Vaccine 27 (2009) 7326–7330

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Vaccine

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Seroconversion, neutralising antibodies and protection in bluetongue serotype 8 vaccinated sheep

C.A.L. Oura a,∗, J.L.N. Wood b, A.J. Sanders a, A. Bin-Tarif a, M. Henstock a, L. Edwards a, T. Floyd b, H. Simmons c, C.A. Batten a a Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU240NF, UK b Cambridge Infectious Diseases Consortium, Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK c VLA Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK article info abstract

Article history: Bluetongue virus serotype 8 (BTV-8) has caused a major outbreak of disease in cattle and sheep in several Received 21 July 2009 countries across northern and western Europe from 2006 to 2008. In 2008 the European Union instigated a Received in revised form mass-vaccination programme in affected countries using whole virus inactivated vaccines. We evaluated 15 September 2009 vaccinal responses in sheep and the ability of the vaccine to protect against experimental challenge. Sheep Accepted 15 September 2009 vaccinated 10 months previously under ﬁeld conditions were challenged with BTV-8. One of 7 vaccinated Available online 26 September 2009 sheep became infected, as evidenced by detection of viral RNA by real-time RT-PCR and by virus isolation. The remaining 6 sheep appeared fully protected from virus replication. None of the vaccinated sheep Keywords: Bluetongue serotype 8 showed clinical signs of BTV and there was a good correlation between the presence of neutralising Inactivated vaccine antibodies on challenge and protection. Commercially available ELISAs were evaluated for their ability to Sheep detect antibodies in sheep vaccinated on a single occasion. The sandwich (double antigen) ELISA assays ELISA were found to be more sensitive at detecting antibodies in vaccinated sheep than the competitive ELISAs.

1. Introduction at the end of April 2008. As more doses of vaccine became available to the UK farmers, the protection zone (PZ) moved northwards Bluetongue serotype 8 (BTV-8) successfully overwintered in and westwards until it covered the whole of England and Wales by many countries in northern and western Europe over the winter September 2008. The combination of high levels of vaccine cov- of 2006/2007 and emerged in the summer of 2007 to cause devas- erage in areas where BTV-8 circulated in 2007 before the 2008 tating effects to the livestock industries of many countries including transmission season and the cold wet summer provided an expla- the Netherlands, France, Belgium and Germany [1,2]. BTV-8 arrived nation for the lack of BTV circulation detected in the UK throughout on the south-eastern shores of the UK in the summer of 2007 [3,4]. 2008 [6]. Once the ability of the virus to overwinter in northern Europe was The BTV-8 inactivated vaccines used across Europe in 2008 were confirmed the need for rapid and immediate control of the dis- produced rapidly and were licenced for emergency use without any ease became apparent before it spread to all corners of Europe and associated efficacy guarantees. Although many millions of animals beyond. in Europe have now been vaccinated, there remain some unan- After much debate about the advantages and disadvantages of swered questions about the immunological mechanisms resulting the use of live attenuated and inactivated vaccines against BTV [5], in vaccine efficacy (ability to protect clinically and to prevent countries in northern and western Europe decided to use inacti- onward transmission), the antibody responses to the products, par- vated as opposed to live attenuated vaccines in their BTV-8 control ticularly in sheep which only receive one dose, the duration of plans. In the autumn of 2007 countries started to place orders for immunity, and both the extent and length of colostral antibody inactivated vaccine against BTV-8. In October 2007 the UK put in an protection in lambs and calves born from vaccinated dams. This is order for 22.5 million doses of the Intervet-manufactured Bovilis- the first published study detailing the efficacy of the BTV-8 Bovilis BTV-8 vaccine. The first 9 million doses of the vaccine were released vaccine in sheep and the first study evaluating the long-term (10 to farmers in the high risk areas of the east and south of the UK months) protective properties of any BTV-8 inactivated vaccine on the market. Soon after the vaccination campaign started in the UK it was ∗ Corresponding author. Tel.: +44 01483 232441. found that many sheep that had received one dose of vaccine did not E-mail address: [email protected] (C.A.L. Oura). have detectable antibodies when tested with competitive ELISAs

