a BTV-16 emergence.
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2. Materials and methods
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2.1. Experimental design
54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94
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Thirty seven sheep were used for the study, randomly allocated to 7 groups of 5 or 6 and housed in group pens in biosafety level 3 animal facilities (Maisons-Alfort Veterinary National School, France). All experimental protocols were reviewed by a state ethics commission and have been approved by the competent authority. On day (D) 0 or D21, 5 groups (G) of sheep received 1 mL each of vaccine (BTVPUR ALSAP® 2, 4, 9, Merial, France; see Table 1). The injections were performed subcutaneously, behind the left elbow on D0 and the right elbow on D21. G4 was vaccinated only once on D21, following the manufacturer’s recommendations for BTV-8 vaccination in sheep (vaccine BTVPUR ALSAP® 8). On D42, sheep were challenged, at two points subcutaneously in the neck, with 3 mL of BTV-16 (G1 and G2) or 3 mL of BTV-8 strain (G3 to G7).
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2.2. Vaccines
98 99 100 101 102 103 104 105 106 107 108 109 110
113 114
Vaccines used (Table 1) are inactivated, monovalent or bivalent preparations, in aqueous solution, adjuvanted with saponins and
aluminium hydroxide (i.e., BTVPUR ALSAP® 2, 4, 9 and 8 commercialized by Merial). 2.3. Challenge viruses The BTV-16 challenge virus GRE2008/11 was obtained from the Orbivirus Reference Collection at The Pirbright Institute (UK). It was isolated in 2008 from blood of an infected Greek sheep and then passaged twice in KC cells (derived from C. sonorensis) [29]. The challenge virus was tested by RT-qPCR targeting Seg-9 (Laboratoire Service International, Lissieu, France) with Cq values ranging from 6.86 to 9.18. The BTV-8 challenge virus UKG2007/77 was isolated in 2007 from blood of a BTV-8 infected cow in UK and passaged three times in KC cells [30]. The challenge virus gave Cq values ranging from 6.69 to 9.65 by RT-qPCR (Laboratoire Service International, Lissieu, France). These challenge viruses were not cell-culture-adapted to mammalian culture cells and the determination of their respective infectious 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 samples were analyzed using VP7 specific cELISA assays (cELISA, ID-Screen Blue Tongue Competition Kit, ID VET, France). Results are expressed as an inhibition-percentage (IP), as follows: IP = (ODsample /ODnegative reference ) × 100. IP < 35 was considered positive. Sera from D0, D21, D42 or D56 were also titrated for specific BTV-2, -4, -8, -9 or -16 NA by serum-neutralization tests (SNT) as described previously [31].
2.5. Virological analyses Viral RNA from EDTA blood samples was extracted using the NucleoSpin RNA Virus kit (Macherey-Nagel, Germany) according to the manufacturer’s instructions and analyzed at the CODACERVA Institute (Belgium) by a “pan-BTV” multiplex RT-qPCR assay [32]. The in-house BTV-8 RT-qPCR was carried out as previously described [33]. The analytical specificities were determined for the in-house BTV-2, 4, 9 and 16 PCRs in a similar fashion (specific data for these PCRs are available on demand). Serotyping of the first and last (day of euthanasia) positive pan-BTV results for each animal was performed using in-house BTV-2, 4, 8, 9 and 16 serotype RT-qPCR (depending on BTV serotypes used for vaccination and challenge).
2.6. Body temperature and clinical signs Rectal temperatures and clinical examination of all animals were recorded at D42 and then daily until D56. Clinical scoring was performed based upon typical BTV clinical signs as already described [31]. The score for each clinical sign is specified in Table 3.
2.7. Statistical analysis Data were analyzed using repeated measures ANOVA, the day post-challenge being treated as a fixed effect and the subject as a random effect. Significance threshold was set to 0.05. Data analysis was performed using R version 3.1.1 [34].
Please cite this article in press as: Breard E, et al. Evaluation of adaptive immune responses and heterologous protection induced by inactivated bluetongue virus vaccines. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.11.053
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117
118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
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Table 1 Vaccination and challenge protocols used in this study. Groups
G1 G2 G3 G4 G5 G6 G7
3. Results
168
3.1. Serology
170 171 172
BTV vaccines
5 6 5 5 5 5 6
167
169
Number of sheep/group
D0
D21
BTV-9 No vaccination BTV-9 No vaccination BTV-2 and BTV-4 BTV-9 No vaccination
BTV-2 and BTV-4 No vaccination BTV-2 and BTV-4 BTV-8 BTV-2 and BTV-4 BTV-9 No vaccination
All sheep were seronegative before vaccination or challenge for groups control (Table 2). Twenty-one days after the first vaccination, IP were reduced in all vaccinated groups, indicating development of anti-VP7 antibodies. On D42, animals vaccinated
BTV challenge D42
Protection level
BTV-16 BTV-16 BTV-8 BTV-8 BTV-8 BTV-8 BTV-8
? None ? Complete ? ? None
twice (G1, G3, G5 and G6) showed a marked boost in VP7 antibody levels and all became ELISA positive (IP < 35). Exception was the sheep 1451 (G3), which remained ‘doubtful’ by ELISA, with an IP = 62 at D56. In the G4 (vaccinated once), 4 ELISA negative sheep have an IP ranged from 58 to 76, suggesting a low seroconversion. The challenge at D42 induced a seroconversion in all unvaccinated animals and an increase of antibodies levels against VP7 in vaccinated sheep.
Table 2 ELISA, SNT and clinical score results after vaccination (at D42; against BTV-2, 4, 8 or -9) and challenge (at D56; against BTV-8 (G1 and G2) or -16 (G3 to G7)) for each sheep. IP: inhibition percentage; +a: positive by BTV-2 and 4 SNT; +b: positive by BTV-2 SNT; +c: positive by BTV-8 SNT; +d: positive by BTV-9 SNT; +e: positive by BTV-16 SNT. NA titre of each serum was defined as the highest dilution (log 10) allowing neutralization of the 100 TCID50. (Titre < 0.6 is considered as negative). SNT titre results by SNT against BTV-2
BTV-4
BTV-9
BTV-8
D42
Lot
No. sheep
D42
D42
D42
G1
137 391 250 568 109
1.2 0.9 0.9 1.2 0.9
0.9 – 0.9 0.9 0.9
– – – – –
G2
493 578 403 306 533 400
G3
1124 1152 1331 1379 1451
G4
791 1138 1407 1455 1466
G5
205 1243 1253 1384 1406
G6
1127 1268 1321 1383 1435
G7
936 1082 1151 1205 1351 1144
0.9 0.6 – 0.9 0.6
2.1 1.2 1.5 1.5 2.1
– – – 0.9 0.6
– – – – –
2.1 1.5 1.5 0.6 2.1 – – – 0.9 –
ELISA results
D56
BTV-16
IP
Results at Day 42
Results at Day 56
D56
D0
D21
D28
D42
D56
ELISA
SNT
ELISA
SNT
Clinical score/sheep
1.8 1.5 0.9 1.5 2.1
114 109 120 106 124
62 57 63 8 65
5 5 8 24 4
2 10 5 20 5
2 6 4 4 4
+ + + + +
+a +b +a +a +a
+ + + + +
+e +e +e +e +e
20 21 17 13 14
0.9 0.6 1.5 2.1 1.2 1.8
115 114 120 110 117 121
115 113 112 106 110 114
109 109 122 108 118 114
109 108 119 105 118 107
7 6 6 15 19 7
– – – – – –
NT NT NT NT NT NT
+ + + + + +
+e +e +e +e +e +e
29 51 34 29 47 62 21 44 46 35 39
– – – – –
1.5 Dead 2.1 >2.4 Dead
107 124 120 123 108
69 96 75 97 102
8 23 25 43 68
11 24 24 13 62
4 Dead 4 4 Dead
+ + + + –
+b +b – +a +a
+
+f
+ +
+f +f
0.6 0.6 1.2 0.6 –
1.5 >2.4 2.1 1.2 1.2
107 107 118 108 107
115 112 114 119 123
60 47 19 43 72
71 65 5 76 58
7 5 4 8 12
– – + – –
+c +c +c +c –
+ + + + +
+f +f +f +f +f
1 4 5 13 3
– – – – –
– >2.4 >2.4 >2.4 >2.4
120 107 112 113 92
4 36 10 33 5
4 5 8 34 4
4 4 5 16 4
Dead 4 4 4 4
+ + + + +
+a +a +a +a +a
+ + + +
+f +f +f +f
15 33 57 25 26
– – – – –
1.8 2.1 2.1 0.9 >2.4
103 103 93 97 129
47 34 54 21 31
11 10 46 5 4
16 6 36 4 5
2 4 4 4 4
+ + Dbt + +
– – – +d –
+ + + + +
+f +f +f +f +f
14 41 40 5 50
– – – – – –
Dead >2.4 >2.4 >2.4 >2.4 0.9
114 120 77 117 97 104
110 112 38 112 123 108
106 107 39 112 108 98
112 112 63 105 106 103
Dead 6 4 8 12 14
– – – – – –
NT NT NT NT NT NT
+ + + + +
+f +f +f +f +f
36 38 65 30 50 4
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B
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G1
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G2
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Cq value means/group
Cq value means/group
A 37
21
37
33
G3 G4
29
G5 G6 G7
25
21 D42
D47
D49
D51
D54
D56
D42
Days Q5
181 182 183 184 185 186 187 188 189 190
191
192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213
214
215 216 217 218 219 220 221
D47
D49
D51
D54
D56
Days Fig. 1.
