Expression of the outer capsid proteins VP2 and VP5 of bluetongue virus in Saccharomyces cereuisiae

Virus ELSEVIER

Virus Research 33 (1994) 11-25

Research

Expression of the outer capsid proteins VP2 and VP5 of bluetongue virus in Saccharomyces cerevisiae John C. Martyn I, Allan R. Gould *, Meng Yu CSIRO, AustralianAnimal Health Laboratory, Geelong, Vktoria, Australia (Received 13 August 1993; revised 14 February 1994; accepted 15 February 1994)

Abstract cDNAs transcribed from bluetongue virus serotype 1 (Australia) ds RNA 2 and ds RNA 6 coding for the major neutralising antigen VP2 and the outer capsid protein VPS, respectively, were amplified in polymerase chain reactions and ligated downstream of the copper-inducible metallothionein promoter in the yeast expression plasmid pYELC5. Saccharomyces cereuisiue transformed with the recombinant plasmid pYELC5-VP2 expressed full-length VP2 only following induction with 1 mM CuSO, and reatihed the maximum level after 6 h. In contrast, S. cerevisiue transformants harboring the recombinant plasmid pYELC5-VP5 expressed VP5 constitutively, although induction increased the level to a maximum after 4 h. A sheep trial was done testing the recombinant proteins, however it was shown that none of these were effective immunogens for eliciting a protective response against a subsequent challenge with bluetongue virus. An analysis of the yeast expression products for the VP2 outer coat protein using a panel of monoclonal antibodies showed that the yeast expressed VP2 was in a conformation different from native VP2 and hence probably unable to elicite an appropriate protective immune response. Key words: Bluetongue virus; VP2; VP5; Neutralisation;

Protective antibody; Vaccine

1. Introduction Bluetongue is a viral disease of sheep, cattle and wild ruminants that occurs in tropical to temperate regions of the world in the range of the insect vector, biting

* Corresponding author. ’ Present address: The Mordoch Institute, Royal Childrens Hospital, Flemington Road, Parkville, Vie. 3052 Australia. 0168-1702/94/$07.00 Q 1994 Elsevier Science B.V. All rights resewed SSDZO168-1702(94)00022-5

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midges of the genus Cc&c&es. Sheep are the most severely affected by the disease, with symptoms ranging from mild fever to severe debilitation, while cattle are subclinically infected and may act as reservoirs for spreading of the virus. The causative agent bluetongue virus (BTV) represents viruses in the Orbivims genus of the Reoviridae family. The BTV genome consists of ten linear, doublestranded (ds) RNA molecules enclosed in an icosahedral capsid composed of two major proteins VP3 and VP7 and three minor proteins VPl, VP4 and VP6 (Verwoerd et al., 1972). This nucleocapsid is contained in an amorphous outer capsid made of VP2 and VP5 In addition, three non-structural proteins NSl, NS2 and NS3 are synthesised in BTV-infected cells. The VP2 protein purified from BTV (Huismans et al., 1987) or present in a crude lysate of insect cells infected with a recombinant baculovirus (Inumaru and Roy, 1987; Roy et al., 1990) elicits antibodies that neutralise infection in plaque reduction tests and protect sheep against infection with the homologous serotype. Serum neutralisation tests have identified 24 BTV serotypes. While little is known of the function of the VP5 protein of BTV, VP5 expressed by a recombinant baculovirus enhanced the ability of VP2 to generate neutralising antibodies (Roy et al., 1990). Plasmid pYELC5 (Macreadie, 1990) is an Escherichia coli-S. cerevisiae shuttle plasmid that contains the metallothionein (CUP11 promoter for high-level, copper-inducible expression of foreign proteins in yeast (German et al., 1986; Macreadie et al., 1989-1991; Fujita et al., 1990; Jagadish et al., 1990). We have now expressed five BTV-1 (Aus) proteins from recombinant pYELC5 plasmids in yeast including VP7, NS3 (Martyn et al., 1990), NSl (Gould et al., 1994) and VP2 and VP5 in this report. This is the first report of the potential of a yeast strain expressing recombinant VP2 to act as a BTV vaccine.

