Protective effect of an ISCOM bovine virus diarrhoea virus (BVDV) vaccine against an experimental BVDV infection in vaccinated and non-vaccinated pregnant ewes

Protective effect of an I S C O M bovine virus diarrhoea virus (BVDV) vaccine against an experimental B V D V infection in vaccinated and non-vaccinated pregnant ewes U . C a r l s s o n *§, S. A l e n i u s * a n d B. S u n d q u i s t ~

Fifteen pregnant ewes were vaccinated twice with an experimental immunostimulating complex (ISCOM) subunit vaccine designed to contain the envelope proteins of a Danish cytopathic bovine virus diarrhoea virus ( B VD V). The serological responses were measured in E L I S A and virus neutralization (VN) tests. All ISCOM-vaccinated ewes developed high VN antibody titres to B V D V in contrast to the 14 non-vaccinated ewes. Both groups of ewes were challenged parenterally when 48-65 days pregnant with a Swedish cytopathic BVD V isolate. In the vaccinated group 26 fetuses out of 29 detected by ultrasound were liveborn, whereas only six out of 26 were liveborn in the non-vaccinated group. It is concluded that the I S C O M vaccine had the potential of eliciting high VN titres as well as protecting fetuses against transplacental infection after challenge with a virulent B VD V isolate. Keywords: Border disease virus; bovine virus diarrhoea virus; ISCOMs; sheep; vaccine

INTRODUCTION Border disease virus (BDV) and bovine virus diarrhoea virus (BVDV) as well as the related hog cholera virus (HCV) belong to the genus Pestivirus and currently hold generic status in the family Togaviridael. Recent findings have shown similarities between pestiviruses and flaviviruses but, before reclassification of the pestiviruses can be considered, further molecular data are needed z. Infections with BDV in sheep and BVDV in cattle are usually subclinical. However, transplacental infection in early and mid-gestation with BDV or BVDV may in susceptible animals lead to abortion, teratogenic defects, or to the birth of lambs or calves persistently infected with a non-cytopathic biotype of the virus. Such animals act as transmitters of the virus and can cause serious losses on individual farms. In ewes, the infection may also lead to the birth of small weak lambs that tremble and/or have abnormal, hairy fleeces. The terms BDV and BVDV usually refer to the host species, indicating that the isolate is of ovine or bovine origin respectively, but cross-infections to alternative host species are known to *The Swedish University of Agricultural Sciences, Faculty of Veterinary Medicine, Department of Cattle and Sheep Diseases, Box 7019, S-750 07 Uppsala, Sweden. tNational Veterinary Institute, Division of Cattle and Swine, Box 7073, S-750 07 Uppsala, Sweden. tNational Veterinary Institute, Division of Vaccine Research, Box 7073, S-750 07 Uppsala, Sweden. ~To whom correspondence should be addressed. (Received 31 January 1991; revised 14 March 1991; accepted 18 March 1991) 0264-410X/91/080577-04 © 1991 Butterworth-Heinemann Ltd

occur 3 and BVDV strains have been shown to induce disease in sheep flocks 4. An effective prophylaxis against disease and reproductive disorders caused by BDV or BVDV would be to vaccinate ewes and cattle before breeding. So far, commercial BVDV vaccines, whether attenuated live or inactivated, have shown various disadvantages 5'6. Therefore, an alternative to conventional vaccines is the use of immunostimulating complexes (ISCOMs), which has proved to be safe and effective and to induce protective immunity in various animal species 7. It has recently been reported that ISCOM-based vaccines can prime M H C class 1-restricted cytotoxic T-lymphocytes (CTL), which suggests that ISCOM vaccines can induce both a CTL and an antibody response to a virus antigen 8. The purpose of the present study was to test the immunogenicity of an ISCOM-vaccine in pregnant ewes and the protective effect was followed up by a challenge infection with a field strain of BVDV. MATERIALS

AND

METHODS

Test animals

Twenty-nine ewes (Swedish landrace, pelt-type), shown to be free of neutralizing antibody to BVDV, were used. They were tupped with a raddled ram, antibody and virus-negative with respect to BVDV, and service dates were recorded. The ewes were fed daily with 1.2 kg of good quality hay and 0.1 1.0 kg of concentrates according to their reproductive stage. All ewes were allocated to

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Protection of pregnant ewes by ISCOM BVDV vaccine: U. Carlsson et al.

one of three groups and each group was penned in a separate ward with no contact until the lambing period was over. The first group consisted of ten vaccinated, the second group of ten non-vaccinated and the third group of five vaccinated and four non-vaccinated ewes, respectively. Diagnosis of pregnancy and number of fetuses were determined by real-time ultrasonic imaging, prior to the experimental infection. Scanning was thereafter performed regularly throughout the trial period.

