Equine arteritis virus-neutralizing antibody in the ... - Semantic Scholar

Journal of General Virology (1995), 76, 1989-1998. Printed in Great Britain

1989

Equine arteritis virus-neutralizing antibody in the horse is induced by a determinant on the large envelope glycoprotein GL E w a n D. Chirnside, 1. Antoine A. F. de Vries, 2 Jennifer A. Mumford ~ and Peter J. M. Rottier 2 1 Department of Infectious Diseases, The Animal Health Trust, Lanwades Park, Kennett, Newmarket CB8 7PN, UK and 2 Department o f Virology, Institute of Infectious Diseases and Immunology, Veterinary Faculty, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands

Complementary DNAs encoding ORFs 2 to 7 of equine arteritis virus (EAV) have been cloned into the expression vector pGEX to produce glutathione-S-transferase fusion proteins. Recombinant proteins were affinity purified and screened in ELISA with equine sera to identify immunoreactive polypeptides. The large envelope glycoprotein (GL) was identified as the most reactive to EAV-positive equine sera and an immuno-

dominant epitope was mapped between amino acids 55 and 98 by subcloning and expression. A fusion protein covering this region and a GL-Specific synthetic peptide (residues 75 through 97) induced EAV-nentralizing antibody in vaccinated horses. The defined antigenic region of G L is likely to be exposed on the surface of the native EAV virion and consequently may be useful in the development of diagnostic tests and vaccines.

Introduction

of 12-14 kDa (Hyllseth, 1973 ; Zeegers et al., 1976). The envelope contains a 16 kDa non-glycosylated protein (M) and N-glycosylated proteins of 25 kDa (Gs) and 30-42 kDa (G 0 (de Vries et al., 1992). The major structural proteins GL, M and N are encoded by ORFs 5, 6 and 7, respectively; the minor envelope glycoprotein G s is specified by O R F 2. ORFs 3 and 4 are both predicted to encode N-glycosylated membrane proteins but the products of these ORFs have not been characterized in detail. EAV G L is a heterogeneously glycosylated envelope protein responsible for the characteristic 30-42 kDa smear seen upon analysis of virus proteins in polyacrylamide gels (van Berlo et al., 1986; de Vries et al., 1992) Minor antigenic differences between EAV isolates have been demonstrated in serological studies (Fukunaga & McCollum, 1977; Fukunaga et al., 1994). Additionally, limited genomic variation was observed by RNase T1 fingerprinting (Murphy et al., 1988, 1992), although sequencing studies have revealed that the EAV M and N proteins are highly conserved between isolates (Chirnside et al., 1994). A long-lived virus-neutralizing (VN) antibody response is induced following viral infection (Gerber et al., 1978) and is believed to be fully protective (McCollum et al., 1988). The observation that colostrum from immune mares, but not from non-immune dams, moderates or prevents equine viral arteritis in young foals (McCollum, 1976) also emphasizes the role of VN antibodies in protection against EAV. A live attenuated vaccine (Doll

Horses and donkeys are the only reported hosts of equine arteritis virus (EAV; Doll et al., 1957; Paweska & Barnard, 1993). In the horse, clinical signs of infection vary widely with the most severe form resulting in fetal abortion from pregnant mares (Doll et al., 1957) and foal death (Golnik et al., 1981; Vaala et al., 1992). Between 30-60 % of seropositive stallions persistently shed virus in their semen (Timoney et al., 1986; Timoney & McCollum, 1991) and infect mares through breeding. The disease then spreads by the respiratory route (McCollum et al., 1961). EAV is a member of the genus Arteriviridae (Cavanagh et al., 1994), a group of small, enveloped, positivestranded RNA viruses with a genome organization, mode of replication and gene expression strategy similar to that of corona- and toroviruses (de Vries et al., 1990, 1992; den Boon et al., 1991; Snijder et al., 1994). The EAV virion has a diameter of 50-70 nm (Hyllseth, 1973) and consists of an isometric nucleocapsid (35 nm) surrounded by an envelope which carries ring-like surface structures with a diameter of 12-15 nm (Murphy, 1980; Horzinek, 1981). The nucleocapsid is composed of an infectious, polyadenylated, single-stranded genomic RNA with a size of 12.7 kb (den Boon et al., 1991) and phosphorylated core protein (N) with a molecular mass

* Author for correspondence. Fax +44 1638750794. 0001-3134 © 1995SGM

1990

E. D. Chirnside and others

et al., 1968; Harry & McCollum, 1981; Timoney et al.,

1988) and inactivated whole virus vaccines (McCollum, 1986; Fukunaga et al., 1990, 1992) induce VN antibody responses in horses. Western blotting and competition ELISA data (Balasuriya et al., 1993) indicate that strongly neutralizing monoclonal antibodies recognize a single epitope on a 29 kDa EAV protein which is likely to correspond to G L. Recently Deregt et al. (1994) demonstrated that from a panel of monoclonal antibodies to the EAV N and GT, proteins all of the neutralizing antibodies recognized G~. Competitive binding assays revealed that they all bound to the same or to closely adjacent sites on the GL protein. However, the specific EAV epitope(s) inducing the protective VN response in horses remain to be identified. In this paper we report the cloning, bacterial expression and serological reactivity of EAV glutathioneS-transferase (GST) fusion proteins to pre- and postinfection equine sera, and the equine antibody response induced by immunization with a GL-derived fusion protein and synthetic peptide.

Methods Materials. Restriction enzymes, dNTP solutions, glutathioneSepharose 4B and the pGEX-2T and pGEX-3X expression vectors (Smith & Johnson, 1988) were obtained from Pharmacia. Calf intestinal

alkaline phosphatase, lysozyme, the Klenow fragment of DNA polymerase I, DNase I, T4 DNA polymerase and T4 DNA ligase were supplied by Boehringer Mannheim. Sequenase (version 2.0) was obtained from United States Biochemical and biotin-labelled affinitypurified goat antiserum to horse IgG and streptavidin peroxidase conjugate were from Kirkegaard and Perry Laboratories.