2. Materials and methods Purification of BTV ds RNA and synthesis of cDNA

SVP cells were passaged and infected with BTV-1 strain CS156 (St. George et al., 1980) as described by Eaton and Gould (1987). Purification of dsRNA segments was as described in Eaton and Gould (19871, while their transcription into complementary DNA was as described by Gould (1988) using ~185 for segment 2 and ~179 for segment 6. Cloning the VP2 and VP5 genes of BTV-1 (Aus)

Two overlapping segments of VP2 DNA were synthesised in polymerase chain reactions (PCR; Saiki et al., 1988) using cDNA transcribed from ds RNA 2 of BTV-1 (Aus) as template, Tuq DNA polymerase (Promega) and a thermal cycler (Perkin Elmer Cetus, Norwalk, CT). The terminal primers for the 5’ gene segment were ~185 (S-CCCGGGATCCTAGTGTCGCGATGGATG-3’) and p41 (5’GAAACAATGCGTAAC-3’) and for the 3’ gene segment were ~404 (Y-GGACAAATGGTGAATGATITG-3’) and ~186 (5’-CCCGGGATCCACG-GTCAAGC-

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J. C. Martyn et al. / Wus Research 33 (1994) 11-25

VP2

BTVlVP2

@9rObpd

A

C Fig. 1. Cloning the complete coding sequence of the VP2 gene of BTV-1 (Aus) into pUC18. (A) Restriction map of the 2.9-kbp VP2 gene showing relevant sites. The location of primer pairs (~41 and ~185, ~186 and ~404) used in polymerase chain reactions with VP2 cDNA to generate two overlapping fragments that span the coding region of the VP2 gene (shaded arrow) is indicated. (B) Recombinant plasmid pUC18-2N comprises the 1.8-kbp BamHI/BgZII fragment excised from the N-terminal PCR product and cloned into the BamHI site of pUC18. (Cl Recombinant plasmid pUC18-VP2 was derived from pUC18-2N by insertion of the 1.5-kbp NsiI fragment excised from the C-terminal PCR product between the NsiI and SmaI sites.

GGGTCATAC3’). The 5’ gene segment PCR product was digested with BumHI and BgZII, purified from a 1.0% agarose gel using Geneclean (BIOlOl, La Jolla, CA) and ligated into pUC18 digested with BumHI and alkaline phosphatase (Promega) to generate pUC18-2N (Fig. 1B). The 3’ gene segment PCR product was digested with MI and ligated into pUC18-2N digested with MI and SmaI to generate the full-length VP2 clone pUC18-VP2 (Fig. 10 cDNA transcribed from ds RNA 6 of BTV-1 (Aus) coding for the VP5 protein was amplified in a PCR using the terminal primers ~179 (5’~CCCGGGATCCAGCGAAGATGG-

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J. C. Martyn et al. / Virus Research 33 (I 994) 1 I-25

GTAAAG-3’) and ~180 (5’-CCCGGGATCCGTCGCTGCGTTCAAG-3’). The blunt-ended PCR product was ligated into the SmaI site of pUC18 to generate pUC18-VPS. The coding sequences of the VP2 and VP5 genes were excised from their respective recombinant pUC18 plasmids with BamHI and subcloned into the BumHI site of the yeast expression plasmid pYELC5. The coding sequence of VP2 was also subcloned into the BarnHI site of the bacterial expression plasmid PET-3b (Novagen, Madison, WI). Primers flanking the BTV-1 VP2 coding sequence (Gould, 1988) and the BTV-1 VP5 coding sequence (Gould and Pritchard, 1988) were designed with BumHI sites to produce in-frame fusions with the six N-terminal amino acids of the yeast metallothionein protein and the 12 N-terminal amino acids of the bacterial gene 10 protein. Transformation of bacteria and yeast E. coli strain HBlOl was transformed with recombinant pUC18 and pYELC5 plasmids and the E. coli lysogen BL21(DE3) was transformed with recombinant

