Chapter 25
Biotechnology in Livestock Production David E. Reed
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Molecular Genetics, Inc., 10320 Bren Road East, Minnetonka, MN 55391
The application of biotechnological advances to food animal production has been far more difficult and costly than was expected. This paper concerns technological approaches to improving veterinary vaccines and preventatives. In particular, some of the successes and failures will be discussed based upon considerations of technology, cost, and intended product use. Examples of biotechnologically derived products currently produced, are a monoclonal antibody product for prevention of calf diarrhea, a genetically modified porcine herpesvirus vaccine, and a subunit vaccine for prevention of calf diarrhea. Products not yet available but in final testing include bovine somatotropin, a subunit vaccine for prevention of pseudorabies in swine, and diagnostic tests which can distinguish infected animals from animals which have been vaccinated.
F o r t h e p u r p o s e s o f t h i s p a p e r , t h e term " b i o t e c h n o l o g y " i s d e f i n e d h e r e as recombinant DNA (rDNA) and hybridoma o r m o n o c l o n a l a n t i b o d y (MAB) t e c h n o l o g i e s . T r a n s l a t i o n o f b i o t e c h n o l o g y i n t o p r o d u c t s has been f a r more d i f f i c u l t t h a n most o f us imagined. A comprehensive l i s t o f t h e b i o t e c h n o l o g i c a l l y d e r i v e d products which a r e being d e v e l o p e d f o r v e t e r i n a r y u s e has been p u b l i s h e d ( 1 ) . T h i s p u b l i c a t i o n s t a t e s t h a t an i m p r e s s i v e number o f v e t e r i n a r y b i o t e c h n o l o g y p r o d u c t s i n c l u d i n g dozens o f v a c c i n e s and numerous p r e v e n t a t i v e s , growth promotants, and d i a g n o s t i c s w i l l be a v a i l a b l e between 1988 and 1996. I n most c a s e s , however, t h e problems i n t r a n s l a t i n g t h e t e c h n o l o g y i n t o p r o d u c t s have been b o t h u n d e r e s t i mated and u n d e r s t a t e d . The purpose o f t h i s paper i s t o g i v e a g e n e r a l overview o f c u r r e n t p r o g r e s s i n a p p l y i n g b i o t e c h n o l o g y t o improve v a c c i n e s , growth promotants, and t h e r a p e u t i c s used i n t h e p r o d u c t i o n o f livestock. T h i s d i s c u s s i o n i s n o t meant t o be an i n c l u s i v e l i s t o f
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t h e p r o d u c t s now i n development but w i l l f o c u s on t h e c o n c e p t s u s i n g examples, w i l l s i t e approaches w h i c h have f a i l e d as w e l l t h o s e w h i c h have succeeded. E a r l y S u c c e s s of
rDNA
and, as
Approach:
One o f t h e e a r l y s u c c e s s e s o f rDNA t e c h n o l o g y i n l i v e s t o c k p r o d u c t i o n was t h e c l o n i n g o f b o v i n e s o m a t o t r o p i n (BST). Expression systems which used t h e b a c t e r i u m E^_ c o l i a l l o w e d p r o d u c t i o n o f n e a r l y l i m i t l e s s q u a n t i t i e s o f BST as i n s o l u b l e a g g r e g a t e s o f p r o t e i n i n the bacterium. A f t e r i n i t i a l problems were s o l v e d by t h e use o f r e d u c i n g agents and s t r o n g c h a o t r o p i c agents t o s o l u b i l i z e t h e a g g r e g a t e d p r o t e i n , t h e E ^ c o l i produced BST was found t o be e q u a l i n p o t e n c y t o n a t u r a l l y d e r i v e d BST ( 2 ) . I t appears t h a t t h e r e m a i n i n g r e s e a r c h problems w i t h BST i n v o l v e development o f n o v e l d e l i v e r y systems which w i l l o b v i a t e t h e need f o r d a i l y i n j e c t i o n s . E x t e n d i n g the