Preparation of the ISCOM vaccine Bovine virus diarrhoea virus was propagated on freshly confluent bovine turbinate (BT) cells grown in suspension culture on Cytodex microcarriers (Pharmacia Fine Chemicals, Uppsala, Sweden) in Eagles minimal medium (MM) supplemented with 5% fetal calf serum free of non-cytopathic BVDV. For vaccine production a cytopathic BVDV isolate (Ug-59) was used 9. The confluent BT cell culture was inoculated with 105 tissue culture infective dosesso (TCIDso) per ml and incubated at 37'C. The cell culture medium was harvested the third day post infection when cytopathic effect was noticed. The virus was affinity purifed on a lectin column of Crotalaria juncea as described by Coria et al. TM. The virus was solubilized with N-decanoyl-N-methyl-glycoside (MEGA-10) 11 at a final concentration of 1% and was incubated at room temperature for 2 h. To the solubilized virus sample Quil A was added to a final concentration of 0.1%. The mixture was dialysed extensively against 0.05 M a m m o n i u m acetate buffer at room temperature. Total protein content was determined with the Coomassie brilliant blue day-binding method according to Bradford 12 and protein analysed by S D S - P A G E and electron microscopy as described previously 13

Serological assays An indirect enzyme linked immunosorbent assay (i-ELISA) was used for detection of antibodies to BVDV according to Juntti et a1.14. The cytopathic BVDV strain Ug-59 was used as antigen in the i-ELISA. Sera from vaccinated ewes were also tested with a microneutralization test 15. Twofold dilutions of sera were each incubated with 100 TCID~o Ug-59 virus at 37~C for 1 h and the mixtures were then inoculated into BT cell culture grown in Nunclon microwell plates (Nunc Intermed, Roskilde, Denmark). The VN titre was read when complete cytopathic effect (CPE) was seen in the control wells. Titres are expressed as the reciprocal of the highest dilution of serum that prevented CPE in 50% of the replicate cultures.

Virus isolation Serum samples were examined for the presence of BVDV by inoculation of 0.15 ml of each sample into two culture tubes containing embryonic BT cells. After one passage, 0.15 ml of the supernatant was inoculated into coverslip cultures of BT cells. The cultures were examined for cytopathic effect, and the presence of pestivirus antigen was determined by indirect immunofluorescence. RESULTS

The protein pattern of the BVDV ISCOM The Crotalaria lectin affinity-purified BVDV material displayed three major bands having estimated molecular weights of 75, 65 and 56 kDa (Figure la) as analysed by

ab

Vaccination and sampling procedures After the service period 15 ewes were given two doses of the B V D V - I S C O M vaccine 3 weeks apart. On each occasion 2 ml vaccine containing 50~g protein was administered, equally divided between the subcutaneous and intramuscular routes. Serum samples were collected from the vaccinated and the 14 non-vaccinated ewes 1 day before vaccination and 4 weeks later. When possible, serum was also collected from lambs born in both groups before colostrum intake. Sixteen lambs in the vaccinated group, as well as the six lambs in the non-vaccinated group, were blood sampled in connection with lambing and again when 6 months old. Clotted blood samples were collected into Vacutainer tubes (Becton-Dickinson). Sera for antibody determination and virus isolation were stored at - 2 0 and -70'~C respectively, until analysis.