In the majority of vaccinated sheep, NA (titres ranging from 0.6 to 2.1) against the BTV-2, -4, and -8 at D42 were detected (Table 2). However, NA against BTV-9 was found at D42 in only 1 (no. 1383 in G6) out the 15 sheep vaccinated (once or twice) with the BTV-9 vaccine. All challenged sheep still alive at D56 had significant levels of NA against BTV-16 or -8 (titres ranging from 0.9 to up than 2.4). Taken together, VP7 and/or neutralizing antibodies were detected in 24/25 animals at D42. Only sheep no. 1466 (G4) was ELISA and NA negative. All challenged sheep, still alive at D56, had developed NA against the BTV-8 or -16 challenge strains (Table 2). 3.2. Detection of BTV RNA in post-challenge sheep by RT-qPCR No BTV RNA was detected in the 37 animals prior to challenges (Fig. 1A and B). RT-qPCR serotyping results confirmed the presence of the challenge virus by consistently identifying the serotype corresponding to the challenge inoculum, but not the vaccine (data not shown). All BTV-16 challenged animals were viraemic at the first sampling post infection (D47). However, the intensity of the BTV-16 viraemia in G1 was reduced (tenfold) compared to unvaccinated G2, showing a significant effect (p < 0.0001) of the heterologous vaccination (against BTV-9, 2 and 4) (Fig. 1A). The average level of viraemia in the vaccinated G3 and G5 challenged with BTV-8 was similar to that of the unvaccinated G7. In contrast, G6 had a reduced level of viraemia when compared with G7 (p = 0.07) (Fig. 1B). This was due to 2 animals (no.1127 and 1383) that developed no viraemia. The three other animals of G6 had a viraemia that was similar to that of G7. The BTV-8 vaccination (G4) blocked viraemia in 4/5 animals, post BTV-8 challenge. Only one animal in G4 (no. 1138) developed a low viraemia (Cq values ranging from 31.7 (D47) to 37.4 (D56)). In the unvaccinated G7, 5/6 sheep had a strong viraemia after BTV-8 challenge, although one sheep (no. 1144) showed no viraemia until D56 with a Cq value of 34.5 from the blood sample. 3.3. Clinical signs and body temperature The clinical signs most frequently observed during BTV-16 infection (G2) were: congestion of skin, conjunctivitis, respiratory difficulties (slight nasal discharge), fever (40–41 ◦ C) and locomotion impairment (stiffness) (Table 3). In heterologous vaccinated G1, conjunctivitis, slight nasal discharge and fever (40–41 ◦ C) were also observed but with a low frequency, while congestion of the skin and locomotion impairment were only observed in one animal. The
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 and in G7 were euthanized for ethical reasons between D51 and 53, due to the severity of disease. They showed severe oedema, depressed behaviour and several days of fever (>40 ◦ C). All sheep in G4 only showed conjunctivitis, with individual clinical scores significantly lower (p < 0.0001) than the other unvaccinated or heterologous vaccinated groups (Tables 2 and 3). The individual clinical score in G7 was the highest (37.2) but was not statistically different from individual scores in the heterologous-vaccinated sheep that were also challenged with BTV-8. The kinetics of clinical signs (Fig. 2A) show a significant reduction in clinical scores and partial protection against BTV-16 challenge in G1 when compared with G2, which can be attributed to heterologous-vaccinations against BTV-9, 2 and 4. Only the sheep in G4 (vaccinated against BTV-8) showed a protection against challenge (Fig. 2B). The other vaccinated groups showed similar clinical patterns to G7. The mean of the individual clinical scores in G6 was slightly lower (30) than in G7 (37.2), although this is due to the 2 animals without viraemia which had low individual clinical scores (Table 2). Interestingly, the duration and intensity of fever reported for all animals challenged with BTV-16 (Fig. 3A) and BTV-8 (Fig. 3B) were mostly similar, except for G4. 4. Discussion After the different vaccination protocols used and before challenge infection, the serological data (ELISA and SNT) demonstrated that 24/25 sheep developed an adaptive immune response against IV detectable by ELISA or/and SNT. Sheep no. 1466 (in G4; ELISA and NA negative at D42) was also protected against the homologous challenge. Following challenge, 33 sheep developed both neutralizing and VP7 antibodies. Four sheep died due to the severity of the clinical signs induced after the BTV-8 challenge. Antibodies levels of the sheep vaccinated once showed a low anti-VP7 antibody response, as previously observed after primo vaccination of ruminants with IV [28,30]. However, it has been shown (experimentally and in the field) that vaccinated sheep, with low or without NA, can be effectively protected against homologous challenges after a single IV dose, [7–11,14]. The sheep in G4 vaccinated once with the BTV-8 IV confirmed these observations, and although one animal showed a low viraemia after challenge, no fever or clinical signs were observed (although some conjunctivis were 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 by inactivated bluetongue virus vaccines. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.11.053
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Table 3 Mean 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
Apathetic Depressed Prostated
1 2 3
0.7
Submandibular Face Nose
1 1 1
0.2
Congestion
Skin
4
Locomotion
Lameness Stiffness
1 2
Respiratory
Slight nasal discharge Important nasal discharge Coughing
1 2 1
3.4
Digestive
Diarrhea
1
0.4
Other
Conjonctivite Ulcers Hypersalivation
1 1 1
Fever
40 to 41 41 to 42 >42
1 2 4
Behaviuor
Oedema
G3
G4
G5
G6
G7
0.2 0.4
0.8
0.2
0.2
0.2 0.8
0.6 1.6
12.7
15.2 0.8
0.4
1.0 2.7
15.2
10.4
0.8
0.4
13.3 1.3
5.8 0.7 0.2
4.2 2.8 1
1
3.6 0.8
5.8 0.4 0.4
4.8 2.3 0.5
6.6 1.2
12.3 0.2 0.3
5.6 0.4
2.8 0.2
5.4
6.8 0.6 0.6
6.7 1.0 0.7
2.8 0.4
4.2 1.0
0.4
3.6 1.2
1.6 2.8
2.0 2.7 0.7
17
41.8
5.2
31.2
30
37.2
0.2
Mean of score/sheep/group
0.8
2.8 2.8 37
7,0
A
4,0
3,0
G1 G2
2,0
1,0
0,0 D41
D44
D47
D50
D53
B
6,0
*
clinical score means/group
clinical score means /group
5,0
5,0
G3 G4 G5 G6 G7
4,0 3,0 2,0 1,0 0,0 D41
D56
D44
D47
D50
D53
D56
Days
Days
A
40,5
40,0
G1 G2
39,5
39,0
Means of temperature/group
temperature means /group
Fig. 2.