Plasmid constructs. Plasmid DNA manipulations were performed as described previously (Sambrook et al., 1989). EAV cDNA sequences encoding ORFs 2 to 7 were excised from cDNA clones 106 and 535 or previously generated recombinant phagemids (de Vries et al., 1990, 1992) and inserted in-frame into pGEX-2T or pGEX-3X as indicated in Table 1. To construct pAVI04, clone 106 was digested with DdeI and the 5' protruding ends repaired with the Klenow fragment of DNA polymerase I. The desired 0.6 kb fragment was then gel-purified and inserted into SmaI-digested pBluescript KS(+). Ligation mixtures were used to transform Escherichia coli strain TG1 (Gibson, 1984) and transformants were selected on Luria broth (LB) agar containing 100 gg/ml ampicillin. Expression of fusion proteins. Colonies were picked into LB medium containing 100 lag/ml ampicillin and grown at 37 °C overnight. A 20fold dilution of this culture was then grown for 2 h at 28 °C and protein expression induced by addition of 0.2 mM-IPTG. After 5 h growth at 28 °C, 1 ml of culture was centrifuged and the bacterial pellet was dissolved in 100 gl sample buffer. The sample was then incubated for 5min at 96°C and 10gl were analysed in an SDS-12.5% polyacrylamide gel (Laemmli, 1970). DNA from clones expressing an altered GST fusion protein was subjected to restriction endonuclease analysis and sequenced by the dideoxynucleotide chain termination method with Sequenase to check the insert size and the GST-EAV cDNA junction and inserted nucleotide sequence. Sequencing was performed using the primer 5" GGCGACCATCCTCCAAA 3', located

Table 1. G S T - E A V fusion proteins: construction, properties and nomenclature Fusion protein (FP)

Number of amino acids in native protein

Amino acid position*

Expected size of fusion protein (kDa)

2

22

227

26227

48-8

2

20

227

68-227

44-3

3 4

30 40

163 152

8-140 3-152

41-5 43-2

5

50

255

28-137

39-7

6

60

162

6-162

44-0

7

70

110

- 3 110t

391

5 5

51 52

255 255

-- 5-54t 38 115

34-2 36.1

5 5 5

54 5Rsal SP25

255 255 255

205-249 55-98 75-97

31-7 31-6 NA

ORF

Restriction sites

EagI~ (9909)-EcoRI§ (10588) (DdeI~, PB535, 10588) Bali (10025)-EcoRI (11494) HaeIII (10328-10726) BgllI (10706)-EcoRI§ (11223) (DdeI~, PB106, 11223) HaelI¶ (11228)-Scai (11556) HinfI~ (11915)-Fspt (12398) HindlII~ (12305)HindlII~§ ( > 12700) RsaI (11132-11309) Sau3AI~ (11258)-EcoRI~ (11493) PvulI (11757-11894) RsaI (1131(~11441) NA

EAV cDNA clone

pGEX vector restriction digest

pAVI02

2T; BamHI'~ EcoRI

535

2T; SmaI EcoRI

535 pAVI04

3X; SmaI 3X; BamHI EcoRI

106

3X; Sinai

106

3X; EcoRI~.

106

3X; Sinai

535 535

3X; SmaI 3X; Sinai

106 535 NA

3X; EcoRI~ 3X; EcoRI~ NA

* EMBL database nucleotide sequence accession number X53459. NA, Not applicable. "~ The negative notation refers to the number of additional amino-terminal residues encoded by the cloned EAV cDNA fragment. 3'-recessed end filled in with the Klenow fragment of DNA polymerase I. § Vector-derived. ¶ 5'-protruding end polished with T4 DNA polymerase.

Equine V N antibodies to E A V recognize G L

approximately 40 bp upstream of the pGEX multiple cloning site, and internal EAV-specific primers.

Purification of fusion proteins. Fusion proteins were affinity purified using glutathion~Sepharose 4B. A 250 ml culture of bacterial cells expressing recombinant antigen was harvested by centrifugation for 15 min at 6000 g and 4 °C, resuspended in 50 ml 10 mN-Tris-HC1 pH 8.0, 1 mM-EDTA and 100 mM-NaC1 (STE), and recentrifuged to pellet the cells. The supernatant was discarded and the cells were resuspended in 3 ml STE, 50 gl lysozyme (50 mg/ml) was added and the tube incubated at 37 °C for 15 min. The mixture was then frozen at 70 °C overnight followed by a short immersion in a boiling water bath to thaw the frozen suspension. After the addition of 30 gl 1 M-MgC12 and 20 lag DNase I the sample was incubated at 37 °C for 15 min. Triton X-100 was added to a final concentration of 1%, the mixture kept on ice for 10 rain and centrifuged for 15 min at 10000 g and 4 °C. glutathione-Sepharose 4B was added to the supernatant and the solution mixed by gentle agitation on a rotary mixer for 20 min. The gel was washed three times with PBS and the recombinant antigen eluted from the gel with reduced glutathione (5 mM-glutathione, 50 mMTris-HC1 pH 8"0). The eluate was collected in 1 ml fractions and analysed by PAGE prior to pooling fractions containing purified protein. Peptide synthesis and coupling to keyhole limpet haemocyanin (KLH). The oligopeptide SP25 (Table 1) was synthesized on a Milligen/ Biosearch 9400 Excell peptide synthesizer by the solid phase Fmoc procedure (Stewart & Young, 1984). To increase its immunogenicity, SP25 was conjugated to KLH using limiting amounts of glutaraldehyde (Gullick et al., 1985).

