A novel subunit ISCOM vaccine against bovine virus diarrhoea virus

Rev. sci. tech. Off int. Epiz., 1992, 11 (3), 873 877 A novel subunit ISCOM vaccine against bovine virus diarrhoea virus S. KAMSTRUP, L. RONSHOLT, M....
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Rev. sci. tech. Off int. Epiz., 1992, 11 (3), 873 877

A novel subunit ISCOM vaccine against bovine virus diarrhoea virus S. KAMSTRUP, L. RONSHOLT, M.H. JENSEN and K. DALSGAARD *

Summary: The preparation and preliminary testing of a subunit ISCOM (immunostimulating complex) vaccine against bovine virus diarrhoea virus (BVDV) is described. Vaccination of calves with this vaccine yields high neutralising titres against a panel of Danish BVDV field isolates. The serological difference between virus isolates and vaccine stram selection is discussed. K E Y W O R D S : Bovine diarrhoea virus - ISCOM - Pestivirus - Vaccines.

Bovine virus d i a r r h o e a ( B V D ) is a n e c o n o m i c a l l y i m p o r t a n t disease of cattle w o r l d ­ wide. In r e c e n t y e a r s , i n s i g h t h a s b e e n g a i n e d i n t o t h e p a t h o g e n e s i s of this d i s e a s e , and t h e i m p o r t a n c e o f p e r s i s t e n t l y i n f e c t e d c a r r i e r a n i m a l s h a s b e e n p r o v e n (2, 9). These c a r r i e r a n i m a l s a r e f o u n d in f r e q u e n c i e s as h i g h as 1 % in d a i r y a n d b e e f h e r d s in D e n m a r k (7). T h e s e a n i m a l s act as s o u r c e s of i n f e c t i o n for p r e g n a n t s e r o n e g a t i v e animals a n d t h e i r f o e t u s e s , t h e r e b y g i v i n g rise t o a b o r t i o n s a n d m a l f o r m e d o r persistently i n f e c t e d c a l v e s . T h e s e p e r s i s t e n t l y infected a n i m a l s will eventually d e v e l o p the m u c o s a l d i s e a s e s y n d r o m e . D e t e c t i o n of carrier a n i m a l s is n o t easy, a n d is possible only b y d e m o n s t r a t i n g p e r s i s t e n t v i r a e m i a . It is n o w a c c e p t e d t h a t i m m u n i s a t i o n of t h e d a m in o r d e r t o p r e v e n t t h e f o e t u s from b e c o m i n g i n f e c t e d w o u l d b e t h e m o s t effective w a y of a v o i d i n g t h e losses c a u s e d by this v i r u s . T h i s i m m u n i s a t i o n m a y b e a c h i e v e d b y v a c c i n a t i n g t h e d a m p r i o r t o mating. F o r this p u r p o s e a new subunit vaccine has been developed, based on the ISCOM (immunostimulating complex) technology. B V D v i r u s ( B V D V ) g r o w s t o o n l y m o d e r a t e titres in cell c u l t u r e , a n d t h e m a s s of s o l u b l e a n t i g e n is g e n e r a l l y l o w c o m p a r e d t o o t h e r v i r u s e s . It h a s b e e n s h o w n t h a t t r e a t m e n t o f v i r u s - i n f e c t e d cells w i t h T r i t o n X - 1 0 0 releases a l a r g e a m o u n t of i m m u n o l o g i c a l l y i m p o r t a n t a n t i g e n s , as m e a s u r e d b y c r o s s e d I m m u n o e l e c t r o p h o r e s i s (4) or by e n z y m e - l i n k e d i m m u n o s o r b e n t a s s a y ( E L I S A ) ( L . R 0 n s h o l t , u n p u b l i s h e d findings). T h i s a m o u n t is c l e a r l y l a r g e r t h a n t h a t r e l e a s e d f r o m t h e cells in t h e f o r m of viral p a r t i c l e s (3). T h e r e f o r e , t h e a p p r o a c h a d o p t e d b y t h e a u t h o r s h a s b e e n t o p r o d u c e l a r g e a m o u n t s o f a n t i g e n in cells, r e l e a s e t h e c e l l - a s s o c i a t e d a n t i g e n w i t h Triton X - 1 0 0 a n d u s e s u c h a n t i g e n for p r e p a r i n g a v a c c i n e . E a r l i e r e x p e r i m e n t s h a v e shown t h a t t h i s a n t i g e n , w h e n p r o p e r l y a d j u v a n t e d , gives at least p a r t i a l p r o t e c t i o n against i n f e c t i o n w i t h B V D V in a c h a l l e n g e e x p e r i m e n t (8). T o i m p r o v e t h e s e r e s u l t s , the a n t i g e n s h a v e n o w b e e n c o n c e n t r a t e d a n d p a r t l y p u r i f i e d .