38,5 D42
D45
D48
D51
41,0
B
40,5
G3 G4 G5 G6 G7
40,0
39,5
39,0
38,5
D54
D42
Days
D45
D48
D51
D54
Days Fig. 3.
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in free range and that they were during this study in rooms in level 3 containment conditions). The BTV-8 and 16 strains used as virulent inocula were field strains with low passages in KC cells. These strains efficiently induced severe clinical signs in the unvaccinated groups, confirming that low passages BTV strains grown in Culicoides cell cultures are more virulent than a mammalian cell-culture derived BTV-8 strain [30]. Although BTV-16 did not kill the challenged sheep, the mean clinical score (41.8) for the unvaccinated sheep (G2), was similar to that observed in the unvaccinated G7 challenged with BTV8 (37.2). The sheep that received heterologous vaccinations (G1) clearly showed reduced viraemia and clinical scores caused by BTV16 (Fig. 1A and Tables 2 and 3) when compared with G2, and specific clinical signs were either not observed (congestion of skin and stiffness) or were highly reduced (nasal discharge or conjunctivis). Even though the ‘curve’ for body temperature (Fig. 3A) in G1 was consistently below the G2 curve, there was no difference in terms of intensity or length of pyrexia. Taken together, these data suggest that IV against serotype 9, 2 and 4 induced a partial protection against challenge with BTV-16, as seen in the clinical and virological data and particularly by a reduction in some of the clinical signs. Group 3, also vaccinated against serotypes 9, 2 and 4, was challenged with BTV-8. The viraemia and the clinical scores were similar to those observed in the unvaccinated G7. Moreover, 2/5 sheep from G3 died after BTV-8 challenge. The partial protection observed in G1 (against BTV-16) is clearly not seen in G3 (against BTV-8), although the vaccination protocols were identical. This indicates that even partial protection against heterologous challenge is highly dependent on the challenge serotype. Furthermore, this partial protection is not due to a cross protection against phylogenetically related serotypes [25] as serotypes 8 and 16 have a very weak interrelationship with serotypes 2, 4 and 9 [27,35]. Two of the 5 sheep in G6 (no. 1127 and 1383), vaccinated twice against BTV-9, showed no viraemia after challenge with BTV-8. However, NA against the BTV-8 challenge were detected in both animals, demonstrating that they were properly challenged at D42. The average clinical score and viraemia were slightly (not statistically significant) reduced in this group when compared with G7, as the response against BTV-8 was heterologous in G6. In this group, the heterologous vaccination seemed to induce a complete protection for 2 of the 5 sheep, while the other three did not show even partial protection. Individual protection therefore seemed to be an “all or nothing” effect. One of the six unvaccinated sheep in G7 showed no viraemia until day 14 after the BTV-8 challenge. This animal developed no fever and no clinical signs (clinical score = 5) but seroconverted (by ELISA and SNT) with similar kinetics for antibody development to the other five animals in the group, demonstrating that the inoculum had been correctly administrated at D42. To our knowledge, no pre-existing condition or remarkable events had affected this experimental result. Taken together, our results suggest 3 different scenarios after multi-type vaccinations and heterologous challenge. The first: partial protection observed in vaccinated animals challenged with a virulent heterologous BTV serotype, with a reduction of the viraemia and a decrease or disappearance of some clinical signs. In this case, a protective effect was not observed on body temperature, showing that fever is not a predictable clinical sign of a protective effect. During BTV spread, one of the requirements for virus transmission is a sufficiently high viraemia in the host for infection of feeding insect vectors [36,37]. The reduction of the viraemia induced by heterologous IV in sheep is an important factor to help control an outbreak and spread. The second: no protection was observed (G3 and G5 challenged against BTV-8) showing that the same heterologous vaccination protocol can generate different
levels of protection against different challenge serotypes (G1 and G3). The third scenario is illustrated by the heterogeneous response against BTV-8 in sheep vaccinated twice against BTV-9 with an individual “all or nothing” protection effect. This study shows that it is difficult to predict if partial or complete heterologous protection will be achieved post inactivated-BTV vaccination. This may reflect different levels of cell-mediated immunity that may be generated by IV against heterologous and phylogenetically unrelated BTV serotypes as well as unpredictable natural resistance against BTV disease in sheep [17,19,38–41]. Acknowledgements We thank for their financial support the European Orbivac Grant Q4 Agreement Project (no.: 245266) coordinated by Prof. Polly Roy (London School of Hygiene and Tropical Medicine). ®: BTVPUR ALSAP is a registered trademark of Merial in the European Union and elsewhere. References [1] Maclachlan NJ, Drew CP, Darpel KE, Worwa G. The pathology and pathogenesis of bluetongue. J Comp Pathol 2009;141:1–16. [2] Mertens PPC, Maan S, Samuel A, Attoui H. Orbivirus reoviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, editors. Virus taxonomy VIIIth report of the ICTV. London: Elsevier/Academic Press; 2005. p. 466–83. [3] Maan S, Maan NS, Nomikou K, Batten C, Antony F, Belaganahalli MN, et al. Novel bluetongue virus serotype from Kuwait. Emerg Infect Dis 2011;17:886–9. [4] Darpel KE, Batten CA, Veronesi E, Williamson S, Anderson P, Dennison M, et al. Transplacental transmission of bluetongue virus 8 in cattle UK. Emerg Infect Dis 2009;15:2025–8. [5] De Clerq K, Vandenbussche F, Vandemeulebroucke E, Vanbinst T, De Leeuw I, Verheyden B, et al. Transplacental bluetongue infection in cattle. Vet Rec 2008;162:64. [6] Backx A, Heutink R, van Rooij E, van Rijn P. Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection. Vet Microbiol 2009;138:235–43. [7] Zientara S, Sánchez-Vizcaíno JM. Control of bluetongue in Europe. Vet Microbiol 2013;165:33–7. [8] Di Emidio B, Nicolussi P, Patta C, Ronchi GF, Monaco F, Savini G, et al. Efficacy and safety studies on an inactivated vaccine against bluetongue virus serotype 2. Vet Ital 2004;40:640–4. [9] Savini G, Monaco F, Citarella R, Calzetta G, Panichi G, Ruiu A, Caporale V. Monovalent modified-live vaccine against bluetongue virus serotype 2: immunity studies in cows. Vet Ital 2004;40:664–7. [10] Zientara S, Maclachlan NJ, Calistri P, Sánchez-Vizcaíno JM, Savini G. Bluetongue vaccination in Europe. Expert Rev Vaccines 2010;9:989–91. [11] Savini G, Maclachlan NJ, Calistri P, Sánchez-Vizcaíno JM, Zientara S. Vaccines against bluetongue in Europe. Comp Immunol Microbiol Infect Dis 2008;31:101–20. [12] Savini G, Hamers C, Conte A, Migliaccio P, Bonfini B, Teodori L, et al. Assessment of efficacy of a bivalent BTV-2 and BTV-4 inactivated vaccine by vaccination and challenge in cattle. Vet Microbiol 2009;133:1–8. [13] Alpar HO, Bramwell VW, Veronesi E, Darpel KE, Pastoret PP, Mertens PPC. Bluetongue virus vaccines past and present. In: Mellor PS, Baylis M, Mertens PPC, editors. Bluetongue. London, United Kingdom: Elsevier Academic Press; 2009. p. 397–428. [14] Eschbaumer M, Hoffmann B, Konig P, Teifke JP, Gethmann JM, Conraths FJ, Probst C, Mettenleiter TC, Beer M. Efficacy of three inactivated vaccines against bluetongue virus serotype 8 in sheep. Vaccine 2009;27:4169–75. [15] Schwartz-Cornil I, Mertens PP, Contreras V, Hemati B, Pascale F, Bréard E, et al. Bluetongue virus: virology, pathogenesis and immunity. Vet Res 2008;39:46. [16] Huismans H, Erasmus BJ. Identification of the serotype-specific and groupspecific antigens of bluetongue virus. Onderstepoort J Vet Res 1981;48:51–8. [17] Mertens PP, Pedley S, Cowley J, Burroughs JN, Corteyn AH, Jeggo MH, et al. Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype. Virology 1989;170:561–5. [18] Huismans H, van der Walt NT, Cloete M, Erasmus BJ. Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep. Virology 1987;157:172–9. [19] Roy P, Urakawa T, Van Dijk AA, Erasmus BJ. Recombinant virus vaccine for bluetongue disease in sheep. J Virol 1990;64:1998–2003. [20] Jeggo MH, Wardley RC. Generation of cross-reactive cytotoxic T lymphocytes following immunization of mice with various bluetongue virus types. Immunology 1982;45:629–35. [21] Jeggo MH, Wardley RC, Brownlie J, Corteyn AH. Serial inoculation of sheep with two bluetongue virus types. Res Vet Sci 1986;40:386–92.