ELISA. Optimum concentrations of reactants were predetermined by chequerboard titration. Immulon 3 microtitre plates (Dynatech) were coated with 0-5 I~gper well of the GST, GST-EAV fusion protein or synthetic peptide diluted in 100 mu-sodium carbonate buffer pH 9"6 overnight at 4 °C. Plates were then washed three times with PBS containing 0"05% Tween 20 (PBST) and blocked by the addition of 100 lal PBST containing 5% goat serum (PBSTG) for 1 h at 37 °C. Following washing, 100 lal of horse sera diluted 100-fold in PBSTG were added to each well and the plates incubated at 37 °C for 90 rain. Plates were washed again and 100 lal of biotin-labelled goat anti-horse IgG (1000-fold dilution in PBSTG) were added for 90 min at 37 °C. After washing, 100 gl of streptavidin peroxidase (1000-fold dilution in PBSTG) were added to each well for 30 min at room temperature. After their final wash, 100 gl of substrate solution (0"5 mg/ml ophenylenediamine dihydrochloride dissolved in 50 raM-sodium phosphate, sodium citrate buffer pH 5.0, 0.03 % sodium perborate) were added for 10 min at room temperature, and the reaction stopped by adding 50 gl 4 M-H2SO4 to each well. The absorbance was read at 490 nm on a Molecular Devices Thermomax plate reader. Equine sera at a 100-fold dilution had a variable background absorbance caused by non-specific binding of the equine sera to GST. Consequently the reactivity of each serum sample to GST was subtracted from that to the GST-EAV fusion protein to derive the EAV-specific A490 value. There was no discernible reactivity of horse serum with uncoated Immulon 3 plates blocked with PBSTG. Reactivities of equine sera to synthetic peptide SP25 are therefore reported as direct readings. Each serum was assayed in duplicate wells against each antigen and the mean A4a0 value calculated. Animal immunizations. New Zealand white rabbits were immunized subcutaneously with 100 ktg of affinity-purified GST or GST-EAV fusion protein in Freund's complete adjuvant, followed 5 weeks later by a second immunization in Freund's incomplete adjuvant. Serum was collected at regular intervals to assess the response to vaccination. KLH-SP25 was also administered subcutaneously; the rabbit was

1991

primed with 200 ~tg of conjugate emulsified in Freund's complete adjuvant and boosted at monthly intervals using 500 ~tg KLH-SP25 and Freund's incomplete adjuvant. The rabbit was bled 2 weeks after the fourth boost. For equine immunizations, affinity purified GST, FP5Rsal or peptide SP25 were mixed with polymer adjuvant (Carbomer-PD; Solvay Duphar) to a final adjuvant concentration of 0-5 %. Three groups of three Welsh ponies were immunized (deep intramuscular) with 400 lag GST (group A), 400 lag FP5Rsal (group B) or 125 lag KLH-SP25 (Group C) in a total volume of 2 ml on day 0. All animals received further immunizations on days 51 and 114, and were bled at intervals throughout the experiment.

Virus neutralization test. The neutralization test was performed as described by Senne et al. (1985) and serum titres calculated according to Karber (1931). In each test three equine sera with stable neutralization titres were included as controls, and a virus titration ensured the correct virus dose was administered to each well. Antisera. The field antisera used for ELISA in this study were submitted to the Animal Health Trust diagnostic laboratory.

Results Design o f fusion proteins I n this s t u d y a p a n e l o f r e c o m b i n a n t f u s i o n p r o t e i n s ( d e s i g n a t e d F P ) d e r i v e d f r o m E A V O R F s 2 - 7 ( T a b l e 1; Fig. 1 a) h a s b e e n u s e d to m a p a n i m m u n o r e a c t i v e v i r a l e p i t o p e . I n F P 2 2 t h e a m i n o - t e r m i n a l signal s e q u e n c e o f t h e O R F 2 p r o t e i n w a s a b s e n t , w h e r e a s in F P 2 0 a n a d d i t i o n a l 42 r e s i d u e s o f t h e G s e c t o d o m a i n (A. de V r i e s et al., u n p u b l i s h e d results) w e r e m i s s i n g . F P 3 0 c o n t a i n e d the e n t i r e O R F 3 c o d i n g s e q u e n c e e x c e p t f o r 7 a m i n o a c i d s o f t h e p u t a t i v e a m i n o - t e r m i n a l signal s e q u e n c e a n d 23 h y d r o p h o b i c r e s i d u e s at t h e c a r b o x y t e r m i n u s . F P 4 0 c o m p r i s e d all e x c e p t 2 a m i n o - t e r m i n a l r e s i d u e s e n c o d e d by O R F 4 a n d F P 5 0 c o n t a i n e d a m i n o a c i d s 28 t h r o u g h 137 o f t h e O R F 5 c o d i n g s e q u e n c e , c o r r e s p o n d i n g to t h e h y d r o p h i l i c p a r t o f t h e G L e c t o d o m a i n (de V r i e s et al., 1992). W e f a i l e d t o p r o d u c e f u s i o n p r o t e i n s c o n t a i n i n g the large internal hydrophobic domain of the G L protein (residues 116 t h r o u g h 191); h o w e v e r , o n t h e basis o f its p h y s i c o c h e m i c a l p r o p e r t i e s w e e x p e c t this d o m a i n to be e m b e d d e d in the v i r a l e n v e l o p e o r to be i n t e r n a l i z e d w i t h i n t h e p r o t e i n , a n d t h e r e f o r e u n l i k e l y to be o f i m p o r t a n c e in t h e i n d u c t i o n o f h o s t a n t i b o d i e s . F P 6 0 c o n t a i n e d all e x c e p t 5 a m i n o - t e r m i n a l r e s i d u e s o f t h e M protein and FP70 contained the complete N protein.

Reactivity o f equine sera with fusion proteins G S T f u s i o n p r o t e i n s w e r e e x p r e s s e d at 28 ° C in o r d e r to o p t i m i z e s o l u b l e f u s i o n p r o t e i n p r o d u c t i o n ( P o g n o n e c et al., 1991). T h e 26.9 k D a G S T p r o t e i n w a s e x p r e s s e d in b a c t e r i a t r a n s f o r m e d w i t h p G E X - 3 X (Fig. 2, l a n e + ) b u t n o t in u n t r a n s f o r m e d T G 1 cells (Fig. 2, l a n e - ) . I n t h e lanes containing material from bacteria transformed w i t h p G E X c o n s t r u c t s t h e 26"9 k D a b a n d w a s r e p l a c e d

1992


(a)

r

i

i

10

11

12

FP50 FP51

3I

kb

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1 FP52

1

I

FP54

I

60

70

D

Fig. 1. Expression cloning of EAV ORFs 2 to 7. (a) The 3' end of the EAV genome. EAV ORFs are shown in the upper part of the diagram with the lower part indicating the regions of the viral genome inserted into the bacterial expression vector pGEX. The non-translated parts of the inserts are indicated by lines. (b) Hydrophobicity plot of the EAV ORF 5 protein (GL) showing the locations of the GST-EAV G L fusion proteins and synthetic peptide. The large white and small hatched boxes represent expressed coding and non-coding EAV sequences, respectively.