pET plasmids by the modified CaCl, method described by Sambrook et al. (1989). Bacterial colonies containing BTV gene inserts were identified directly by PCR (Gussow and Clackson, 1989) and recombinant plasmids were extracted from positive colonies by the modification of the alkaline lysis method described by Sambrook et al. (1989) and characterised by digestion with restriction endonucleases (Promega). Saccharomyces cerevisiue strain 6657-4D was transformed by the lithium acetate method (Ito et al., 1983) with recombinant pYELC5 plasmids prepared in E. coli and purified on CsCl gradients. Yeast transformants were confirmed to be genuine by PCR of gene insertions in whole cells (Sathe et al., 1991). Bacterial and yeast transformants were selected on agar plates as described by Martyn et al. (19901. Construction and induction of yeast diploids S. cerevisiae 6657-4D (a leu2-3,112 his3-11,15 CUPlR) haploid transformants were mated with S. cerevisiae JRY188 (a sir3-8 leu2-3 leu2-112 trpl ura3-52 hisl)

haploid yeast by cross-streaking the two strains on YEPD agar at 30°C and transferred to minimal agar for selection of diploid colonies. Diploids were inoculated into minimal broth, grown and induced with 1 mM CuSO, as described by Martyn et al. (1990). Extraction of BTVproteins

from yeast

BTV proteins were extracted from pelleted yeast cells by either: (1) incubation in three volumes of lysis buffer (0.1% SDS, 0.2 N NaOH, 0.2% &mercaptoethanol) for 15 min at 25°C or (2) resuspension in 1.2 M sorbitol and incubation with 100 U/ml lyticase (Sigma) for 30 min at 30°C followed by centrifugation (3000 rpm, 10 min), a wash in 0.01 M phosphate-buffered saline (PBSA), pH 7.2 and sonication of the cell pellet resuspended in three volumes of PBSA containing the protease inhibitors aprotinin (1 pg/ml; Boehringer) and phenylmethylsulfonyl fluoride (100 pg/ml; Sigma) for 10 x 30 s bursts on ice.

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1.5

Sheep immunisation regime

Three pairs of sheep were inoculated intramuscularly with a crude lysate of yeast in Freund’s Incomplete Adjuvant (FIA) containing either: (1) 300 pg VP2, (2) 300 pg VP2 + 300 pg VP5 or (3) no BTV protein (controls). Booster inoculations were given at three weeks in FIA and at six weeks without adjuvant. All sheep were challenged at eight weeks with virulent BTV-1 (Aus) and monitored for clinical signs (temperature, rash, coronitis, etc.) of BTV infection for the next two weeks. Antisera collected three weeks after the initial inoculation, three weeks after the first booster and two weeks after the second booster were tested for BTV antibody by ELISA and Western blot. Serum neutralisation tests were done to determine whether elicited antibodies were neutralising. Western blot analysis and ELISA

Western blot analysis of BTV-1 yeast-expressed proteins of BTV-1 was as described in Gould et al., (1994), while the detection of sheep polyclonal antibodies was as described by Lunt et al. (1988a,b). The VP2 protein of BTV-1 was detected by incubation with the mouse monoclonal antibody (mAb) DS/A12/D2 (White and Eaton, 1990) or antisera raised in sheep or rabbits to yeast-expressed VP2. The VP5 protein of BTV-1 was detected by incubation with a rabbit anti-peptide antiserum (Wade-Evans et al., 1988) or antisera raised in sheep or rabbits to yeast-expressed VP5 The VP2 mAb was diluted 1: 10, the anti-VP5 peptide antiserum was diluted 1: 200 and sheep and rabbit antisera to VP2 and VP5 were diluted 1: 20 in blotto buffer.