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[22] Jones LD, Williams T, Bishop D, Roy P. Baculovirus-expressed nonstructural protein NS2 of bluetongue virus induces a cytotoxic T-cell response in mice which affords partial protection. Clin Diagn Lab Immunol 1997;4:297–301. [23] Wade-Evans AM, Romero CH, Mellor P, Takamatsu H, Anderson J, Thevasagayam J, et al. Expression of the major core structural protein (VP7) of bluetongue virus, by a recombinant capripox virus, provides partial protection of sheep against a virulent heterotypic bluetongue virus challenge. Virology 1996;220:227–31. [24] Umeshappa CS, Singh KP, Pandey AB, Singh RP, Nanjundappa RH. Cell-mediated immune response and cross-protective efficacy of binary ethylenimine-inactivated bluetongue virus serotype-1 vaccine in sheep. Vaccine 2010;28:2522–31. [25] Roy P, Bishop DH, LeBlois H, Erasmus BJ. Long-lasting protection of sheep against bluetongue challenge after vaccination with virus-like particles: evidence for homologous and partial heterologous protection. Vaccine 1994;12:805–11. [26] Calvo-Pinilla E, Navasa N, Anguita J, Ortego J. Multiserotype protection elicited by a combinatorial prime-boost vaccination strategy against bluetongue virus. PLoS One 2012:e34735. [27] Maan S, Maan NS, Samuel AR, Rao S, Attoui H, Mertens PP. Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes. J Gen Virol 2007;88:621–30. [28] Savini G, Ronchi GF, Leone A, Ciarelli A, Migliaccio P, Franchi P, Mercante MT, Pini A. An inactivated vaccine for the control of bluetongue virus serotype 16 infection in sheep in Italy. Vet Microbiol 2007;124:140–6. [29] Wechsler SJ, McHolland LE, Tabachnick WJ. Cell lines from Culicoides variipennis (Diptera: Ceratopogonidae) support replication of bluetongue virus. J Invertebr Pathol 1989;54:385–93. [30] Moulin V, Noordegraaf CV, Makoschey B, van der Sluijs M, Veronesi E, Darpel K, et al. Clinical disease in sheep caused by bluetongue virus serotype 8, and prevention by an inactivated vaccine. Vaccine 2012;30:2228–35.
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[31] Bréard E, Belbis G, Hamers C, Moulin V, Lilin T, Moreau F, et al. Evaluation of humoral response and protective efficacy of two inactivated vaccines against bluetongue virus after vaccination of goats. Vaccine 2011;29:2495–502. [32] Vandenbussche F, Vandemeulebroucke E, De Clercq K. Simultaneous detection of bluetongue virus RNA, internal control GAPDH mRNA, and external control synthetic RNA by multiplex real-time PCR. Methods Mol Biol 2010;630:97–108. [33] Vandenbussche F, De Leeuw I, Vandemeulebroucke E, De Clercq K. Emergence of bluetongue serotypes in Europe, part 1: description and validation of four real-time RT-PCR assays for the serotyping of bluetongue viruses BTV-1, BTV-6, BTV-8 and BTV-11. Transboundary Emerging Dis 2009;56:346–54. [35] (a) R Core Team. R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2014; (b) Dungu B, Gerdes T, Smit T. The use of vaccination in the control of bluetongue in southern Africa. Vet Ital 2004;40:616–22. [36] Jennings DM, Mellor PS. Variation in the responses of Culicoides variipennis (Diptera, Ceratopogonidae) to oral infection with bluetongue virus. Arch Virol 1987;95:177–82. [37] Fu H, Leake CJ, Mertens PPC, Mellor PS. The barriers to bluetongue virus infection, dissemination and transmission in the vector, Culicoides variipennis (Diptera: Ceratopogonidae). Arch Virol 1999;144:747–61. [38] Andrew M, Whiteley P, Janardhana V, Lobato Z, Gould A, Coupar B. Antigen specificity of the ovine cytotoxic T lymphocyte response to bluetongue virus. Vet Immunol Immunopathol 1995;47:311–22. [39] Janardhana V, Andrew ME, Lobato ZI, Coupar BE. The ovine cytotoxic T lymphocyte responses to bluetongue virus. Res Vet Sci 1999;67:213–21. [40] Jones LD, Chuma T, Hails R, Williams T, Roy P. The non-structural proteins of bluetongue virus are a dominant source of cytotoxic T cell peptide determinants. J Gen Virol 1996;77:997–1003. [41] Lobato ZI, Coupar BE, Gray CP, Lunt R, Andrew ME. Antibody responses and protective immunity to recombinant vaccinia virus-expressed bluetongue virus antigens. Vet Immunol Immunopathol 1997;59:293–309.