Fusion protein (FP) kDa

-

+

22

20

30

40

50

97~ 66~ 45~

31 m

21.5 m

by a higher molecular mass band (lanes FP22 to FP70). The position of these bands corresponded approximately to the expected sizes of the recombinant fusion proteins (Table 1). A panel of equine sera consisting of one nonneutralizing and 10 post-infection sera with EAVneutralizing antibody titres ranging from log10 0-6 to 2.5

Fig. 2. Expression of GST-EAV fusion proteins. Bacterial lysates were run through SDS-12.5% polyacrylamide gels and stained with Coomassie Brilliant Blue R250. Expression products are marked with an arrow within each lane. The lanes designated ' - ' and ' + ' contain lysates from untransformed and pGEX-3X-transformed bacteria respectively. For the derivation of each fusion protein see Table 1. The sizes in kDa of marker proteins analysed in the same gel (left lane) are indicated.

was selected at random and assayed for reactivity in Western blots to peptides FP20 to FP70. These blots produced conflicting results as individual VN sera bound the GST-EAV fusion proteins extremely poorly, and in many cases failed to recognize any of them. As a consequence the reactivity patterns of the sera to proteins FP20 to FP70 were re-investigated by ELISA using

1993

Equine VN antibodies to E A V recognize G L

Table 2. Equine sera reactivity to EA V fusion proteins in E L I S A

,:f

1'6[-

ELISA reactivity~ (A490) to: Serum titre*

FP22

FP20

FP30

FP40

FP50

FP60

FP70

0 0.6 0.8 0.9 1.4 1.6 1-9 2.2 2"3 2-4 2.5

0 0-069 0.564 0 0 0-417 0.072 0 0.229 0.090 0.101

0.185 0 0-225 0 0.080 0.177 0.009 0.123 0.047 0 0-156

0.030 0.130 0.156 0 0.077 0.008 0.219 0.003 0.178 0.049 0.014

0.099 0 0.040 0 0 0.218 0 0.033 0.033 0.002 0.109

0.186 0-218 0.554 0.506 0.792 0-692 0.733 0.593 0.849 1.021 0.863

0 0.002 0 0.030 0 0 0 0.043 0.021 0 0

0.091 0.046 0.133 0.297 0.281 0.008 0.218 0'013 0.114 0.299 0

* Log10 VN titre. "~ ELISA plates were coated with 0-5 gg per well of purified fusion protein or GST. Sera were tested in duplicate wells to each antigen and the absorbance read at 490 nm. The GST absorbance was subtracted from the fusion protein absorbance to derive the EAV-specific value.

affinity-purified fusion proteins as antigen (Table 2). The highest ELISA A490 readings were obtained against FP50, with a clear-cut absorbance difference between post-infection VN and non-neutralizing equine sera (Table 2). FP20, FP30, FP40 and FP60 were not specifically recognized by the post-infection sera. FP22, which contains almost the entire ectodomain of Gs, was bound at a significant level by three of the VN sera (VN titre 0.8, 1.6 and 2.3) and FP70 was recognized to a limited extent by four individual VN sera (VN titres 0"9, 1.4, 1.9 and 2.4).

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In order to examine the reactivity of a wider range of equine sera to FP50 a panel of 192 field sera, comprising 96 VN (VN +) and 96 non-neutralizing (VN-) equine sera, was evaluated by ELISA. All VN + sera produced a specific A490 ~> 0.4 at a 100-fold dilution in direct ELISA to FP50 (Fig. 3 a). A linear correlation existed between VN antibody titre and ELISA A490 value (r = 0.848), although considerable variation existed between different individuals with similar serum neutralizing antibody titres. Using the intercept value (0.326) as the cut-off point for an ELISA positive value, 33/96 VN sera were positive and no VN + sera were negative.

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Although the membrane topology of the G L protein has not been formally established, its hydropathy profile (de Vries et al., 1992) and the position of its N-glycosylation site at amino acid residue 56 (den Boon et al., 1991)

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Reactivity o f field sera to FP50 in E L I S A

Determination of a major epitope on EA V G L

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Fig. 3. ELISA absorbance values of 96 VN + and 96 VN equine sera to (a) FP50, (b) FP5Rsal and (c) SP25. Each point represents the mean ELISA A490 values from duplicate wells plotted against the VN titre calculated from a doubling dilution series repeated in quadruplicate for each serum sample. The solid line is the best fit as calculated by linear regression analysis (r = 0.848, 0.833 and 0.634 for FP50, FP5Rsal and SP25, respectively).

imply that residues 19 to 115 are contained within the ectodomain and may be recognized by neutralizing equine sera. To test this hypothesis small parts of the ORF 5 gene were subcloned into pGEX and the individual reactivities of the purified fusion proteins were assayed in ELISA. Initially three pGEX constructs, FP51, FP52 and FP54, were made (Fig. lb; Table 1),

1994


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Time post-injection(days)

Time post-injection(days) Time post-injection (days) Fig. 4. The equine immuneresponseto vaccinationwith GST (a-c), FP5Rsal (d J) and syntheticpeptide SP25 (g-i). VN antibody titres were determined in quadruplicate,ELISA absorbancesare plotted as the mean value from duplicate wells. Pony numbers are shown in the top right corner of each graph. VN titre (*), ELISA with FP5Rsal (Vq)and ELISA with SP25 (11)excluding the large internal hydrophobic domain which is likely to be involved in anchoring the GL protein in the membrane. These fusion proteins were screened by ELISA, with only FP52 being specifically recognized by V N equine antisera. Although abundantly expressed in bacteria, FP52 bound to glutathione-Sepharose 4B very poorly and was extremely difficult to purify to homogeneity. Consequently, a further expression construct located within GL, FP5Rsal, was made. This protein proved extremely reactive with the V N ÷ field sera in ELISA (Fig. 3 b). The correlation coefficient determined by regression analysis for this ELISA was 0"833 with an intercept value of 0"322. Using this as the cut-off point for ELISA positive sera, 25/96 V N - sera were positive and no V N + sera were negative. Using the algorithm of Jameson & Wolf (1988) the most immunogenic region of EAV G L was predicted to be between amino acid residues 75 through 97. A synthetic peptide, SP25, covering these residues was consequently used to study the reactivity of this part of the G L ectodomain. SP25 is located within FP5Rsal and was recognized less specifically by the panel of V N + equine antisera in ELISA (r = 0.634) than either FP50 or FP5Rsal (Fig. 3c); 10/96 V N - sera were positive in ELISA and 22/96 V N + sera were negative using the intercept (0-372) as the cut-off point for positive results.