0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.09.070 C.A.L. Oura et al. / Vaccine 27 (2009) 7326–7330 7327 that had been validated for the detection of BTV antibodies in nat- 2.4. Serology tests urally infected animals [7,8]. In this paper the sensitivities of the commercially available ELISAs (sandwich and competitive formats) 2.4.1. ELISA assays in detecting BTV antibodies following vaccination in sheep vacci- Whole blood samples were centrifuged at 2400 × g for 5–10 min nated on one and two occasions have been compared. to obtain serum. The detection of BTV specific antibodies in serum was carried out using three competitive ELISA assays (Pourquier 2. Materials and methods C-ELISA kit (IDEXX, UK), ID-Screen Bluetongue Competition ELISA (ID-Vet. France), Bluetongue antibody test kit C-ELISA (VMRD, USA) 2.1. Animals and experimental design and two sandwich (double antigen) ELISA assays (ID-Screen Blue- tongue Early detection ELISA, ID-Vet, France and INGEZIM BTV Fifty-three (53) adult healthy Cheviot and poll Dorset sheep DR 12.BTV.K0, Ingenasa, Spain) according to the manufacturer’s were selected for the study from a farm in East Anglia in the UK. All instructions. animals were tested negative for BTV antibodies by serology and for viral RNA by real-time RT-PCR prior to inclusion in the study. 2.4.2. Serum Neutralisation Test (SNT) SNT was performed according to the method of Haig et al. The sheep were vaccinated for the first time in May 2008 and a [10] using the BTV-8 South African reference virus and serotype- group of 33 sheep was sampled (whole blood) the day before vac- specific BTV-8 positive control antisera. Briefly, sera were diluted cination and at 7, 14 and 56 days post-vaccination (dpv). At 56 dpv (1:10–1:1280) and titrated against 100 TCID of the BTV-8 South only 28 out of the 33 sheep were presented for bleeding by the 50 African reference virus. Plates were incubated for 1 h at 37 ◦C and farmer. A second group of 20 sheep, vaccinated in May 2008, was then transferred to 4 ◦C overnight. The following day 50 ␮l of a Vero sampled 82 dpv. These sheep were vaccinated for a second time cell (African green monkey kidney) suspension 2 × 105/ml were in September 2008 and sampled again 24 days after the second added per well and, after incubation for 4–7 days at 37 ◦C, the wells vaccination. Samples were tested throughout the study by com- were scored for cytopathic effect (CPE) observed. The neutralisation petitive ELISA (cELISA), sandwich ELISA (sELISA) and samples from titre was determined as the dilution of serum giving a 50% neu- the second group of 20 sheep were tested by Serum Neutralisa- tralisation end point SNT antibody titres were expressed as log10 tion Test (SNT). EDTA blood samples were taken from the 33 sheep reciprocal of the highest positive serum dilution. at both 1 and 7 dpv and were tested for viral RNA by real-time RT-PCR. 2.5. Virus isolation Vaccine/Challenge Study Group 1: Three (3) unvaccinated poll Dorset sheep between 6 and 9 months of age, born from unvac- EDTA blood samples were washed 3× with PBS and sonicated as cinated dams, were used as unvaccinated controls in the study. described in the OIE manual [11].KC(C. variipennis) cells were inoc- Vaccine/Challenge Study Group 2: Seven (7) vaccinated poll ulated with 200–500 ␮l of washed blood and incubated overnight Dorset sheep older than 12 months of age were selected from a at 26 ◦C. The following day the inoculum was removed and replaced farm in East Anglia in the UK. The sheep were vaccinated subcuta- with fresh media (Schneiders (Sigma, UK), 1% pen/strep, 1% Ampho- neously according to the manufacturers instructions in May 2008, tericin B, 10% FCS). Cells were incubated 26 ◦C for 7 days and then 10 months prior to challenge. harvested by centrifugation (2400 × g for 5–10 min), supernatant One week prior to challenge the sheep in groups 1 and 2 were was tested by real-time RT-PCR for the presence of BTV RNA as transferred to the Veterinary Laboratories Agency (VLA) BSL-3 facil- described in Section 2.3. ity. The sheep were inoculated subcutaneously in the neck with 1 ml of the Netherlands 2006 strain that was passaged twice in KC 2.6. Statistical analyses (Culicoides variipennis) cells (NET2006/02). The BTV challenge pro- tocol used avoids the growth of virus on mammalian cells as this Proportions of sheep with detectable antibodies at different has been found to reduce viral virulence. Due to a lack of cytopathic points in time following vaccination were compared using a stan- effect (cpe) in KC cells it was not possible to provide a viral titre for dard 2 approach. The levels of neutralising antibodies in sheep the inoculum although, when tested by real-time RT-PCR, it gave a before and after booster vaccination were compared using a stan- Ct of 15. dard paired Student t-test approach. EDTA and whole blood (serum) samples were taken from all the sheep on days—1, 2, 4, 6, 8, 10, 14, 18 and 22 post-challenge 3. Results infection (dpi). Samples were tested throughout the study by 3.1. A comparison of the ability of commercial ELISA assays to real-time RT-PCR, virus isolation, ELISA and SNT. Throughout the detect antibodies in sheep following a single dose of inactivated study the body temperature of the animals was monitored daily BTV-8 vaccine and the sheep were examined by a veterinarian daily for clinical signs. Comparison between the different ELISAs at different dpv 2.2. Vaccine (Table 1) show significantly lower percentage seroconversion with the cELISAs compared to the sELISAs at each of the 3 time-points 2 = . 2 = . The sheep were vaccinated with the Intervet-manufactured (7 dpv: 4d.f. 44 4, P < 0.0001; 14 dpv: 4d.f. 47 4 P < 0.0001; 2 = . Bovilis-BTV-8 inactivated vaccine (Batch number 00018201, expiry 56 dpv: 4d.f. 32 9, P < 0.0001), consistent with the sELISAs being 02/2009) according to the manufacturer’s instructions. more sensitive than the cELISAs at detecting antibodies in single- vaccinated sheep. In addition all the sheep that were positive with 2.3. Molecular tests the cELISAs were also positive with the sELISAs.