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Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Evaluation of adaptive immune responses and heterologous protection induced by inactivated bluetongue virus vaccines
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Emmanuel Breard a,∗,1 , Guillaume Belbis b,1 , Cyril Viarouge a , Kyriaki Nomikou c , Andy Haegeman d , Kris De Clercq d , Pascal Hudelet e , Claude Hamers f , Francis Moreau g , Thomas Lilin g , Benoit Durand h , Peter Mertens c , Damien Vitour a , Corinne Sailleau a , Stéphan Zientara a a
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ANSES, UMR 1161 Virologie ANSES-INRA-ENVA, 23 avenue du Général de Gaulle, 94704 Maisons-Alfort, France Université Paris-Est, Ecole Nationale Vétérinaire d’Alfort, Unité de Pathologie du Bétail, 7 avenue du Général de Gaulle, 94704 Maisons-Alfort, France c Vector-Borne Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey GU24 0NF, United Kingdom d CODA–CERVA, Department of Virology, Ukkel, Belgium e MERIAL S.A.S., 254 Rue Marcel Mérieux, 69007 Lyon, France f MERIAL S.A.S., P.I. Plaine de l’Ain, Allée des Cyprès, 01150 Saint-Vulbas, France g Université Paris-Est, Ecole Nationale Vétérinaire d’Alfort, Centre de recherche biomédicale, 7 avenue du Général de Gaulle, 94704 Maisons-Alfort, France h ANSES, unité Epidémiologie, 23 avenue du Général de Gaulle, 94704 Maisons-Alfort, France b
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Article history: Received 22 October 2013 Received in revised form 20 November 2014 Accepted 28 November 2014 Available online xxx
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Keywords: Bluetongue virus disease Inactivated vaccines Experimental infection Heterologous challenges Sheep
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1. Introduction
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Eradication of bluetongue virus is possible, as has been shown in several European countries. New serotypes have emerged, however, for which there are no specific commercial vaccines. This study addressed whether heterologous vaccines would help protect against 2 serotypes. Thirty-seven sheep were randomly allocated to 7 groups of 5 or 6 animals. Four groups were vaccinated with commercial vaccines 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 of either BTV-16 (2 groups) or BTV-8 (5 groups). Taken together, 24/25 sheep from the 4 experimental groups developed detectable antibodies against the vaccinated viruses. Furthermore, sheep that received heterologous vaccines showed significantly reduced viraemia and clinical scores for BTV-16 when compared to unvaccinated controls. Reductions in clinical signs and viraemia among heterologously vaccinated sheep were not as common after challenge with BTV-8. This study shows that heterologous protection can occur, but that it is difficult to predict if partial or complete protection will be achieved following inactivated-BTV vaccination. © 2014 Published by Elsevier Ltd.
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Bluetongue (BT) is an infectious, OIE-listed arboviral disease that infects ruminants [1]. The disease is caused by viruses belonging to Bluetongue virus species [2]. There are 26 bluetongue virus (BTV) serotypes, which raise type specific neutralising antibodies (NA) during infection of their hosts [3]. BTV is transmitted primarily by biting midges (Culicoides spp.) but can be transmitted vertically or via an oral route [4–6]. Since the 20th century, 10 serotypes
∗ Corresponding author. Tel.: +33 695063201; fax: +33 143689762. E-mail address: [email protected] (E. Breard). 1 Both authors contributed equally.
have been detected in Europe, some of them on several different occasions [7]. After the emergence of BTV in Europe (1998), the first successful inactivated vaccine (IV) protected against BTV-2 [7–11]. Subsequently, monovalent and bivalent IV were developed, used in the field [9,10] and efficient for eradication of different homologous BTV serotypes [12–14]. Since 2011, several countries that were previously infected with serotype 1, 2, 4 or 8 returned to freedom-from-disease after mass vaccination campaigns [7,11]. Animals vaccinated with these IV develop protective immune responses against the homologous serotype(s). The BTV outercapsid proteins VP2 and VP5 (particularly VP2) are the BTV proteins inducing NA [15]; VP2 is also the major protein involved in serotype determination [16,17]. Sheep inoculated with VP2 alone also
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produced NA and were protected against challenge with homologous BTV serotype [18,19]. A vaccine that protects against multiple BTV types would provide a valuable defence against incursions of new BTV serotypes. However, such vaccines seem difficult to be create, despite evidence that cytotoxic T cells play a role in protection against BTV infection [20–22]. Few studies report complete or even partial protection after immunization and heterologous challenge. However, a significant level of cross-protection against a BTV challenge was obtained after immunization with a single BTV protein (VP7) without NA response against virus particles [23]. Studies show that inactivated or virus-like particle vaccines can reduce the severity of heterologous BTV infections, even though the BTV serotypes used were not closely related [24,25]. One study using a recombinant vaccine against BTV-4 in IFNAR(−/−) knockout mice, showed effective cross-protection against heterologous lethal challenges [26]. Little is known about the mechanism(s) of protection after pan- or multi-serotype vaccinations and heterologous challenge. Earlier studies of sheep serially infected with BTV-3 and 4 demonstrated resistances to challenge with BTV-6 [21]. However, the level of protection induced in sheep against heterologous serotypes by simultaneous multivalent vaccination with IV has not been reported. The nucleotype grouping for BTV segment 2 illustrates the ‘relatedness’ of different strains [27]. Different serotypes within a single BTV nucleotype are more likely to generate crossreactive responses to other viruses within the same nucleotype [27]. The possibility that animals can be protected and to restrict the emergence of new BTV serotypes in ‘free’ regions, using currently available IV in a polyvalent vaccination strategy, needs to be further assessed. The only IV registered within Europe are for serotypes 1, 2, 4, 8, and 9. No vaccine against BTV-16 is available in the market even if a study demonstrated that two doses of BTV-16 IV protect sheep [28]. Those would be the immediate solution in the case of an outbreak of an exotic serotype. In this study we evaluated the level of heterologous protection offered by sequential vaccinations in sheep using different BTV serotypes (-2, -4 or -9) against virulent European strains of BTV-8 and -16. These 3 inactivated vaccines were available in 2006 when BTV-8 appeared in north of Europe. In this study, we try to evaluate the potential protector effect (or not) of these 3 IV in a BTV-8 spread context (no IV against this serotype was available) and also against a BTV-16 emergence.
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2. Materials and methods
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2.1. Experimental design
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Thirty seven sheep were used for the study, randomly allocated to 7 groups of 5 or 6 and housed in group pens in biosafety level 3 animal facilities (Maisons-Alfort Veterinary National School, France). All experimental protocols were reviewed by a state ethics commission and have been approved by the competent authority. On day (D) 0 or D21, 5 groups (G) of sheep received 1 mL each of vaccine (BTVPUR ALSAP® 2, 4, 9, Merial, France; see Table 1). The injections were performed subcutaneously, behind the left elbow on D0 and the right elbow on D21. G4 was vaccinated only once on D21, following the manufacturer’s recommendations for BTV-8 vaccination in sheep (vaccine BTVPUR ALSAP® 8). On D42, sheep were challenged, at two points subcutaneously in the neck, with 3 mL of BTV-16 (G1 and G2) or 3 mL of BTV-8 strain (G3 to G7).
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2.2. Vaccines
98 99 100 101 102 103 104 105 106 107 108 109 110
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Vaccines used (Table 1) are inactivated, monovalent or bivalent preparations, in aqueous solution, adjuvanted with saponins and
aluminium hydroxide (i.e., BTVPUR ALSAP® 2, 4, 9 and 8 commercialized by Merial). 2.3. Challenge viruses The BTV-16 challenge virus GRE2008/11 was obtained from the Orbivirus Reference Collection at The Pirbright Institute (UK). It was isolated in 2008 from blood of an infected Greek sheep and then passaged twice in KC cells (derived from C. sonorensis) [29]. The challenge virus was tested by RT-qPCR targeting Seg-9 (Laboratoire Service International, Lissieu, France) with Cq values ranging from 6.86 to 9.18. The BTV-8 challenge virus UKG2007/77 was isolated in 2007 from blood of a BTV-8 infected cow in UK and passaged three times in KC cells [30]. The challenge virus gave Cq values ranging from 6.69 to 9.65 by RT-qPCR (Laboratoire Service International, Lissieu, France). These challenge viruses were not cell-culture-adapted to mammalian culture cells and the determination of their respective infectious 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 samples were analyzed using VP7 specific cELISA assays (cELISA, ID-Screen Blue Tongue Competition Kit, ID VET, France). Results are expressed as an inhibition-percentage (IP), as follows: IP = (ODsample /ODnegative reference ) × 100. IP < 35 was considered positive. Sera from D0, D21, D42 or D56 were also titrated for specific BTV-2, -4, -8, -9 or -16 NA by serum-neutralization tests (SNT) as described previously [31].