Immunogenicity of G L The immunogenicity of the first series of fusion proteins and peptide SP25 was initially tested in rabbits. FPs 20, 30, 40, 50, 60, 70 and GST failed to elicit a V N response even after multiple immunizations. However, sera from rabbits immunized with affinity-purified FP5Rsal or KLH-SP25 blocked EAV infection in virus neutralization tests. The immunogenicity of GST, FP5Rsal and SP25 was subsequently studied in a small group of Welsh ponies. Immunizations were performed on three groups of horses each comprised of three animals. Horses were vaccinated on days 0, 51 and 114, bled at regular intervals and the sera tested for EAV-specific antibodies by microneutralization assay, and ELISA to SP25 and FP5Rsal (Fig. 4a-i). Immunization with GST (horses 165, 270 and 310) failed to elicit any specific antibody response to EAV detectable by V N tests or ELISA (Fig. 4 a-c). In contrast, all three animals immunized with SP25 (236, 259 and 276) produced both V N and ELISA antibodies (Fig. 4g-i). V N antibody levels peaked directly after the second and third immunizations, and decayed within 60 days; an increased peak antibody titre followed each immunization. Horses immunized with SP25 exhibited high ELISA antibody levels specific to both SP25 and


FP5Rsa 1 which peaked directly after each immunization and persisted, even in the absence of VN antibody. The antibody response in horses immunized with FP5Rsal was more varied than that for SP25 (Fig. 4d-J); horse 232 did not produce neutralizing antibody to EAV in response to vaccination. However, both horses 168 and 235 had neutralizing antibody induced following the second and third immunizations with FP5Rsal. The peak VN antibody titre was lower after the third immunization compared to the response induced by the second immunization. All three animals had consistently high ELISA antibody levels to SP25, which peaked following each vaccination, but the high reactivity of these sera to GST after the second immunization masked any direct antibody response to the EAV-specific domain of FP5Rsal. As with the three horses immunized with SP25, ELISA antibody levels to SP25 in the FP5Rsal-vaccinated group persisted in the absence of specific EAV-neutralizing antibody.

Discussion The pGEX bacterial expression system allows the production of abundant, easily purified fusion proteins (Smith & Johnson, 1988) which are useful for defining virus epitopes (Lopez de Turiso et al., 1991) and as ELISA and vaccine antigens (Johnson et al., 1989; Thomas et al., 1992). In this study we have utilized GST fusion proteins to identify the EAV G L protein as an important viral antigen; post-infection, VN equine sera predominantly recognized bacterial fusion proteins containing GL. The approach was further used to map the immunodominant domain to a particular region within the G L ectodomain. Finally, this domain, as well as a synthetic oligopeptide contained within it, successfully induced a VN antibody response in horses. Fusion proteins comprised of Gs, M, and N, and of polypeptides encoded by EAV ORFs 3 and 4, were poorly immunoreactive with post-infection equine sera in ELISA. In contrast, G L fusion proteins, uniquely amongst the EAV structural protein repertoire, reacted strongly. Coarse mapping of the immunoreactive epitope(s) was accomplished by subcloning of O R F 5 to produce a series of smaller recombinant polypeptides. Fusion proteins including G L residues 55 through 98 were reactive in the ELISA; in the absence of these residues post-infection, VN equine sera did not bind to the fusion protein. A synthetic peptide containing residues 75 through 97 was also reactive in ELISA with these sera, although with decreased specificity and sensitivity compared to the immunoreactive fusion proteins FP50 and FP5Rsal. The observations suggest that an important linear epitope exists between residues 55 and 98 of the G L protein, although other linear

1995

and/or conformational epitopes in the protein cannot be excluded. The single N-glycosylation site present within GL lies within the immunoreactive region at residue 56. The poor reactivity of the fusion proteins derived from the other EAV ORFs both in Western blots and in direct ELISA does not preclude that the corresponding viral proteins have a role in promoting the equine immune response to EAV. In fact, in addition to G L, the viral N and M proteins are recognized in Western blots to whole virus antigen and can also be immunoprecipitated from lysates of EAV-infected cells by post-infection horse sera (data not shown). Prokaryotic expression products may fail to react for many reasons. Fusion proteins may not effectively mimic the native viral proteins due to differences in polypeptide folding, disulphide bond formation or in post-translational modification, notably N-glycosylation. Also, expression of incomplete proteins may have conformational effects. With one exception (FP70) all the fusion proteins in our study omitted portions of terminal or internal domains of the EAV proteins. Finally, altered ELISA conditions, optimized for the individual fusion proteins, may have made some differences to equine serum reactivity to individual polypeptides. Finding G L the immunodominant EAV protein was not unexpected in view of its structure, position and abundance in the virion. GL is the major viral envelope glycoprotein which, together with M and N comprises more than 95 % of the total virion protein (de Vries et al., 1992). N is contained within the virion interior while the M protein is largely embedded in the envelope membrane. In contrast, the large hydrophilic amino-terminal half of the G~ protein is exposed and apparently protrudes from the virion surface. The strong reactivity of the GL-based fusion proteins with VN ÷ field sera from horses in ELISA may form the basis for a reliable, rapid and cheap serological test (Chirnside et al., 1995) to replace virus neutralization (Senne et al., 1985) and complement fixation tests (Fukunaga & McCollum, 1977). Such an assay to detect previously EAV-infected animals will allow more widespread screening of horses than at present, and reduce the possibility of persistently infected animals being used for breeding. Previous EAV ELISA tests, based on the use of purified whole virus as antigen, have suffered from poor specificity (Cook et al., 1989). The significance of antigenic variation for the reliability of a G~-based ELISA will have to be established. Complement fixation (Fukunaga & McCollum, 1977) and cross-neutralization studies using monovalent equine antisera in homologous and heterologous virus neutralization tests (Fukunaga et al., 1994) have indicated that minor antigenic variation between EAV isolates does exist. At the genetic level, RNase T1 oligonucleotide fingerprinting of viral