r D N A A p p r o a c h to
Vaccine
Development
S u b u n i t v a c c i n e s . R e s e a r c h e r s i n s e v e r a l commercial l a b o r a t o r i e s , buoyed w i t h t h e rDNA s u c c e s s e s o f BST and o t h e r s m a l l ( * 2 0 , 0 0 0 D a l t o n MW) p e p t i d e s , p r e d i c t e d s i m i l a r s u c c e s s i n p r o d u c i n g v i r a l subunit proteins i n l i m i t l e s s q u a n t i t i e s . S u c c e s s i n c l o n i n g and e x p r e s s i n g a number o f v i r a l s u r f a c e p r o t e i n s has been a c h i e v e d . In most c a s e s , t h e i m m u n o l o g i c a l l y i m p o r t a n t s u r f a c e p o l y p e p t i d e s were i d e n t i f i e d and t h e genes s p e c i f y i n g t h o s e p e p t i d e s were i s o l a t e d , sequenced, and p l a c e d i n t o E^_ c o l i e x p r e s s i o n systems. Unfortunatel y , i n a l m o s t a l l t h e c a s e s , t h e immunizing p o t e n t i a l o f E^_ c o l i produced p o l y p e p t i d e s was s i g n i f i c a n t l y l e s s t h a n t h a t o f t h e n a t i v e v i r a l p o l y p e p t i d e and v a c c i n e s f o r m u l a t e d from t h e s e p o l y p e p t i d e s g e n e r a l l y were n o t e f f i c a c i o u s . The e x a c t r e a s o n s f o r t h e s e f a i l u r e s were d i f f i c u l t t o d e t e r m i n e . I t i s known t h a t d e n a t u r a t i o n of p o l y p e p t i d e s by s o l u b i l i z a t i o n p r o c e s s e s ( e . g . 1 0 M u r e a and 0 . 1 M d i t h i o t h r e i t o l t r e a t m e n t ) can d e s t r o y confirmâtion-dependent antigenic sites. I n a d d i t i o n , u n l i k e mammalian c e l l systems i n w h i c h mammalian v i r u s e s a r e produced, c e r t a i n p o s t - t r a n s c r i p t i o n a l or p o s t - t r a n s l a t i o n a l p r o c e s s i n g e v e n t s such as c l e a v a g e o r g l y c o s y l a t i o n do n o t o c c u r o r o c c u r d i f f e r e n t l y i n E^_ c o l i e x p r e s s i o n systems. I n our l a b o r a t o r y , we were s u c c e s s f u l i n e x p r e s s i n g i n E ^ c o l i c a n i n e and p o r c i n e p a r v o v i r u s s t r u c t u r a l proteins. These p r o t e i n s , when used t o immunize a n i m a l s , would produce a n t i b o d y w h i c h was r e c o g n i z e d i m m u n o l o g i c a l l y by a u t h e n t i c v i r u s p r o t e i n from i n f e c t e d mammalian c e l l s b u t , u n l i k e a u t h e n t i c v i r u s p r o t e i n , f a i l e d t o produce p r o t e c t i v e o r n e u t r a l i z i n g a n t i b o d y (3). I n a p o r c i n e h e r p e s v i r u s system, our l a b o r a t o r y compared t h e e f f i c a c y o f v a c c i n e s p r e p a r e d from E ^ c o l i produced v i r a l p r o t e i n w i t h t h a t o f v a c c i n e s made from a u t h e n t i c v i r a l p r o t e i n ( L I t a l i e n , Zamb, R o b b i n s , M a r s h a l l , u n p u b l i s h e d d a t a ) . Compared t o a u t h e n t i c v i r a l p r o t e i n , a p p r o x i m a t e l y 1 0 0 0 t i m e s more E ^ c o l i produced v i r a l p r o t e i n t h a n a u t h e n t i c v i r a l p r o t e i n was r e q u i r e d t o immunize a p i g . 1
The k i l l e d E ^ c o l i - BPV recombinant b a c t e r i n i s more s u c c e s s f u l example o f rDNA t e c h n o l o g y . T h i s p r o d u c t has been t e s t e d i n e x t e n s i v e immunogenicity t r i a l s i n c a t t l e and found t o be h i g h l y e f f i c a c i o u s i n p r e v e n t i n g warts (4).