* Ministry of Agriculture, State Veterinary Institute for Virus Research, Lindholm, DK 4771 Kalvehave, Denmark.

874 T h e adjuvant system used has also been i m p r o v e d . W h e n f o r m u l a t i n g inactivated v a c c i n e s , it is v e r y i m p o r t a n t t o c h o o s e a p o t e n t a d j u v a n t s y s t e m . F o r t h i s p u r p o s e Q u i l A h a s b e e n c h o s e n , as this s a p o n i n h a s p r o v e d t o b e o n e of t h e s t r o n g e s t a d j u v a n t s for p r e s e n t i n g m e m b r a n e a s s o c i a t e d a n t i g e n s t o t h e i m m u n e s y s t e m (1). T h e a n t i g e n e x t r a c t e d u s i n g T r i t o n X - 1 0 0 is c h r o m a t o g r a p h e d o n a n e w t y p e of c h r o m a t o g r a p h i c m e d i u m called " M a t r e x Cellufine S u l p h a t e " ( A m i c o n ) . T h i s m a t e r i a l preferentially binds the i m m u n o g e n i c p r o t e i n s of B V D V p r o t e i n s r e n d e r e d soluble b y T r i t o n X 100 t r e a t m e n t a n d c o m p l i e s w i t h g o o d m a n u f a c t u r i n g p r a c t i c e . T h e m a t r i x is a u t o c l a v a b l e , w i t h s t a n d s 0.1 M s o d i u m h y d r o x i d e a n d f o r m a l d e h y d e , a n d d o e s not bind pyrogens, bovine serum a l b u m i n or bovine i m m u n o g l o b u l i n s , which m e a n s t h a t t h e s e c o n t a m i n a n t s c a n b e w a s h e d a w a y easily. T h e p r o c e s s is e a s y t o scale u p . Once the antigens are b o u n d to the c o l u m n a n d w a s h e d , they m a y be eluted by a single b u f f e r s t e p . By e x c h a n g i n g t h e T r i t o n X - 1 0 0 b u f f e r w i t h a b u f f e r c o n t a i n i n g Q u i l A , a n e l u t i o n step h a s b e e n successfully d e v e l o p e d d u r i n g w h i c h I S C O M s are f o r m e d . A n I S C O M c o n s i s t s of a n t i g e n s i n s e r t e d i n t o a m a t r i x f o r m e d b y Q u i l A , c h o l e s t e r o l a n d p h o s p h a t i d y l c h o l i n e (6). T h e t e c h n i q u e u s e d is a s i m p l e i n d u s t r i a l p r o c e s s for t h e p r o d u c t i o n of I S C O M s . It h a s m a j o r t e c h n i c a l a d v a n t a g e s o v e r t h e m o r e s o p h i s t i c a t e d t e c h n i q u e s c o m m o n l y a p p l i e d in l a b o r a t o r y - s c a l e p r o d u c t i o n s . T o d a t e , t h e r e is o n l y o n e c o m m e r c i a l l y a v a i l a b l e I S C O M v a c c i n e ( a g a i n s t e q u i n e influenza virus). T h e a u t h o r s h a v e p r e p a r e d I S C O M v a c c i n e s a g a i n s t B V D V , u s i n g t w o different D a n i s h s t r a i n s of B V D V for a n t i g e n p r o d u c t i o n . T h e t w o s t r a i n s ( U g 5 9 a n d 258) are b o t h of t h e c y t o p a t h o g e n i c b i o t y p e . T h e y w e r e selected b e c a u s e t h e y r e p r e s e n t quite different v a r i a n t s of t h e a n t i g e n