1996


genomes (Murphy et al., 1988) and sequence analysis of EAV M and N genes from different isolates (Chirnside et al., 1994) have also demonstrated the existence of variation. Both a GL subunit (residues 55 through 98) and a GLspecific oligopeptide (residues 75 through 97) induced VN antibodies in horses following immunization. This is consistent with the recent observation that neutralizing monoclonal antibodies to EAV are invariably directed to the G L protein. Balasuriya et al. (1993) described a panel of six such monoclonal antibodies that recognized a single site on a 29 kDa glycoprotein. Deregt et al. (1994), in addition to identifying G L as the viral protein with which neutralizing antibodies reacted, also demonstrated that one monoclonal antibody still recognized the G L protein when most of the carboxy-terminal half (residues 117 through 211) was deleted. This antibody competed in ELISA with all the other GL-Specific monoclonal antibodies. These data support our finding that the G L ectodomain is the immunogenic and biologically most relevant domain. Although the importance of the host cellular immune response to EAV remains to be investigated, equine vaccination schedules with both live attenuated and inactivated whole virus preparations probably rely on the induction of neutralizing antibodies. Host neutralizing antibodies are known to persist for many years after infection (Gerber et al., 1978) and correlate closely with protection from re-infection (McCollum, 1986; Fukunaga et al., 1990, 1992). The subunit-vaccinated horses in our study maintained high levels of GL-specific antibody over long time periods. In contrast, VN activity was only intermittently detectable for short periods after each booster immunization, and the peak levels and duration decreased with each successive immunization. Although ELISA and VN antibody peaks concurred, the rapid turnover and low levels of the neutralizing activity as well as the occurrence of one non-responder animal in virus neutralization tests suggest that the whole virus antigen responsible for inducing VN antibody in horses differs in composition or presentation from the subunits studied. The Arteriviridae is currently comprised of EAV, lactate dehydrogenase-elevating virus (LDV), simian haemorrhagic fever virus and porcine respiratory and reproductive syndrome virus (PRRSV) (Plagemann & Moennig, 1992; Cavanagh et al., 1994). These intracellular budding viruses are very similar both structurally and with respect to their replication (Plagemann & Moennig, 1992; Conzelmann et al., 1993). Interestingly, comparative analysis of the predicted ORF 5 products of these viruses revealed a number of conserved features indicating that despite the absence of significant sequence similarities these proteins are the structural homologues

of the EAV G L protein. Serologically no cross-reactivity exists between EAV, LDV and PRRSV (van Berlo et al., 1983; Wensvoort et al., 1992). The host immune responses to these viruses, in particular the temporal induction and biological activity of neutralizing antibody, are also quite different. LDV- and PRRSVneutralizing antibodies appear 1 5 months after infection and are relatively inefficient in neutralization (Harty & Plagemann, 1988; Frey et al., 1992). In contrast, effective neutralizing antibodies are circulating in the horse within 14 days of infection with EAV (Gerber et al., 1978) and efficiently clear circulating virus. However, a carrier state exists for EAV (Timoney et al., 1986), with virus present in the accessory sex cells and semen of persistently shedding stallions (Neu et al., 1988). The primary target cells of other arteriviruses are host macrophages where these viruses can establish asymptomatic, persistent infections unaffected by the host immune response (Plagemann & Moenning, 1992). However, the specific mechanism that allows EAV to escape immune surveillance in the presence of high titres of neutralizing antibody requires further study. This work was supported in part by grants from the European Breeders Fund, the New York Jockey Club and the Ministry of Agriculture, Fisheries and Food Contract Research Award No. CSA 2311. Animal work was performed under UK Home Office licence regulations and all experimental work involving horses was carried out by qualified veterinarians. The use of G L amino acid sequences is covered by Patent No. GB 9400656.4. The authors thank Eric Snijder and Fred Wassenaar for kindly providing plasmids expressing FP20 and FP30 and Matthew Binns for providing sequencing oligonucleotides.

References BALASURIYA, U . B . R . , ROSSITTO, P.V., DEMAULA, C.D. & MACLACHLAN, N. J. (1993). A 29K envelope glycoprotein of equine arteritis virus expresses neutralization determinants recognized by murine monoclonal antibodies. Journal of General Virology 74, 2525-2529. CAVANAGH, D., BRIAN, D. A., BRINTON, M., ENJUANES, L., HOLMES, K.V., HORZINEK, M. C., LAI, M. M. C., LAUDE, H., PLAGEMANN, P. G. W., SIDDELL,S., SPAAN,W. J. M., TAGUCHI,F. & TALBOT, P. J. (1994). Revision of the taxonomy of the Coronavirus, Torovirus and Arterivirus genera. Archives of Virology 135, 227-237. CHIRNSIDE, E. D., WEARING, C. M., BINNS, M. M. & MUMFORD, J. A. (1994). Comparison of M and N gene sequences distinguishes variation amongst equine arteritis virus isolates. Journal of General Virology 75, 1491 1497. CHIRNSIDE, E. D., FRANCIS, P. M., DE VRIES, A. A. F., SINCLAIR, R. & MUMFORD, J. A. (1995). Development and evaluation of an ELISA using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus (EAV). Journal of Virological Methods (in press). CONZELMANN, K.-K., VISSER, N., VAN WOENSEL, P. & ThaEL, H.-J. (1993). Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group. Virology 193, 329-339. COOK, R. F., GANN, S.J. & MUMFORD, J.A. (1989). The effects of vaccination with tissue culture-derived viral vaccines on detection of antibodies to equine arteritis virus by enzyme-linked immunosorbent assay (ELISA). Veterinary Microbiology 20, 181 189.