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I f rDNA approaches t o s u b u n i t v a c c i n e s a r e t o be s u c c e s s f u l , t h e r e a r e some p o s s i b i l i t i e s t o i n c r e a s e t h e e f f i c a c y . A s t r a i g h t f o r w a r d a p p r o a c h i s t o i n t r o d u c e t h e genes f o r t h e immunogenic p r o t e i n s i n t o mammalian c e l l e x p r e s s i o n systems. U n f o r t u n a t e l y , t h i s has n o t been e a s y and p r o d u c t s produced by t h i s method have n o t y e t been c o m m e r c i a l i z e d . We can c o n c l u d e t h a t i t w i l l be, t e c h n i c a l l y f e a s i b l e t o p r o d u c e v i r a l s u b u n i t v a c c i n e s u s i n g rDNA t e c h n o l o g y . However, t h e c o s t o f t h e r e s e a r c h n e c e s s a r y t o complete development o f many o f t h e v e t e r i n a r y v a c c i n e s w i l l make i t e c o n o m i c a l l y n o t f e a s i b l e . The development o f t h e f i r s t rDNA v a c c i n e f o r f o r use i n a n i m a l s , f o o t & mouth d i s e a s e (FMD) o f c a t t l e , r e q u i r e d t h a t t h e immunizing s u b u n i t be d e f i n e d , and t h e gene c l o n e d and e x p r e s s e d . T h i s took a number o f y e a r s and t h e i n v e s t m e n t s o f s e v e r a l r e s e a r c h g r o u p s . In order t o make t h e rDNA v a c c i n e f o r FMD a p r a c t i c a l v a c c i n e , t h e r e s e a r c h , development, and c l i n i c a l t e s t i n g must be r e p e a t e d f o r each t y p e o f FMD v i r u s and p o s s i b l y f o r each s u b t y p e because t h e f i n a l FMD v a c c i n e p r o d u c t needs t o be m u l t i v a l e n t . T h i s w i l l p r e s e n t a r a t h e r f u n d a m e n t a l dilemma when t h e c o s t o f t h e r e s e a r c h b e g i n s t o exceed t h e p o t e n t i a l revenues f o r t h e p r o d u c t . The s u b u n i t v a c c i n e s seem t o be h i g h l y dependent upon a d j u v a n t s i n o r d e r t o be e f f i c a c i o u s . U n f o r t u n a t e l y , t h e a d o p t i o n o f new a d j u v a n t s has been slow. One i m p o r t a n t r e a s o n f o r t h i s i n v o l v e s t h e h i g h c o s t o f t a k i n g a p r o d u c t t h r o u g h t h e Food S a f e t y I n s p e c t i o n Service approval process. Because t h e mechanisms a r e so v a r i e d by w h i c h a d j u v a n t s p o t e n t i a t e immunologic r e s p o n s e t o an i n j e c t e d a n t i g e n , a d j u v a n t s s t i l l a r e p i c k e d by l a r g e l y e m p i r i c a l means. A d d i t i o n a l l y , t h e s p e c i e s - s p e c i f i c i t y o f many a d j u v a n t s makes i t d i f f i c u l t to e x t r a p o l a t e r e s u l t s obtained i n l a b o r a t o r y animals to r e s u l t s expected i n food animals. Live Virus Vaccines. T h e r e a r e two a r e a s i n l i v e v i r u s v a c c i n e development where t h e rDNA r e s e a r c h has been s u c c e s s f u l . T h a t i s i n p r o d u c t i o n o f l i v e v i r u s v a c c i n e s i n which a v i r u l e n c e gene has been d e l e t e d and p r o d u c t i o n o f l i v e v i r u s v a c c i n e s which a r e g e n e t i c recombinants between v a c c i n i a v i r u s and t h e immunizing s u b u n i t ( s ) genes o f another v i r u s . The t r a d i t i o n a l e m