i c s p e c t r u m o f B V D V . T r a d i t i o n a l l y , n o distinct s e r o t y p e s h a v e b e e n d e f i n e d for B V D V , a l t h o u g h i n d i v i d u a l i s o l a t e s m a y s h o w c o n s i d e r a b l e differences in c r o s s n e u t r a l i s a t i o n t e s t s . In testing these vaccines, t h r e e g r o u p s of t h r e e a n i m a l s e a c h w e r e v a c c i n a t e d twice, t h r e e w e e k s a p a r t , w i t h v a c c i n e b a s e d o n e i t h e r m o n o v a l e n t a n t i g e n ( U g 5 9 o r 258) o r b i v a l e n t a n t i g e n ( U g 5 9 a n d 258 v a c c i n e s in c o m b i n a t i o n ) . S e r u m s a m p l e s t a k e n t w o w e e k s after t h e s e c o n d injection w e r e t e s t e d in a v i r u s n e u t r a l i s a t i o n a s s a y against a n u m b e r of D a n i s h B V D V i s o l a t e s ( F i g . 1). C o n s i d e r a b l e differences in n e u t r a l i s a t i o n o f i n d i v i d u a l D a n i s h field i s o l a t e s of B V D V were observed, with serum neutralising titre (SNT) values for the Ug59-based vaccine differing b y u p t o 30 t i m e s against t h e serologically m o s t d i s t a n t i s o l a t e s (Ug59 a n d 10039). H o w e v e r , t h e n e u t r a l i s i n g titres a r e g e n e r a l l y m u c h h i g h e r t h a n p r e v i o u s l y o b t a i n e d w i t h i n a c t i v a t e d v a c c i n e s , a n d t h e v a c c i n e s u s e d i n t h i s s t u d y w e r e active against all isolates tested. T h e a n i m a l s v a c c i n a t e d w i t h b o t h s t r a i n s o f B V D V (bivalent vaccine) s h o w S N T v a l u e s as h i g h o r h i g h e r t h a n t h e a n i m a l s in t h e o t h e r t w o g r o u p s . C o m p a r i n g t h e t w o m o n o v a l e n t v a c c i n e s , it s e e m s t h a t t h e v a c c i n e b a s e d o n strain 258 i n d u c e s a slightly b e t t e r overall r e a c t i o n a c r o s s t h e r a n g e o f B V D V i s o l a t e s used in t h e test. F u r t h e r e x p e r i m e n t s a r e n e e d e d t o d e t e r m i n e w h e t h e r a m o n o v a l e n t v a c c i n e is sufficiently effective. T h e u l t i m a t e test for this v a c c i n e is a c h a l l e n g e e x p e r i m e n t in p r e g n a n t c a t t l e , to determine whether the vaccine can protect against intrauterine infection. H o w e v e r , t h e r e is c i r c u m s t a n t i a l e v i d e n c e t h a t significant levels o f n e u t r a l i s i n g a n t i b o d i e s in the d a m are capable of protecting the foetus ( 5 , 8 ) . Since B V D V p r o d u c e s a viraemic

Serum neutralising titres (SNT) *

875

Virus isolate neutralised

vaccine based on Ug59 vaccine based on Ug59 and 258 vaccine based on 258 * SNT obtained with immunostimulating complex (ISCOM) vaccines, measured against a number of Danish field isolates (titres given as the reciprocal o f the highest dilution neutralising 100 T C I D ) 50

FIG.