2.5. Virological analyses Viral RNA from EDTA blood samples was extracted using the NucleoSpin RNA Virus kit (Macherey-Nagel, Germany) according to the manufacturer’s instructions and analyzed at the CODACERVA Institute (Belgium) by a “pan-BTV” multiplex RT-qPCR assay [32]. The in-house BTV-8 RT-qPCR was carried out as previously described [33]. The analytical specificities were determined for the in-house BTV-2, 4, 9 and 16 PCRs in a similar fashion (specific data for these PCRs are available on demand). Serotyping of the first and last (day of euthanasia) positive pan-BTV results for each animal was performed using in-house BTV-2, 4, 8, 9 and 16 serotype RT-qPCR (depending on BTV serotypes used for vaccination and challenge).
2.6. Body temperature and clinical signs Rectal temperatures and clinical examination of all animals were recorded at D42 and then daily until D56. Clinical scoring was performed based upon typical BTV clinical signs as already described [31]. The score for each clinical sign is specified in Table 3.
2.7. Statistical analysis Data were analyzed using repeated measures ANOVA, the day post-challenge being treated as a fixed effect and the subject as a random effect. Significance threshold was set to 0.05. Data analysis was performed using R version 3.1.1 [34].
Please cite this article in press as: Breard E, et al. Evaluation of adaptive immune responses and heterologous protection induced by inactivated bluetongue virus vaccines. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.11.053
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117
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Table 1 Vaccination and challenge protocols used in this study. Groups
G1 G2 G3 G4 G5 G6 G7
3. Results
168
3.1. Serology
170 171 172
BTV vaccines
5 6 5 5 5 5 6
167
169
Number of sheep/group
D0
D21
BTV-9 No vaccination BTV-9 No vaccination BTV-2 and BTV-4 BTV-9 No vaccination
BTV-2 and BTV-4 No vaccination BTV-2 and BTV-4 BTV-8 BTV-2 and BTV-4 BTV-9 No vaccination
All sheep were seronegative before vaccination or challenge for groups control (Table 2). Twenty-one days after the first vaccination, IP were reduced in all vaccinated groups, indicating development of anti-VP7 antibodies. On D42, animals vaccinated
BTV challenge D42
Protection level
BTV-16 BTV-16 BTV-8 BTV-8 BTV-8 BTV-8 BTV-8
? None ? Complete ? ? None
twice (G1, G3, G5 and G6) showed a marked boost in VP7 antibody levels and all became ELISA positive (IP < 35). Exception was the sheep 1451 (G3), which remained ‘doubtful’ by ELISA, with an IP = 62 at D56. In the G4 (vaccinated once), 4 ELISA negative sheep have an IP ranged from 58 to 76, suggesting a low seroconversion. The challenge at D42 induced a seroconversion in all unvaccinated animals and an increase of antibodies levels against VP7 in vaccinated sheep.
Table 2 ELISA, SNT and clinical score results after vaccination (at D42; against BTV-2, 4, 8 or -9) and challenge (at D56; against BTV-8 (G1 and G2) or -16 (G3 to G7)) for each sheep. IP: inhibition percentage; +a: positive by BTV-2 and 4 SNT; +b: positive by BTV-2 SNT; +c: positive by BTV-8 SNT; +d: positive by BTV-9 SNT; +e: positive by BTV-16 SNT. NA titre of each serum was defined as the highest dilution (log 10) allowing neutralization of the 100 TCID50. (Titre < 0.6 is considered as negative). SNT titre results by SNT against BTV-2
BTV-4
BTV-9
BTV-8
D42
Lot
No. sheep
D42
D42
D42
G1
137 391 250 568 109
1.2 0.9 0.9 1.2 0.9
0.9 – 0.9 0.9 0.9
– – – – –
G2
493 578 403 306 533 400
G3
1124 1152 1331 1379 1451
G4
791 1138 1407 1455 1466
G5
205 1243 1253 1384 1406
G6
1127 1268 1321 1383 1435
G7
936 1082 1151 1205 1351 1144
0.9 0.6 – 0.9 0.6
2.1 1.2 1.5 1.5 2.1
– – – 0.9 0.6
– – – – –
2.1 1.5 1.5 0.6 2.1 – – – 0.9 –
ELISA results
D56
BTV-16
IP
Results at Day 42
Results at Day 56
D56
D0
D21
D28
D42
D56
ELISA
SNT
ELISA
SNT
Clinical score/sheep
1.8 1.5 0.9 1.5 2.1
114 109 120 106 124
62 57 63 8 65
5 5 8 24 4
2 10 5 20 5
2 6 4 4 4
+ + + + +
+a +b +a +a +a
+ + + + +
+e +e +e +e +e
20 21 17 13 14
0.9 0.6 1.5 2.1 1.2 1.8
115 114 120 110 117 121
115 113 112 106 110 114
109 109 122 108 118 114
109 108 119 105 118 107
7 6 6 15 19 7
– – – – – –
NT NT NT NT NT NT
+ + + + + +
+e +e +e +e +e +e
29 51 34 29 47 62 21 44 46 35 39
– – – – –
1.5 Dead 2.1 >2.4 Dead
107 124 120 123 108
69 96 75 97 102
8 23 25 43 68
11 24 24 13 62
4 Dead 4 4 Dead
+ + + + –
+b +b – +a +a
+
+f
+ +
+f +f
0.6 0.6 1.2 0.6 –
1.5 >2.4 2.1 1.2 1.2
107 107 118 108 107
115 112 114 119 123
60 47 19 43 72
71 65 5 76 58
7 5 4 8 12
– – + – –
+c +c +c +c –
+ + + + +
+f +f +f +f +f
1 4 5 13 3
– – – – –
– >2.4 >2.4 >2.4 >2.4
120 107 112 113 92
4 36 10 33 5
4 5 8 34 4
4 4 5 16 4
Dead 4 4 4 4
+ + + + +
+a +a +a +a +a
+ + + +
+f +f +f +f
15 33 57 25 26
– – – – –
1.8 2.1 2.1 0.9 >2.4
103 103 93 97 129
47 34 54 21 31
11 10 46 5 4
16 6 36 4 5
2 4 4 4 4
+ + Dbt + +
– – – +d –
+ + + + +
+f +f +f +f +f
14 41 40 5 50
– – – – – –
Dead >2.4 >2.4 >2.4 >2.4 0.9
114 120 77 117 97 104
110 112 38 112 123 108
106 107 39 112 108 98
112 112 63 105 106 103
Dead 6 4 8 12 14
– – – – – –
NT NT NT NT NT NT
+ + + + +
+f +f +f +f +f
36 38 65 30 50 4
Please cite this article in press as: Breard E, et al. Evaluation of adaptive immune responses and heterologous protection induced by inactivated bluetongue virus vaccines. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.11.053
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4
B
33
G1
29
G2
25
Cq value means/group
Cq value means/group
A 37
21
37
33
G3 G4
29
G5 G6 G7
25
21 D42
D47
D49
D51
D54
D56
D42
Days Q5
181 182 183 184 185 186 187 188 189 190
191
192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213
214
215 216 217 218 219 220 221
D47
D49
D51
D54
D56
Days Fig. 1.