95 64

45

30

Challenge procedure A Swedish cytopathic strain of BVDV, designated EC-82, was used for challenge. It was isolated from the gut of a bull that died of typical mucosai disease in 1982. The isolate was propagated on BT cells and was used at the third cell culture passage level. Three weeks after the second vaccination, at 47 64 days gestation, vaccinated and non-vaccinated ewes were challenged. Each ewe received 3 ml of the challenge virus (105 TCIDso ml-1); l ml was administered intramuscularly and 2 ml subcutaneously.

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20

Figure 1 SDS-PAGE showing the polypeptide pattern of BVDV proteins purified by Crotalaria juncea affinity chromatography; (a) molecular weight reference, (b) BVDV proteins. Electron micrograph of ISCOMs prepared with the same Crotalaria-selected BVDV proteins, b a r : 100 nm

Protection of pregnant ewes by ISCOM BVDV vaccine: U. Carlsson e t al.

SDS-PAGE, thus closely resembling the protein pattern demonstrated by others 1°. Electron microscopy of the vaccine revealed the presence of ISCOM-Iike particles measuring 35 55 nm in diameter (Figure Ib).

well with those obtained by the i-ELISA (250-1250), whereas the control animals remained seronegative

(Table 1). Clinical evaluation of vaccine study

Development of ELISA and VN titres after vaccination All ewes were shown to be free of antibodies to BVDV before vaccination. The 15 vaccinated ewes seroconverted and 1 week after the second vaccination the serum neutralizing titres (ranging from 1 : 128-1 : 2048) correlated Table 1 Development of ELISA and neutralizing antibodies in e w e s following vaccination with a BVDV-ISCOM vaccine ELISA

VN antibodies a

Ewe

no.

T1

T2

T1

T2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16-29 b

< 10 < 10 < 10 < 10 < 10 <10 <10 < 10 <10 < 10 <10 < 10 < 10 < 10 <10 <10

1250 250 1250 250 250 1250 250 1250 250 250 1250 1250 1250 250 250 <10

< 1:4 < 1:4 < 1:4 < 1:4 < 1:4 <1:4 <1:4 < 1:4 <1:4 < 1:4 <1:4 < 1:4 < 1:4 < 1:4 <1:4 <1:4

1:256 1:256 1:1024 1:256 1:256 1:512 1:128 1:256 1:256 1:512 1:1024 1:2048 1:1024 1:128 1:128 <1:4

Ewes 1-15 were vaccinated twice, 3 weeks apart. Titres 1 day before the first vaccination (T1) and 1 week after the second vaccination (T2). aThe titre was determined by serum neutralization test and was expressed as the reciprocal of the highest dilution of serum that prevented cytopathogenic effect in 50% of the replicate cultures. bEwes 16-29 served as non-vaccinated control animals

Group 1 (vaccinated). Before challenge 29 fetuses were detected by ultrasound. Twenty-six viable lambs were born (Table 2). One ewe, no. 8, was killed because of dystocia. At autopsy, two lambs were found, one fully developed and one mummified. Another ewe, no. 15, lambed one liveborn and one stillborn. Precolostral sera from these two lambs proved antibody-positive and virus-negative. Sera obtained before colostrum intake from seven other lambs in this group had no detectable antibodies and were not viraemic. In 16 lambs out of 26 that were tested, maternal antibodies were detected and when retested at 6 months of age they were both antibody and virus negative. Group 2 (non-vaccinated). In the control group, 26 fetuses were recorded before challenge (Table 2). Fetal death was already observed 14 days alter the experimental infection. The lambing results in this group were six liveborn and one stillborn. The remaining 19 fetuses in the group were either resorbed, mummified, aborted or autolysed and expelled as vaginal discharge. Sera obtained before suckling from five liveborns were assayed for antibodies to BVDV. All were found to be seropositive and virus-negative, as was the stillborn lamb. The six liveborn lambs in this group still had antibody titres when tested 6 months later. DISCUSSION In the present study we have shown that a BVDVI S C O M vaccine was effective in inducing VN titres in

Table 2

Results of a challenge infection with a cytopathic strain of BVDV in pregnant sheep. Fifteen ewes were vaccinated and 14 ewes served as non-vaccinated controls Vaccinated

Ewe no. 1 2 3 4 5 6 7 8~ 9 10 11 12 13 14 15

Non-vaccinated

No. of fetuses"

Challenge (days post conception)

No. of liveborn lambs/outcome of pregnancy

3 2 2 2 2 2 2 2

54 54 47 63 49 62 49 48

2 2 1 1 2 2 2

49 59 48 63 60 62 47

3 2 2 2 2 2 2 1 fully developed 1 mummified 2 2 1 1 2 2 1 1 stillbirth

Total no. of liveborn lambs

26

Ewe no.