DEN BOON, J. A., SNIJDER, E. J., CHIRNSIDE, E. D., DE WRIES, A. A. F., HORZINEK,M. C. & SPAAN,W. J. M. (1991). Equine arteritis virus is not a togavirus but belongs to the coronavirus-like superfamily. Journal of Virology 65, 2910-2920. DEREGT, D., DE VRIES,A. A. F., RAAMSMAN,M. J. B., ELMGREN,U D. & ROTTIER, P . J . M . (1994). Monoclonal antibodies to equine arteritis virus proteins identify the G~ protein as a target for virus neutralization. Journal of General Virology 75, 2439-2444. DE VRIES,A. A. F., CHIRNSIDE,E. D., BREDENBEEK,P. J., GRAVESTEIN, L. A., HORZINEK,M. C. & SPAAN,W. J. M. (1990). All subgenomic RNAs of equine arteritis virus contain a common leader sequence. Nucleic Acids Research 18, 3241-3247. DE VRIES, A. A. F., CHIRNSIDE,E. D., HORZINEK, M. C. & RoTrIER, P. J. M. (1992). The structural proteins of equine arteritis virus. Journal of Virology 66, 6294-6303. DOLL, E.R., KNAPPENBERGER, R.E. & BRYANS, J.T. (1957). An outbreak of abortion caused by the equine arteritis virus. Cornell Veterinarian 47, 69-75. DOLL, E. R., BRYANS,J. T., WILSON,J. C. & McCOLLUM,W. H. (1968). Immunization against equine viral arteritis using modified live virus propagated in cell cultures of rabbit kidney. Cornell Veterinarian 58, 497-524. FREY, M.L., EARNISSE,K.A., LANDGRAF, J.G. & PEARSON, J.E. (1992). Diagnostic testing for swine infertility and respiratory syndrome virus at the National Veterinary Services Laboratories. In Proceedings of International Symposium on SIRS/PRRS/PEARS, Minnesota, 1992, p. 25. FUKUNAGA, Y. & McCOLLUM, W.H. (1977). Complement fixation reactions in equine viral arteritis. American Journal of Veterinary Research 38, 2043-2046. FUKUNAGA,Y., WADA,R., MATSUMURA,T., SUGIURA,T. & IMAGAWA, H. (1990). Induction of immune response and protection from equine viral arteritis (EVA) by formalin-inactivated virus vaccine for EVA in horses. Journal of Veterinary Medicine 37, 135-141. FUKUNAGA,Y., WADA, R., MATSUMURA,T., ANZAI, T., IMAGAWA,H., SUGIURA, T., KUMANOMIDO,T., KANEMARU,T. & KAMADA, M. (1992). An attempt to protect against persistent infection of equine viral arteritis in the reproductive tract of stallions using formalin inactivated-virus vaccine. In Proceedings of the Sixth International Conference on Equine Infectious Diseases, Cambridge, 1991, pp. 239-244. Edited by W. Plowright, P.D. Rossdale & J. F. Wade. Newmarket: R&W Publication. FUKUNAGA,Y., MATSUMURA,T., SUGIURA,T., WADA, R., IMAGAWA, H., KANEMURA, T. & KAMADA, M. (1994). Use of the serum neutralization test for equine viral arteritis with different virus strains. Veterinary Record 134, 574-576. GERBER,H., STECK,F., HOFER,B., WALTHER,L. & FRIEDLI, U. (1978). Serological investigations on equine viral arteritis. In Proceedings of the Fourth International Conference on Equine Infectious Diseases, Lyon, 1976, pp. 461-465. Edited by J.T. Bryans & H. Gerber. Princeton: Veterinary Publications. GmSON, T. J. (1984). Studies on the Epstein-Barr virus genome. PhD thesis, University of Cambridge. GOLNIK, W., MICHALSKA,Z. & MICHALAK,T. (1981). Natural equine viral arteritis in foals. Schweizer Archiv fiir Tierheilkunde 123, 523-533. GULLICK, W.J., DOWNWARD, J. & WATERFIELD, M.D. (1985). Antibodies to the autophosphorylation sites of the epidermal growth factor receptor protein-tyrosine kinase as probes of structure and function. EMBO Journal 4, 2869-2877. HARRY, T. O. & McCOLLUM, W. H. (1981). Stability of viability and immunizing potency of lyophilized, modified equine arteritis livevirus vaccine. American Journal of Veterinary Research 42, 1501 1505. HARTY, J. T. & PLAGEMANN,G. W. (1988). Formalin inactivation of the lactate dehydrogenase-elevating virus reveals a major neutralizing epitope not recognized during natural infection. Journal of Virology 62, 321~3216. HORZINEK, M.C. (1981). Equine arteritis virus. In Non-Arthropod Borne Togaviruses, pp. 30-31. London: Academic Press. HYLLSETH, B. (1973). Structural proteins of equine arteritis virus. Archiv fiir die gesamte Virusforschung 40, 17%188.