1

V a c c i n a t i o n o f cattle against b o v i n e virus diarrhoea virus w i t h a subunit Q u i l A / I S C O M vaccine stage early a f t e r i n f e c t i o n , s u c h a c o r r e l a t i o n w o u l d s e e m r e a s o n a b l e . A t p r e s e n t , no m i n i m u m p r o t e c t i v e S N T v a l u e h a s b e e n a s c e r t a i n e d , a l t h o u g h it h a s b e e n suggested that t h e r e m i g h t b e a c e r t a i n c r i t i c a l level b e l o w w h i c h t h e r e is n o p r o t e c t i o n (8). In c o n c l u s i o n , a s u b u n i t I S C O M v a c c i n e a g a i n s t B V D V h a s b e e n d e v e l o p e d , capable o f i n d u c i n g h i g h s e r u m n e u t r a l i s i n g titres n o t o n l y a g a i n s t h o m o l o g o u s strains but also a g a i n s t a n u m b e r o f different D a n i s h field i s o l a t e s . T h e p r e p a r a t i v e p r o c e d u r e is s u i t a b l e f o r s c a l i n g u p a n d is b a s e d o n o n l y a few s t e p s . T h e v a c c i n e is c u r r e n t l y being t e s t e d i n c l i n i c a l t r i a l s .

UN N O U V E A U V A C C I N SOUS-UNITAIRE A COMPLEXE IMMUNOSTIMULANT (ISCOM) CONTRE LE VIRUS D E LA DIARRHÉE VIRALE BOVINE. - S. Hamstrup, L. R0nsholt, M . H . Jensen et K. Dalsgaard. Résumé : Les auteurs décrivent la préparation et les premiers essais concernant un vaccin sous-unitaire à complexe immunostimulant (ISCOM) contre le virus de la diarrhée virale bovine (VDVB). L'administration de ce vaccin à des veaux entraîne l'apparition de titres d'anticorps neutralisants élevés contre toute une

876 série de souches du VDVB trouvées au Danemark. Dans la discussion, les auteurs abordent les différences sérologiques entre les souches virales isolées sur le terrain et les souches vaccinales sélectionnées. MOTS CLÉS : ISCOM - Pestivirus

Vaccins - Virus de la diarrhée virale bovine.

*

UNA NUEVA VACUNA COMPLEJA INMUNOESTIMULANTE (ISCOM) SUBUNITARIA CONTRA EL VIRUS D E LA DIARREA VÍRICA BOVINA. S. Hamstrup, L. Ronsholt, M.H. Jensen y K. Dalsgaard. Resumen: Los autores describen la preparación y las pruebas preliminares de una vacuna compleja ininunoestimulante (ISCOM) subunitaria contra el virus de la diarrea vírica bovina. La vacunación de terneros con esta vacuna produce elevados títulos neutralizantes contra un grupo de virus de la diarrea vírica bovina daneses aislados en el campo. El artículo también comenta la diferencia serológica entre los virus aislados y una selección de cepas vacunales. PALABRAS CLAVE: ISCOM bovina.

Pestivirus - Vacunas - Virus de la diarrea vírica

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T h e efficacy

of an experimental inactivated BVD-MD vaccine. In Agriculture. Pestivirus infections of ruminants ( J . W . Harkness, ed.). C E C publication EUR 10238 E N , 233-250. 6. H O G L U N D S., D A L S G A A R D K., L O V G R E N K., S U N D Q U I S T B . , O S T E R H A U S A . & M O R E I N B .

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7. MEYLING A. (1984). Detection of BVD virus in viremic cattle by a n indirect immunoperoxidase technique. In Recent advances in virus diagnosis ( M . S . McNulty & J . B . McFerran, eds). Martinus Nijhoff, The Hague, 37-46.

877 8. M E Y L I N G A . , R O N S H O L T L . , D A L S G A A R D K . & J E N S E N A . M . ( 1 9 8 7 ) .

Experimental

exposure of vaccinated and non-vaccinated pregnant cattle to isolates of bovine viral diarrhoea virus ( B V D V ) . In Agriculture. Pestivirus infections of ruminants ( J . W . Harkness, ed.). CEC publication E U R 1 0 2 3 8 E N ,

225-231.

9. M O E N N I G V . , F R E Y H . R . , L I E B L E R E . , P O H L E N Z J . & L I E S S B . ( 1 9 9 0 ) .

Reproduction

of mucosal disease with cytopathogenic bovine viral diarrhoea virus selected in vitro. Vet. Rec.,

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