p i r i c a l methods o f a t t e n u a t i o n ( m u t a g e n e s i s , m u l t i p l e c e l l c u l t u r e passage, t e m p e r a t u r e s e l e c t i o n , and passage i n non-host a n i m a l s ) , e v e n t u a l l y s h o u l d be r e p l a c e d by t h e more e x a c t i n g rDNA methods whereby a v i r u l e n c e gene i s i d e n t i f i e d and deleted. An example o f t h i s i s t h e c o m m e r c i a l l y a v a i l a b l e p o r c i n e h e r p e s v i r u s v a c c i n e i n w h i c h t h e v i r a l t h y m i d i n e k i n a s e (TK) gene has been d e l e t e d ( 5 ) . The TK" v a c c i n e i s s a f e r t h a n t h e p a r e n t TK v a c c i n e by b e i n g l e s s n e u r o v i r u l e n t . L i v e v i r u s v a c c i n e s , however, a r e l e s s s a f e t h a n t h e n o n - l i v i n g v a c c i n e s because t h e e f f i c a c y o f the l i v e v i r u s vaccine r e q u i r e s that the vaccine v i r u s r e p l i c a t e i n the host animal. I n a d d i t i o n , the l i v e v i r u s v a c c i n e s , i n comparison t o t h e k i l l e d v a c c i n e s , a r e much more l i k e l y t o be c o n t a m i n a t e d w i t h p a s s e n g e r v i r u s e s o r mycoplasmas. Research success w i t h l i v e v i r u s v a c c i n e s which are recombinants w i t h v a c c i n i a has been r e p o r t e d commonly. F o r example, a recombinant v a c c i n i a v i r u s c a r r y i n g t h e R i f t V a l l e y f e v e r v i r u s (RVFV) G l and G2 g l y c o p r o t e i n genes c o n f e r r e d 90 - 100% p r o t e c t i o n +
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of mice challenged with v i r u l e n t v i r u s (6). In contrast, a RVFV subunit vaccine produced by rDNA methods i n E^ c o l i conferred only 56 - 70% protection. Unfortunately, because of the v a c c i n i a pathogenicity f o r humans, the recombinant v a c c i n i a approach to veterinary vaccine development i s burdened with safety r i s k s beyond that of conventional l i v e v i r u s vaccines. I t i s unclear whether or not U.S. Department of A g r i c u l t u r e regulatory clearance w i l l be forthcoming for v a c c i n i a recombinants. Monoclonal Antibody (MAB)
Technology
MABs f o r Treatment or Prevention of Disease. One MAB product curr e n t l y being sold f o r the prevention of animal disease i s a MAB against the K99 p i l u s of E^_ c o l i (7). Newborn calves infected with the K99 E^ c o l i often die of diarrhea. The product i s a s i n g l e o r a l dose of MAB given to calves within 12 hours of b i r t h . The mechanism of action i s not p r e c i s e l y determined but presumably the MAB blocks the attachment of p i l i a t e d K99 E^ c o l i to the i n t e s t i n a l w a l l . The e f f i c a c y of t h i s product i n f i e l d use appears to be very high. An a d d i t i o n a l MAB product has been developed f o r conferring passive immunity to young pigs i n the face of an outbreak of porcine herpesvirus. This product i s intended to reduce the death losses from pseudorabies. +
+