2.3.1. Real-time RT-PCR 3.2. Seroconversion and neutralising antibody titres in RNA was extracted from EDTA blood using either the MagnaPure vaccinated sheep (Roche) extraction robot using the ‘total NA/External lysis’ proto- col. Real-time RT-PCR was performed using a modiﬁed version of Of the 20 sheep vaccinated once, 16 had detectable neutralis- the procedure described by Shaw et al. [9]. ing antibodies measured by SNT with titres (log10) ranging from 7328 C.A.L. Oura et al. / Vaccine 27 (2009) 7326–7330 sELISA 2 = IDvet Table 1 Rates of detection of antibodies in sheep at different times following administration of one dose of inactivated vaccine, using 5 commercially available ELISAs.

−1 dpv 7 dpv 14 dpv 56 dpva

cELISA 1 0/33 (0%) 2/33 (6%) 10/33 (30%) 7/28 (25%) cELISA 2 0/33 (0%) 3/33 (9%) 12/33 (36%) 13/28 (46%) cELISA 3 0/33 (0%) 4/33 (12%) 22/33 (66%) 10/28 (36%) sELISA 1 0/33 (0%) 17/33 (52%) 30/33 (90%) 21/28 (75%) sELISA 2 0/33 (0%) 20/33 (60%) 30/33 (90%) 25/28 (89%) sELISA–Sandwich (double antigen) ELISA. cELISA–Competetive ELISA. dpv–days post-vaccination. a At 56 dpv only 28 out of the 33 sheep were bled.

Table 2 Rates of detection of antibodies in sheep at different times following administration of a ﬁrst and second dose of inactivated vaccine, using 5 commercially available ELISAs.

82 days post ﬁrst vaccine 24 days post second vaccine

cELISA 1 4/20 (20%) 20/20 (100%) cELISA 2 9/20 (45%) 20/20 (100%) cELISA 3 7/20 (35%) 20/20 (100%) sELISA 1 20/20 (100%) 20/20 (100%) sELISA 2 20/20 (100%) 20/20 (100%) sELISA—sandwich (double antigen) ELISA. cELISA—competetive ELISA.