1997

JAMESON, B.A. & WOLF, H. (1988). The antigenic index: a novel algorithm for predicting antigenic determinants. Computer Applications in the Biosciences 4, 181-186. JOHNSON, K. S., HARRISON, G. B. L., LIGHTOWLERS,M.W., O'HOY, K. L., COUGLE,W. G., DEMPSTER,R. P., LAWRENCE,S. B., VINTON, J. G., HEATH,D. D. & RICKAR3, M. D. (1989). Vaccination against ovine cysticercosis using a defined recombinant antigen. Nature 338, 585-587. KARBER, G. (1931). Beitrag zur kollektiven Behandlung pharmakologischer. Reihenversuche. Naunyn-Schmiedebergs Archiv fiir experimentele Pathologie und Pharmakologie 162, 480-483. LAEMMLI,U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 277, 680-685. LOPEZ DE TURISO,J. A., CORTES,E., RANZ, A., GARCIA,J., SANZ, A., VELA, C. & CASAL,J. I. (1991). Fine mapping of canine p arvovirus B cell epitopes. Journal of General Virology 72 2445-2456. M¢COLLUM, W.H. (1976). Studies of passive immunity in foals to equine viral arteritis. Veterinary Microbiology 1, 45-54. M¢COLLUM, W. H. (1986). Responses of horses vaccinated with an avirulent modified-live equine arteritis virus propagated in E. derm (NBL-6) cell line to nasal inoculations with virulent virus. American Journal of Veterinary Research 47, 1931-1934. M¢COLLUM, W. H., DOLL, E. R. & WILSON,J. C. (1961). The recovery of virus from horses with experimental cases of equine arteritis using monolayer cell cultures of equine kidney. American Journal of Veterinary Research 23, 465-469. MCCOLLUM,W. H., TIMONEY,P. J., ROBERTS,m. W., WILLARD,J. E. & CARSWELL, G.D. (1988). Responses of vaccinated and nonvaccinated mares to artificial insemination with semen from stallions persistently infected with equine arteritis virus. In Proceedings of the Fifth International Conference on Equine Infectious Diseases, Lexington, 1991, pp. 13 18. Edited by D. G. Powell. Lexington: University Press of Kentucky. MURPHY, F. A. (1980). Togavirus morphology and morphogenesis. In The Togaviruses: Biology, Structure and Replication, pp. 241-316. Edited by R. W. Schlesinger. London: Academic Press. MURPHY, T. W., TIMONEY,P. J. & McCOLLUM,W. H. (1988). Analysis of genetic variation among strains of equine arteritis virus. In Proceedings of the Fifth International Conference on Equine Infectious Diseases, Lexington, 1987, pp. 3-12. Edited by D.G. Powell. Lexington: University Press of Kentucky. MURPHY, T.W., McCOLLUM, W. H., TIMONEY, P.J., KLINGEBORN, B. W., HYLLSETH,B., GOLNIK,W. & ERASMUS,B. (1992). Genomic variability among globally distributed isolates of equine arteritis virus. Veterinary Microbiology 32, 101-115. NEU, S. M., TIMONEY,P. J. & McCoLLUM, W. H. (1988). Persistent infection of the reproductive tract of stallions persistently infected with equine arteritis virus. In Proceedings of the Fifth International Conference on Equine Infectious Diseases, Lexington, 1987, pp. 149-154. Edited by D. G. Powell. Lexington: University Press of Kentucky. PAWESKA, J. T. & BARNARD,B. J. H. (1993). Serological evidence of equine arteritis virus in donkeys in South Africa. Onderstepoort Journal of Veterinary Research 60, 155-158. PLAGEMANN,P. G. W. & MOENNIG,V. (1992). Lactate dehydrogenaseelevating virus, equine arteritis virus and simian hemorrhagic fever virus: a new group of positive-strand RNA viruses. Advances in Virus Research 41, 99-192. POGNONEC,P., KATO, H., SUMIMOTO,H., KRETZSCHMAR,M. & ROEDER, R. G. (1991). A quick procedure for the purification of functional recombinant proteins over-expressed in E. coll. Nucleic Acids Research 19, 6650. SAMBROOK, J., FRITSCH, E.F. & MANIATIS, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. New York: Cold Spring Harbour Laboratory. SENNE, D. A., PEARSON,J. E. & CARBP~Y, E.A. (1985). Equine viral arteritis: a standard procedure for the virus neutralization test and comparison of results of a proficiency test performed at five laboratories. In Proceedings of the 89th Annual Meeting of the United States Animal Health Association, Milwaukee, Wisconsin, 1985, pp. 29-34. SMITH, D.B. & JOHNSON, K.S. (1988). Single-step purification of

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polypeptides expressed in Escherichia coli as fusion proteins with glutathione-S-transferase. Gene 67, 31~40. SNIJDER, E.J., HORZINEK, M.C. & SVAAN, W . J . M . (1994). The coronaviruslike superfamily. In Coronaviruses: Molecular Biology and Virus-Host Interactions, pp. 235--244. Edited by H. Laude & J.-F. Vautherot. New York: Plenum Press. STEWART,J. M. & YOUNG,J. D. (1984). Solid Phase Peptide Synthesis, 2nd edn. Rockford: Pierce Chemical. THOMAS, L.M., HUNTINGTON,P.J., MEAD, L.J., WINGATE, D.L., ROGERSON, B.A. & LEW, A.M. (1992). A soluble recombinant fusion protein of the transmembrane envelope protein of equine infectious anemia virus for ELISA. Veterinary Microbiology 31, 127 137. TIMONEY, P.J. & MCCOLLUM, W.H. (1991). Equine viral arteritis: current clinical and economical significance. Proceedings of the American Association of Equine Practitioners 36, 403-409. TIMONEY,P. J., MCCOLLUM,W. H., ROBERTS,A. W. & MURPHY,T. W. (1986). Demonstration of the carrier state in naturally acquired equine arteritis virus infection in the stallion. Research in Veterinary Science 41, 279-280. TIMONEY, P.J., UMPHENOUR, N.W. & McCOLLUM, W.H. (1988). Safety evaluation of a commercial modified live equine arteritis virus vaccine for use in stallions. In Proceedings of the Fifth International Conference on Equine Infectious Diseases, Lexington, 1987, pp.

19-27. Edited by D.G. Powell. Lexington: University Press of Kentucky. VAALA,W. E., HAMIR,A. N., DUBOVI,E. J., TIMONEY,P. J. & RUIZ, B. (1992). Fatal, congenitally acquired infection with equine arteritis virus in a neonatal thoroughbred. Equine Veterinary Journal 24, 155-158. VAN BERLO, M. F., ZEEGERS,J. J. W., HORZINEK, M. C. & VAN DER ZEIJST, B. A. M. (1983). Antigenic comparison of equine arteritis virus (EAV) and lactic dehydrogenase virus (LDV); binding of staphylococcal protein A to the nucleocapsid protein of EAV. Zentralblatt fiir Veterinaermedizin Beiheft 30, 297-304. VAN BERLO, M. F., ROTTIER, P. J. M., SPAAN, W. J. m. 8z HORZINEK, M. C. (1986). Equine arteritis virus-induced polypeptide synthesis. Journal of General Virology 67, 1543-1549. WENSVOORT, G , KLUYVER, E.P., POL, J. M.A., WAGENAAR, F., MOORMAN, R. J. M., HULST, M. M., BLOEMRAAD,R., DEN BESTEN, A., ZETSTRA,T. • TERPSTRA,C. (1992). Lelystad virus, the cause of porcine epidemic abortion and respiratory syndrome: a review of mystery swine disease research at Lelystad. Veterinary Microbiology 33, 185-193. ZEEGERS,J. J. W., VANDER ZEIJST, B. A. M. & HORZINEK,M. C. (1976). The structural proteins of equine arteritis virus. Virology 73, 200-205.

(Received 16 January 1995; Accepted 31 March 1995)