In the majority of vaccinated sheep, NA (titres ranging from 0.6 to 2.1) against the BTV-2, -4, and -8 at D42 were detected (Table 2). However, NA against BTV-9 was found at D42 in only 1 (no. 1383 in G6) out the 15 sheep vaccinated (once or twice) with the BTV-9 vaccine. All challenged sheep still alive at D56 had significant levels of NA against BTV-16 or -8 (titres ranging from 0.9 to up than 2.4). Taken together, VP7 and/or neutralizing antibodies were detected in 24/25 animals at D42. Only sheep no. 1466 (G4) was ELISA and NA negative. All challenged sheep, still alive at D56, had developed NA against the BTV-8 or -16 challenge strains (Table 2). 3.2. Detection of BTV RNA in post-challenge sheep by RT-qPCR No BTV RNA was detected in the 37 animals prior to challenges (Fig. 1A and B). RT-qPCR serotyping results confirmed the presence of the challenge virus by consistently identifying the serotype corresponding to the challenge inoculum, but not the vaccine (data not shown). All BTV-16 challenged animals were viraemic at the first sampling post infection (D47). However, the intensity of the BTV-16 viraemia in G1 was reduced (tenfold) compared to unvaccinated G2, showing a significant effect (p < 0.0001) of the heterologous vaccination (against BTV-9, 2 and 4) (Fig. 1A). The average level of viraemia in the vaccinated G3 and G5 challenged with BTV-8 was similar to that of the unvaccinated G7. In contrast, G6 had a reduced level of viraemia when compared with G7 (p = 0.07) (Fig. 1B). This was due to 2 animals (no.1127 and 1383) that developed no viraemia. The three other animals of G6 had a viraemia that was similar to that of G7. The BTV-8 vaccination (G4) blocked viraemia in 4/5 animals, post BTV-8 challenge. Only one animal in G4 (no. 1138) developed a low viraemia (Cq values ranging from 31.7 (D47) to 37.4 (D56)). In the unvaccinated G7, 5/6 sheep had a strong viraemia after BTV-8 challenge, although one sheep (no. 1144) showed no viraemia until D56 with a Cq value of 34.5 from the blood sample. 3.3. Clinical signs and body temperature The clinical signs most frequently observed during BTV-16 infection (G2) were: congestion of skin, conjunctivitis, respiratory difficulties (slight nasal discharge), fever (40–41 ◦ C) and locomotion impairment (stiffness) (Table 3). In heterologous vaccinated G1, conjunctivitis, slight nasal discharge and fever (40–41 ◦ C) were also observed but with a low frequency, while congestion of the skin and locomotion impairment were only observed in one animal. The
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 and in G7 were euthanized for ethical reasons between D51 and 53, due to the severity of disease. They showed severe oedema, depressed behaviour and several days of fever (>40 ◦ C). All sheep in G4 only showed conjunctivitis, with individual clinical scores significantly lower (p < 0.0001) than the other unvaccinated or heterologous vaccinated groups (Tables 2 and 3). The individual clinical score in G7 was the highest (37.2) but was not statistically different from individual scores in the heterologous-vaccinated sheep that were also challenged with BTV-8. The kinetics of clinical signs (Fig. 2A) show a significant reduction in clinical scores and partial protection against BTV-16 challenge in G1 when compared with G2, which can be attributed to heterologous-vaccinations against BTV-9, 2 and 4. Only the sheep in G4 (vaccinated against BTV-8) showed a protection against challenge (Fig. 2B). The other vaccinated groups showed similar clinical patterns to G7. The mean of the individual clinical scores in G6 was slightly lower (30) than in G7 (37.2), although this is due to the 2 animals without viraemia which had low individual clinical scores (Table 2). Interestingly, the duration and intensity of fever reported for all animals challenged with BTV-16 (Fig. 3A) and BTV-8 (Fig. 3B) were mostly similar, except for G4. 4. Discussion After the different vaccination protocols used and before challenge infection, the serological data (ELISA and SNT) demonstrated that 24/25 sheep developed an adaptive immune response against IV detectable by ELISA or/and SNT. Sheep no. 1466 (in G4; ELISA and NA negative at D42) was also protected against the homologous challenge. Following challenge, 33 sheep developed both neutralizing and VP7 antibodies. Four sheep died due to the severity of the clinical signs induced after the BTV-8 challenge. Antibodies levels of the sheep vaccinated once showed a low anti-VP7 antibody response, as previously observed after primo vaccination of ruminants with IV [28,30]. However, it has been shown (experimentally and in the field) that vaccinated sheep, with low or without NA, can be effectively protected against homologous challenges after a single IV dose, [7–11,14]. The sheep in G4 vaccinated once with the BTV-8 IV confirmed these observations, and although one animal showed a low viraemia after challenge, no fever or clinical signs were observed (although some conjunctivis were 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 by inactivated bluetongue virus vaccines. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.11.053
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247
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Table 3 Mean 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
Apathetic Depressed Prostated
1 2 3
0.7
Submandibular Face Nose
1 1 1
0.2
Congestion
Skin
4
Locomotion
Lameness Stiffness
1 2
Respiratory
Slight nasal discharge Important nasal discharge Coughing
1 2 1
3.4
Digestive
Diarrhea
1
0.4
Other
Conjonctivite Ulcers Hypersalivation
1 1 1
Fever
40 to 41 41 to 42 >42
1 2 4
Behaviuor
Oedema
G3
G4
G5
G6
G7
0.2 0.4
0.8
0.2
0.2
0.2 0.8
0.6 1.6
12.7
15.2 0.8
0.4
1.0 2.7
15.2
10.4
0.8
0.4
13.3 1.3
5.8 0.7 0.2
4.2 2.8 1
1
3.6 0.8
5.8 0.4 0.4
4.8 2.3 0.5
6.6 1.2
12.3 0.2 0.3
5.6 0.4
2.8 0.2
5.4
6.8 0.6 0.6
6.7 1.0 0.7
2.8 0.4
4.2 1.0
0.4
3.6 1.2
1.6 2.8
2.0 2.7 0.7
17
41.8
5.2
31.2
30
37.2
0.2
Mean of score/sheep/group
0.8
2.8 2.8 37
7,0
A
4,0
3,0
G1 G2
2,0
1,0
0,0 D41
D44
D47
D50
D53
B
6,0
*
clinical score means/group
clinical score means /group
5,0
5,0
G3 G4 G5 G6 G7
4,0 3,0 2,0 1,0 0,0 D41
D56
D44
D47
D50
D53
D56
Days
Days
A
40,5
40,0
G1 G2
39,5
39,0
Means of temperature/group
temperature means /group
Fig. 2.
38,5 D42
D45
D48
D51
41,0
B
40,5
G3 G4 G5 G6 G7
40,0
39,5
39,0
38,5
D54
D42
Days
D45
D48
D51
D54
Days Fig. 3.