MABs f o r Producing Subunit Vaccines. Monoclonal a f f i n i t y chromatography technology has provided the t o o l s to make vaccines of unprecedented p u r i t y . Our company i s developing a vaccine f o r porcine herpesvirus using a f f i n i t y chromatography technology. The product i s prepared by extracting a s i n g l e surface glycoprotein from infected c e l l s . Beyond the obvious safety benefits of a highly pure product, the product has an a d d i t i o n a l advantage which i s c o m p a t i b i l i t y with a serologic t e s t f o r pseudorabies. Because pseudorabies i s a c o n t r o l l e d disease, any animal which i s s e r o l o g i c a l l y p o s i t i v e i s subject to r e s t r i c t i o n s on sale or shipment. A diagnostic t e s t which detects serologic response to a surface glycoprotein not included i n the vaccine w i l l detect infected pigs but w i l l not detect pigs vaccinated with the a f f i n i t y p u r i f i e d vaccine. Summary
In hindsight i t appears that s c i e n t i s t s and administrators i n both the public and p r i v a t e sectors have expected too much and too soon from rDNA technology. I t i s time to reassess both the technology and the needs. The research goals must be l i n k e d to products to improve l i v e s t o c k performance and not l i n k e d to a p a r t i c u l a r technology. For example, we must f i r s t ask what i s needed i n a foot & mouth disease (FMD) vaccine before we decide that rDNA technology can improve the product. One of the great promises of the rDNA technology i s reduced cost of vaccines. With FMD, cost of the vaccine has not been a major problem.
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The most p r e s s i n g problems o f FMD v a c c i n e s have been i n s a f e t y ( a l l e r g i c r e a c t i o n s and i n c o m p l e t e i n a c t i v a t i o n o f t h e v i r u s ) . O t h e r t e c h n o l o g i e s b e s i d e s rDNA can be used t o s o l v e s a f e t y problems. B i o t e c h n o l o g y has j u s t begun t o impact f o o d a n i m a l p r o d u c t i o n . However, because t h e c o s t s o f b i o t e c h n o l o g y a r e g r e a t and t h e markets i n v e t e r i n a r y b i o l o g i c a l s ( v a c c i n e s , d i a g n o s t i c s , and a n t i b o d y p r o d u c t s ) a r e s m a l l r e l a t i v e t o t h e human market, a d o p t i o n o f t h e rDNA and MAB t e c h n o l o g y f o r t h e improvement o f f o o d a n i m a l p r o d u c t s i s l i k e l y t o be slow.
Literature Cited 1. Emerging Developments in Veterinary Biotechnology. PB86-222379. U.S. Food and Drug Administration, Rockville, MD. U.S. Department of Commerce, National Technical Information Service: Springfield, VA. July, 1986. 2. George, H. J . ; L'Italien, J. J . ; Pilacinski, W. P. DNA 1985, 4, 273-281. 3. Halling, S.M. & Smith, S. Gene 1984, 29, 263-269. 4. DeLorbe, W.; Pilacinski, W. P.; Lum, Μ. Α.; et al. in Vaccines 87 Modern Approaches to New Vaccines: Prevention of Aids and other Viral, Bacterial, and Parasitic Diseases; Chanock, R. M.; Lerner, R. Α.; Brown, F.; Ginsberg, H. (Eds.) Cold Springs Harbor Laboratory Publications: Cold Springs Harbor, NY, 1987, pp 431-434. 5. Kit, S.; Kit, M.; Pirtle, E. C. Am. J. Vet. Res. 1985, 46, 1359-1367. 6. Collett, MS.; Keegan, K.; Hu, S.-L.; et al. in The Biology of Negative Strand Viruses; Mahy, B.; Kolakofsky, D. (Eds.) Elsevier: New York, 1987, pp 321-329. 7. Sherman, D. M.; Acres, S. D.; Sadowski, P. L.; et al. Infection & Immunity 1983, 42, 653-658. RECEIVED August 17,
1987
Phillips et al.; The Impact of Chemistry on Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