1.0 to 1.9 (Fig. 1). When these sheep were sampled 24 days after a second dose of vaccine, all animals had antibodies detectable Fig. 2. (a) Real-time RT-PCR threshold cycle (Ct) values for the three sheep in the by all assays (Table 2). Significantly higher levels of neutralising control unvaccinated group 1. (b) Real-time RT-PCR threshold cycle (Ct) values antibodies (Fig. 1) were present after the second compared to the for the seven sheep in group 2 vaccinated with the BTV-8 inactivated vaccine 10 first vaccination (paired t-test, P < 0.0001). The four sheep that had months prior to challenge with BTV-8. Negative animals are shown with a Ct value undetectable levels of neutralising antibodies after the first vacci- of 45. nation had high titres (log10) of neutralising antibodies (2.4, 2.8, 3.0, >3.1) after the second vaccination, consistent with a strong anamnestic response. 3.4. Protection of sheep following virulent BTV-8 challenge 10 months after immunisation with BTV-8 inactivated vaccine 3.3. Absence of BTV genome in sheep post-vaccination Following challenge, viral RNA was detected in the control In order to determine whether BTV RNA (measured by real-time unvaccinated animals from 2 dpi (Fig. 2a) and antibodies were RT-PCR) persists in animals vaccinated with inactivated BTV vac- detected from 6 dpi (Table 3). The levels of BTV RNA as mea- cines, the group of 33 sheep were vaccinated and then tested by sured by the threshold cycle (Ct) peaked at around 4–6 dpi and real-time RT-PCR [9] at 1 and 7 dpv. No viral RNA was detected in then slowly reduced up to 21 dpi when the study was terminated any of the 33 sheep at either time-point. (Fig. 2a). Virus was detected in blood from all 3 unvaccinated controls from 4 up to 21 dpi. These control sheep showed mild to moderate signs of BTV, including inappetance, facial oedema, hyperaemia of the gums, ulcerous lesions in the mouth, nasal discharge and laboured breathing and all had elevated body temperatures (Fig. 3). Six of 7 vaccinates were seropositive by both sELISA and SNT at the time of challenge (Table 3). Not all these sheep had cELISA antibodies. All 6 seropositive vaccinated sheep were completely protected both clinically and virologically in that no viral RNA was detected in any of them (Fig. 2b). The seronegative vaccinated sheep (sheep 4: Table 3) did not show any clinical signs of BTV following challenge and its temperature remained below 40 ◦C, but did develop a viraemia from 2 dpi up to the end of the study (Fig. 2b). Slightly lower levels of viral RNA (as measured by Ct values) were seen in this animal compared to the sheep in the unvaccinated control group (Fig. 2a and b). Interestingly when samples from this animal were tested by both cELISA, sELISA and SNT at various times post-vaccination (7, 14, 21, 28 and 56 dpv) the animal did not seroconvert and no neutralising antibodies Fig. 1. Box and whisker plot (produced using R, version 2.9.0) showing the distribu- were detected at any of these time-points. Neutralising antibodies tion of serum neutralisation titres (log 10) in sheep after first and second vaccination increased significantly in all vaccinated sheep following challenge with an inactivated BTV-8 vaccine. Titres of <1.0 were rounded to 1.0 and titres of >3.1 were rounded to 3.1. (Table 3). C.A.L. Oura et al. / Vaccine 27 (2009) 7326–7330 7329 sELISA + + + + + + + + + + cELISA + + + + + + + + + + 10 Log dpi 2.7 2.4 2.4 2.8 2.8 2.7 2.5 3.0 2.7 >3.1 SNT 21 sELISA + + + + + + + + + + Fig. 3. Mean rectal temperatures of control unvaccinated sheep (group 1) and vaccinated sheep (group 2) after challenge with BTV-8. cELISA + + + + + + + + + + 4. Discussion 10 Inactivated vaccines have been used in many countries across Log Europe to combat outbreaks of disease and to control virus circula- dpi SNT 8 1.9 1.3 1.6 2.4 3.0 3.0 2.5 3.0 1.8 2.2 tion caused by various BTV serotypes. A double dose of monovalent inactivated vaccine directed against BTV-2, 4 and 16 and a bivalent vaccine for BTV-2 and 4 have been shown to protect sheep sELISA + + + + + + + + + + from both viral replication and clinical disease [12,13]. There has been some debate as to whether a single dose of inactivated vaccine is sufficient to provide long-term protection. However, a single cELISA − − − + + + + + + + dose of BTV-2 inactivated vaccine was shown to fully protect sheep against both clinical signs and viraemia for at least 12 months [14]. 10 With the rapid expansion of BTV-8 across Europe came the