3. Results Construction of clones of BTV1

genes VP2 and VP5

Full-length VP2 (pUC18-VP21 and VP5 (pUC18-VP51 clones were inserted into pUC18 and characterised by restriction endonuclease cleavage, PCR analysis, direct nucleotide sequencing as well as expression in PET and pGEX baterial expression vectors (Gould et al., 1994) (not shown). An internal BamHI site at was identified 159 bp from the 5’-terminus of the VP5 insert as a G to C transition and should be amended in the sequence of Gould and Pritchard (1988). Full-length BamHI clones of VP2 and VP5 in plasmid pYELC5 were identified by PCR screening and the correct orientation for transcription of each gene from the metallothionein promoter was determined by restriction endonuclease digestion (not shown). Yeast expression of VT’2 and VT?‘5proteins of BTV-1

Protein extracts of BTV-1, yeast induced to express the VP2 protein of BTV-1 from pYELCS-VP2 and control yeast harboring the parent plasmid pYELC5 were resolved by electrophoresis through an 8% SDS-polyacrylamide gel (Fig. 2A). Coomassie brilliant blue staining revealed a protein band with a Mr of 105 kDa as

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J. C. Martyn et al. / Virus Research 33 (I 994) II-25

A

97kD66 45 31 -

C 1

2

3

M M

1

2

3

200kD92c VP5

Fig. 2. Expression of the VP2 and VP5 proteins of BTV-1 (Aus) in S.cereuisiae. Yeast proteins were extracted by lyticase treatment and sonication before SDS-polyactylamide gel electrophoresis. (A) The VP2 proteins of BTV-1 and yeast resolved on an 8% gel. Lane 1, control yeast harboring pYELCS; lane 2, yeast harboring pYELC5-VP2; lane 3, BTV-1 (Aus); lane M, Bio-Rad low Mr markers. The position of the VP2 band is indicated. (B) Western blot of a similar gel to A. Lane 1, BTV-1 (Aus); lane 2, yeast harboring pYELCS-VP2; lane 3, control yeast harboring pYELC5; lane M, Amersham high Mr rainbow markers. (0 Western blot of the VP5 proteins of BTV-1 and yeast resolved on a 10% gel. Lane 1, BTV-1 (Aus); lane 2, yeast harboring pYELCS-VP5; lane 3, control yeast harboring pYELC5; lane M, Amersham high Mr rainbow markers. The position of the VP5 band is indicated. A faint band migrating close to that of VP5 was seen in some of the western blots, however this was attributed to non-specific binding.

present in the VP2 expression strain (but not in the control) which co-migrated with VP2 of BTV-1 that has a predicted Mr of 112 kDa (Gould, 1988). A Western blot of a similar gel (Fig. 2B) using mAb D8/A12/D2 confirmed that the protein in the yeast expression strain co-migrating with VP2 of BTV-1 was authentic VP2 and that some degradation, notably a band of about 35 kDa, occurred during the extraction procedure that involved lyticase treatment and sonication. Analysis of

J. C. Martynet al./ KITAS Research33 (1994) 1I-25

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protein extracts of BTV-1, yeast induced to express the VP5 protein of BTV-1 from pYELCS-VP5 and control yeast harboring the parent plasmid pYELC5, were resolved on a 10% SDS-polyacrylamide gel. It was not possible to detect a band corresponding to the VP5 protein of BTV-1 in the expression strain by protein staining as many yeast proteins were also present in this region (compare Fig. 2A and C). A Western blot of a similar gel (Fig. 2C) using anti-VP5 peptide antiserum identified a major protein band with an estimated Mr of 57.5 kDa in the VP5 expression strain but not in the control that co-migrated with VP5 of BTV-1 [that has a predicted Mr of 59 kDa (Gould and Pritchard, 198811. Three minor VP5 bands of 48,45 and 38 kDa in the virus preparation and one band of 46 kDa in the VP5 expression strain may indicate that degradation may have occurred during the extraction procedure that involved lyticase treatment and sonication. To determine the characteristics of BTV VP2 and VP5 production in induced yeast cells, hourly time samples were taken, lysed with lysis buffer and analysed by Western blot. VP2 reached a maximum 6 h post-induction. There was virtually no degradation of VP2 (estimated Mr 105 kDa) extracted rapidly from yeast in lysis buffer (not shown). In contrast synthesis of VP5 detected with an anti-VP5 peptide antiserum was constitutive but could be induced to a maximum concentration by 4 h post-induction. A major band (estimated Mr 57.5) and three minor bands of 50, 47 and 46 kDa corresponding to products of VP5 degradation or premature termination of translation increased in concentration with time after induction. Two minor bands estimated to be 68 and 65 kDa were higher in Mr than VP5 and increased in concentration with time after induction (not shown). Immune response of sheep and rabbits to W2 and VP5 synthesised in yeast