Please cite this article in press as: Breard E, et al. Evaluation of adaptive immune responses and heterologous protection induced by inactivated bluetongue virus vaccines. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.11.053
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in free range and that they were during this study in rooms in level 3 containment conditions). The BTV-8 and 16 strains used as virulent inocula were field strains with low passages in KC cells. These strains efficiently induced severe clinical signs in the unvaccinated groups, confirming that low passages BTV strains grown in Culicoides cell cultures are more virulent than a mammalian cell-culture derived BTV-8 strain [30]. Although BTV-16 did not kill the challenged sheep, the mean clinical score (41.8) for the unvaccinated sheep (G2), was similar to that observed in the unvaccinated G7 challenged with BTV8 (37.2). The sheep that received heterologous vaccinations (G1) clearly showed reduced viraemia and clinical scores caused by BTV16 (Fig. 1A and Tables 2 and 3) when compared with G2, and specific clinical signs were either not observed (congestion of skin and stiffness) or were highly reduced (nasal discharge or conjunctivis). Even though the ‘curve’ for body temperature (Fig. 3A) in G1 was consistently below the G2 curve, there was no difference in terms of intensity or length of pyrexia. Taken together, these data suggest that IV against serotype 9, 2 and 4 induced a partial protection against challenge with BTV-16, as seen in the clinical and virological data and particularly by a reduction in some of the clinical signs. Group 3, also vaccinated against serotypes 9, 2 and 4, was challenged with BTV-8. The viraemia and the clinical scores were similar to those observed in the unvaccinated G7. Moreover, 2/5 sheep from G3 died after BTV-8 challenge. The partial protection observed in G1 (against BTV-16) is clearly not seen in G3 (against BTV-8), although the vaccination protocols were identical. This indicates that even partial protection against heterologous challenge is highly dependent on the challenge serotype. Furthermore, this partial protection is not due to a cross protection against phylogenetically related serotypes [25] as serotypes 8 and 16 have a very weak interrelationship with serotypes 2, 4 and 9 [27,35]. Two of the 5 sheep in G6 (no. 1127 and 1383), vaccinated twice against BTV-9, showed no viraemia after challenge with BTV-8. However, NA against the BTV-8 challenge were detected in both animals, demonstrating that they were properly challenged at D42. The average clinical score and viraemia were slightly (not statistically significant) reduced in this group when compared with G7, as the response against BTV-8 was heterologous in G6. In this group, the heterologous vaccination seemed to induce a complete protection for 2 of the 5 sheep, while the other three did not show even partial protection. Individual protection therefore seemed to be an “all or nothing” effect. One of the six unvaccinated sheep in G7 showed no viraemia until day 14 after the BTV-8 challenge. This animal developed no fever and no clinical signs (clinical score = 5) but seroconverted (by ELISA and SNT) with similar kinetics for antibody development to the other five animals in the group, demonstrating that the inoculum had been correctly administrated at D42. To our knowledge, no pre-existing condition or remarkable events had affected this experimental result. Taken together, our results suggest 3 different scenarios after multi-type vaccinations and heterologous challenge. The first: partial protection observed in vaccinated animals challenged with a virulent heterologous BTV serotype, with a reduction of the viraemia and a decrease or disappearance of some clinical signs. In this case, a protective effect was not observed on body temperature, showing that fever is not a predictable clinical sign of a protective effect. During BTV spread, one of the requirements for virus transmission is a sufficiently high viraemia in the host for infection of feeding insect vectors [36,37]. The reduction of the viraemia induced by heterologous IV in sheep is an important factor to help control an outbreak and spread. The second: no protection was observed (G3 and G5 challenged against BTV-8) showing that the same heterologous vaccination protocol can generate different
levels of protection against different challenge serotypes (G1 and G3). The third scenario is illustrated by the heterogeneous response against BTV-8 in sheep vaccinated twice against BTV-9 with an individual “all or nothing” protection effect. This study shows that it is difficult to predict if partial or complete heterologous protection will be achieved post inactivated-BTV vaccination. This may reflect different levels of cell-mediated immunity that may be generated by IV against heterologous and phylogenetically unrelated BTV serotypes as well as unpredictable natural resistance against BTV disease in sheep [17,19,38–41]. Acknowledgements We thank for their financial support the European Orbivac Grant Q4 Agreement Project (no.: 245266) coordinated by Prof. Polly Roy (London School of Hygiene and Tropical Medicine). ®: BTVPUR ALSAP is a registered trademark of Merial in the European Union and elsewhere. References [1] Maclachlan NJ, Drew CP, Darpel KE, Worwa G. The pathology and pathogenesis of bluetongue. J Comp Pathol 2009;141:1–16. [2] Mertens PPC, Maan S, Samuel A, Attoui H. Orbivirus reoviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, editors. Virus taxonomy VIIIth report of the ICTV. London: Elsevier/Academic Press; 2005. p. 466–83. [3] Maan S, Maan NS, Nomikou K, Batten C, Antony F, Belaganahalli MN, et al. Novel bluetongue virus serotype from Kuwait. Emerg Infect Dis 2011;17:886–9. [4] Darpel KE, Batten CA, Veronesi E, Williamson S, Anderson P, Dennison M, et al. Transplacental transmission of bluetongue virus 8 in cattle UK. Emerg Infect Dis 2009;15:2025–8. [5] De Clerq K, Vandenbussche F, Vandemeulebroucke E, Vanbinst T, De Leeuw I, Verheyden B, et al. Transplacental bluetongue infection in cattle. Vet Rec 2008;162:64. [6] Backx A, Heutink R, van Rooij E, van Rijn P. Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection. Vet Microbiol 2009;138:235–43. [7] Zientara S, Sánchez-Vizcaíno JM. Control of bluetongue in Europe. Vet Microbiol 2013;165:33–7. [8] Di Emidio B, Nicolussi P, Patta C, Ronchi GF, Monaco F, Savini G, et al. Efficacy and safety studies on an inactivated vaccine against bluetongue virus serotype 2. Vet Ital 2004;40:640–4. [9] Savini G, Monaco F, Citarella R, Calzetta G, Panichi G, Ruiu A, Caporale V. Monovalent modified-live vaccine against bluetongue virus serotype 2: immunity studies in cows. Vet Ital 2004;40:664–7. [10] Zientara S, Maclachlan NJ, Calistri P, Sánchez-Vizcaíno JM, Savini G. Bluetongue vaccination in Europe. Expert Rev Vaccines 2010;9:989–91. [11] Savini G, Maclachlan NJ, Calistri P, Sánchez-Vizcaíno JM, Zientara S. Vaccines against bluetongue in Europe. Comp Immunol Microbiol Infect Dis 2008;31:101–20. [12] Savini G, Hamers C, Conte A, Migliaccio P, Bonfini B, Teodori L, et al. Assessment of efficacy of a bivalent BTV-2 and BTV-4 inactivated vaccine by vaccination and challenge in cattle. Vet Microbiol 2009;133:1–8. [13] Alpar HO, Bramwell VW, Veronesi E, Darpel KE, Pastoret PP, Mertens PPC. Bluetongue virus vaccines past and present. In: Mellor PS, Baylis M, Mertens PPC, editors. Bluetongue. London, United Kingdom: Elsevier Academic Press; 2009. p. 397–428. [14] Eschbaumer M, Hoffmann B, Konig P, Teifke JP, Gethmann JM, Conraths FJ, Probst C, Mettenleiter TC, Beer M. Efficacy of three inactivated vaccines against bluetongue virus serotype 8 in sheep. Vaccine 2009;27:4169–75. [15] Schwartz-Cornil I, Mertens PP, Contreras V, Hemati B, Pascale F, Bréard E, et al. Bluetongue virus: virology, pathogenesis and immunity. Vet Res 2008;39:46. [16] Huismans H, Erasmus BJ. Identification of the serotype-specific and groupspecific antigens of bluetongue virus. Onderstepoort J Vet Res 1981;48:51–8. [17] Mertens PP, Pedley S, Cowley J, Burroughs JN, Corteyn AH, Jeggo MH, et al. Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype. Virology 1989;170:561–5. [18] Huismans H, van der Walt NT, Cloete M, Erasmus BJ. Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep. Virology 1987;157:172–9. [19] Roy P, Urakawa T, Van Dijk AA, Erasmus BJ. Recombinant virus vaccine for bluetongue disease in sheep. J Virol 1990;64:1998–2003. [20] Jeggo MH, Wardley RC. Generation of cross-reactive cytotoxic T lymphocytes following immunization of mice with various bluetongue virus types. Immunology 1982;45:629–35. [21] Jeggo MH, Wardley RC, Brownlie J, Corteyn AH. Serial inoculation of sheep with two bluetongue virus types. Res Vet Sci 1986;40:386–92.
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