Log urgent need to develop safe and effective inactivated BTV-8 vac- dpi 1.0 1.6 2.2 2.2 2.4 3.0 1.9 2.4 <1 SNT 6 cines. Various pharmaceutical companies have produced vaccines that came onto the market throughout 2008. Some vaccine produc- BTV-8. ers started to produce inactivated BTV vaccines for the ﬁrst time, with sELISA + + + + + + using different protocols and adjuvants, so it was essential that each individual vaccine underwent appropriate safety and efﬁcacy testing prior to use. However, due to the urgency of the situa- tion and the need to vaccinate animals early in 2008, prior to the cELISA −− −− <1 −− −− − − + + + + challenged BTV transmission season, many of these vaccines were licensed 10

sheep for use without the usual rigorous efﬁcacy testing. Some vaccine companies recommended a single initial dose for sheep whereas Log dpi

1.6 other companies recommended an initial course of two doses. A 1.5 1.5 2.5 3.0 1.9 1.8 SNT 4 very important question that needed to be addressed was whether vaccinated sheep would be protected, following a single shot of vaccine, for

and the entire length of the midge season which is up to 10 months in sELISA + + + + + + some southern European countries. A vaccine trial was carried out in Germany to assess the efﬁcacy of three inactivated monovalent vaccines selected for the Ger- −− <1 cELISA −− <1 −− <1 −− − − + + − + unvaccinated man compulsory vaccination programme in 2008 [15]. This study of showed that all the vaccinated sheep that seroconverted were fully 10 protected against clinical disease and viral replication and only one groups Log

dpi of the vaccinated animals was weakly positive in the real-time RT- in 1 1 1.2 1.6 1.6 1.5 1.9 1.3 <1 <1 SNT − 1 PCR. However, the inactivated BTV-8 vaccine selected for use in the

tests UK in 2008 was not included in the German study.

ID In this study, there was a poor correlation between the absence

ELISA of antibodies, measured using a cELISA, and protection, as 3 out of 7 1 2< 3< 4 5 6 7 8 9 and Animal 10 sheep that were negative in the cELISA after vaccination were pro- IDvet tected fully both clinically and virologically. Antibodies detected BTV − 8. Test. (SNTs) by the ELISA kits are directed against the group-speciﬁc inner Sheep with capsid protein (VP-7) whereas neutralising antibodies are raised Tests Sheep

ELISA. against serotype-speciﬁc VP-2 and VP-5 [16]. There was however a ELISA. good correlation between the presence of neutralising antibodies infection at the time of challenge and both clinical and virological protection. Neutralisation result. unvaccinated vaccinated post result. This study suggests that, if an animal has neutralising antibod- 1 2

Neutralisation ies to a particular serotype present at the time of challenge, it 3 is likely to be protected against that serotype. However, it has Negative Group Group Positive sELISA—sandwich SNT—Serum dpi—days cELISA—competitive + Table Serum − also been reported that animals are protected in the absence of 7330 C.A.L. Oura et al. / Vaccine 27 (2009) 7326–7330 neutralising antibodies [13] and that animals which seroconvert Acknowledgements following vaccination, although negative at the time of challenge, are still protected against both viraemia and clinical signs when This work was funded by the Department for the Environment, challenged with the homologous virulent serotype [17]. In this Food and Rural Affairs (DEFRA). The authors would like to thank study the vaccinated animal (sheep 4) that was not fully protected the staff involved in the Veterinary Laboratories Agency (VLA) BSL- on challenge with BTV-8 showed no evidence of serconversion 3 facility especially Derek Clifford and Angela Burke and the staff or neutralising antibody titres at any time-point post-vaccination. from ADAS Arthur Rickwood for provision of the sheep in the study. This sheep was protected from clinical signs and its body temperature remained below 40 ◦C (in contrast to the unvaccinated control References sheep) however we cannot exclude the possibility that this ani- [1] Saegerman C, Berkvens D, Mellor PS. Bluetongue epidemiology in the European mal was improperly vaccinated which could have resulted in only Union. Emerging Infectious Diseases 2008;14(4):539–44. partial protection. [2] Hoffmann B, Sasserath M, Thalheim S, Bunzenthal C, Strebelow G, Beer M. 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