Sera from sheep immunised three times with crude lysates of yeast containing either 300 pg VP2, a cocktail of 300 pg VP2 and 300 pg VP5 or no BTV protein were tested for BTV antibodies by Western blot and ELISA using BTV-1 as antigen. Western blots showed that one of two sheep injected with VP2 elicited an immune response, of the two sheep injected with VP2 and VP5, both had elicited an immune response to VP5 and one a slight response to VP2, and two sheep injected with control yeast did not elicit an immune response to BTV proteins as expected (Fig. 3). In an indirect ELISA, BTV-specific antibodies could be detected in one sheep immunised with VP2 and VP5 (not shown). Protection of immunised sheep following challenge with virulent BTV-1 (Am)

None of the sheep with antibodies to VP2 or VP5 expressed in yeast were protected after challenge with virulent BTV-1 (Aus). The four sheep injected with yeast-expressed BTV proteins developed clinical signs of bluetongue disease (Table 1) including fever, rash and coronitis. Control and vaccinated sheep showed typical immune responses to BTV infections with a temperature spike at day 8-9 with a drop in circulating platelet cells occurring at approximately the same time (Fig. 4A). This corresponded well with the appearance of anti-VP7 antibodies in both the control and immunised sheep (Fig. 4B) upon challenge with BTVl.

J.C. Martyn et al. / Virus Research 33 (1994) 11-25

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ABC VP2. VP5.

IPIP

IP

Fig. 3. Immune response of sheep to the VP2 and VP5 proteins synthesised in yeast. Equivalent quantities of BTV-1 proteins were electrophoresed in a 10% SDS-polyacrylamide gel beside Amersham high Mr rainbow markers and transferred to a sheet of nitrocellulose. The nitrocellulose was cut into strips for Western blots. Two strips were used per sheep: one was incubated with pre-immune serum (P) and the other with serum taken after three injections of a crude yeast Iysate (I) diluted 1:5 in blotto buffer. Six sheep were tested for BTV antibodies: A, sheep immunised with yeast-expressed VP2 and VP5; B, sheep immunised with yeast-expressed VP5; C, sheep immunised with control yeast.

Synthesis of a recombinant protein in a foreign environment can lead to inappropriate folding or di-sulphide bond formation that in turn can lead to an inappropriate presentation of important epitope(s). The ability of yeast expressed VP2 protein, native BTV and SDS-treated BTV antigen to bind to a panel of

Table 1 Clinical signs follo~ng inoculation Site Ears Eyes Face Mouth Thigh L. front leg R. front leg L. hind leg R. hind Ieg Lameness Scratch test Total

challenge of immunised sheep with virulent BTV-I (Aus. by day 9 after

Animal number 1

2

3

4

5

6

2-t 1+ If 3+ 3+ 3+ 3+ 3+ 2f 0 4f

0

0

0

0

0

0 1+ 0

0 0 0 0 0 1+ 0 0 0

2+ 2+ 2+ 3f 1+ 1+ 2+ 2-l. 1+ 0 2+

0

0 0

1

20

3

27

1+ 0 1+ 1+ 1+ 2+ 0 1+ 7

2+ 2+ 2+ 0 0 2+ 11

0 0 0 0 0 0

1+ 0 0

1+

Sheep 2 and 4 were the control animals inoculated with yeast proteins from a Saccaromyces cerevisae carrying pfasmid pYELC5. Sheep 1 and 3 were immunised with yeast expressed VP2 and VP5, while sheep 5 and 6 were immunised with yeast expressed VP2 alone.