No. of fetuses

Challenge (days post conception)

No. of liveborn lambs/outcome of pregnancy

16 17

2 2

62 52

18 19

2 2

61 61

20

2

50

21 22

2 2

56 61

23

2

52

24 25 26 27 28 29

2 2 1 1 2 2

48 48 62 54 48 64

2 Vaginal discharge 1 aborted Aborted 1 1 resorbed Vaginal discharge 1 aborted Vaginal discharge 1 1 resorbed 1 1 mummified Aborted Resorbed Resorbed Aborted Resorbed 1 1 stillbirth 6

aDetected by ultrasound prior to challenge. bThis e w e w a s killed at full term

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P r o t e c t i o n o f p r e g n a n t e w e s b y I S C O M B V D V vaccine: U. Ca'rlsson et al.

all vaccinated ewes. The titres ranged between 1 : 128 and 1:2048 and protected against fetal death following challenge with a cytopathic BVDV isolate. In the vaccinated group, 13 pregnant ewes out of 15 were fully protected against fetal death and they gave birth to 25 viable and healthy lambs with normal birth weights. Furthermore, a decline in antibody titres consistent with those maternally derived against BVDV was seen in the 16 tested lambs. However, the protective effect in ewe no. 15 was incomplete, since sera from her live- and stillborn lambs contained precolostral antibodies to BVDV indicative of a transplacental infection. The lack of protection in this ewe might have been caused by a lower VN titre (1:128) than was seen in the majority of the ewes. In ewe no. 8 (titre 1:256), protection was probably also incomplete, since one of her lambs was mummified. However, due to the parenteral challenge procedure the infection dose might have been too large to give a satisfactory protection in ewes 15 and 8. Ewes with even lower titres would probably have been protected had they been exposed to the infection under natural conditions. In group 2, the non-vaccinated pregnant ewes lost 20 out of 26 viable fetuses during the trial period. None of the six liveborn lambs were found to be persistently infected, but five had precolostral antibodies. These results agree with the findings of a recent study made on cattle using a cytopathic BVDV isolate for experimental infection. It was shown that heifers infected in early pregnancy did not produce persistently infected offspring, although stillbirths were recorded 16. However, the challenge isolate used in the present study was not plaque-purified and was most probably contaminated with a non-cytopathic biotype of the virus, explaining the large number of fetal deaths after challenge. The precolostral antibodies found in lambs in this group persisted after 6 months, which indicates an in utero infection. Infections with BDV and BVDV can cause considerable losses within sheep and cattle populations 1~'~8 and persistently infected animals are the most important transmitters of these infections. As has been shown in the present study, the ISCOM vaccine can safely be used in pregnant ewes, and effectively prevents transplacental infection. However, antigen variation is known to exist among BVDV isolates 19'2°. Similar suggestions have been reported regarding BDV 21'22. Therefore, it is of great importance that a vaccine containing a single strain should respond serologically with high VN titres capable of neutralizing a wide spectrum of strains. In the current study the ISCOM vaccine elicited VN titres which are comparable to those induced by a natural infection. Most importantly, a significant difference in the number of liveborn lambs between the two groups was evident. This experiment also showed that although the vaccine used was prepared from a Danish BVDV isolate, it protected ewes that were infected with a heterologous Swedish isolate. However, further investigations need to be carried out in order to ascertain whether the vaccine affords effective protection against various strains and challenge infection should preferably be carried out by bringing vaccinated animals in contact with those persistently infected. In such an experimental model the animals

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would be exposed to an appropriate amount of challenge virus, thus resembling infection under natural conditions. ACKNOWLEDGEMENTS The authors are grateful for the laboratory assistance of Ms Eva Blomkvist. This work was supported by grants from the Swedish Council for Forestry and Agricultural Research. REFERENCES 1

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