J.C. Martyn et al. /@us

m

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Research 33 (1994111-25

PLATELET

TEMP

4 1.20 2 ci

40.40

f5 5

39.60

460

38.80

280

I+

100

38.00

0

1 2

3

4

5

6

DAYS +

0

SHEEP1

01

7

8

9 10111213141516

POST-CHALLENGE

--A--

SHEEP2

05 DAYS

07

-o--

SHEEP3

09

12

+

SHEEP4

14

16

POST-CHALLENGE

Fig. 4. Protective effect of yeast expressed VP2 against BTV 1 challenge. A: Temperature and platelet levels in experimental sheep challenged with BTVl after immunisation with yeast expressed VP2. Control sheep showed essentially the same general responses (not shown) B: presense of anti-VP7 anti~dies as measured by competitive ELISA in controi sheep (numbers 2 and 4) or sheep pre-immunised with yeast expressed VP2 and VP5 (numbers 1 and 3).

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Monoclonal antibody binding to VP2 antigen

SDS-treated virus

Fig. 5. Ability of neutral&kg mAbs to bind to either yeast expressed VP2, SDS-denatured native virus as measured by indirect ELBA.

VP2 or

monoclonal antibodies was tested using an indirect-ELISA (Fig. 5). The monoclonal antibodies were directed against VP2 and were a mixture of both conformational and linear epitopes (White and Eaton, 1990). The purpose of these tests was to see if the yeast expressed protein appeared to be more like a denatured protein or like the native protein in the complete virus. It was immediately apparent that VP2 was being inappropriately expressed by the yeast system.

4. Discussion The PCR was used to amplify and clone cDNAs of two genes of BTV-1 (Aus) coding for the outer capsid proteins VP2 and VP5. Previously BTV genes coding for the core protein VP7 (Martyn et al., 1990) and the non-st~ctural proteins NSl (Gould et al., 1994) and NS3 (Martyn et al., 1990) were cloned by this method. All of these genes, except VP2, could be cloned from a single PCR product. Although a fuIl-length PCR product with terminal &mHI sites was synthesised for VP2, all attempts to clone the DNA into pUC18 following digestion with BumHI failed (Stoker, 1990; Jung et al., 1990; Kaufman and Evans, 1990). Therefore we resorted to cloning two overlapping fragments of VP2 DNA, synthesised in separate PCRs, to construct a full-Iength cIone in the BarnHI site of pUC18. This method has been sucessfuIly used to construct another full-length clone of BTV23 VP2 in the Sal1 site of pUC18 and may prove useful in cloning other large genes of BTV-1. BTV-1 gene coding sequences for VP2 and VP5 were subcloned into yeast expression plasmids to provide a plentiful source of BTV protein for investigation as potential diagnostic or immunological reagents and in structure/function studies. We have previously used plasmid pGEX (Smith and Johnson, 1988) to

J.C. Martynet al./ Wu.s Research 33 (I 994) 1I-25

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overexpress VP7 (Eaton et al., 1990) and NS3 (Gould, unpublished) in E.coli as fusions with the 26-kDa glutathione S-transferase regulated by the IPTG-inducible tuc promoter. However, these hybrid proteins formed insoluble aggregates on expression in bacterial cells. Soluble VP2 expressed from the PET plasmid (Gould et al., 1994) appeared to be rapidly degraded even in the protease-deficient strain BL21(DE3) (not shown). This was perhaps to be expected for a high Mr, hydrophilic protein such as VP2 to be susceptible to proteolysis in the reducing environment inside E. coli cells. Western blot analysis of yeast-expressed VP2 showed virtually no degradation when the cells were rapidly lysed in lysis buffer but minor degradation bands appeared when cells were lysed by lyticase treatment and sonication (Fig. 2B), a procedure designed to extract yeast proteins in a native state for immunisation. Western blots of yeast-expressed VP5 showed additional minor bands irrespective of the lysis procedure used. This phenomenon has also been observed in lysates of Spodopteru fmgiperdu cells infected with a recombinant baculovirus expressing the VP5 protein of BTV-10 (Marshall and Roy, 1990) and in SVP cells infected with BTV-1 (Fig. 2C). These lower Mr VP5 products may be the result of the susceptibility of the hydrophilic VP5 protein to proteolysis; however, there are no data to dismiss the argument that they arise from initiation at an internal methionine residue or indeed different conformational forms of the protein as has been suggested for AHSV VP5 (Grubman and Lewis, 1992). Maximum levels of VP2 were reached 6 h post-induction and maximum levels of VP5 were reached 4 h post-induction. VP2 was only expressed following induction with 1 mM CuSO,, whereas VP5 was expressed constitutively although induction increased the level several-fold. Other BTV-1 genes expressed constitutively are VP7 (Martyn et al., 1990) and NSl (Gould et al., 1994), while NS3 (Martyn et al., 1990) requires induction for expression. Clearly, whether expression of a gene is inducible or constitutive in this system depends upon the nucleotide sequence being expressed. The immune responses of sheep to crude lysates of yeast containing about 300 pg VP2 or a mixture of 300 pg VP2 and 300 pg VP5 per injection were variable in a pilot study in which six sheep were given two booster injections. Western blots showed that antibodies to both VP2 and/or VP5 were elicited; however, neither antisera contained antibodies capable of neutralising virus infection in serum neutralisation assays or in vivo (Table 1). This may be the result of differences in the conformation of VP2 made in yeast compared to VP2 in the virion given that neutralising epitopes are almost universally conformational rather than linear. This last supposition appears to have some validity as when a panel of virus-neutralising monoclonal antibodies were tested for their ability to bind to either native VP2 (in intact virus), SDS-treated antigen or native yeast expressed VP2, it was observed that the yeast antigen reacted in all cases as if it were in a denatured form. This we have assumed to be due to the very large number of internal cysteine residues (of which six of the 19 are conserved in the VP2s from all BTV serotypes analysed (Gould, 1989.). It appears that the major neutralising epitope(s) of BTV is highly conformational and that the presentation of this site(s) to the immune system of an

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J. C. Martyn et al. / Virus Research 33 (I 994) I I-25

infected animal in subtly different arrangements is responsible for an effective protective response (Gould and Eaton, 1990; White and Eaton, 1990). Recent studies by DeMaula et al. (1993) lend support to this hypothesis. These studies showed that the neutralisation determinants of BTVlO US were identical for two of the three regions mapped when compared to those identified for BTVl Aus (Gould et al., 1988; Gould and Eaton, 1990). This was despite the fact that the two viruses were antigenically distinct and have quite divergent VP2 sequences. Furthermore Heidner et al. (1990) showed that these BTVlO sites were conformationally dependent at at least two sites. Vaccine trials using VP2 and VP5 proteins purified from S. fmgiperdu cells infected with recombinant baculoviruses (Roy et al., 1990) have shown that two injections of 50 pug VP2 alone can protect sheep against challenge with virulent BTV-10 but that less of VP2 is required when VP5 is included in the vaccine. The VP2 protein is the major neutralising antigen of BTV and evidence suggests that VP5 assists the presentation of VP2 to the immune system although little is known of the other functions of VP5. Obviously this system is expressing these outer coat proteins in an appropriate conformational form to act as competant immunogens. Capsid proteins expressed from recombinant plasmids in yeast can assemble into non-infectious, virus-like particles. Examples of this new generation of vaccines include the hepatitis B virus surface antigen (Valenzuela et al., 1982; Miyanohara et al., 1983) and core antigen Kniskern et al., 1986; Miyanohara et al., 1986) and the potyvirus coat protein (Jagadish et al., 1991). The potential exists for co-expression and assembly of two viral capsid proteins in Scerevisiae as functional hemoglobin was assembled from its constituent (Y- and P-chains (Wagenbach et al., 1991) and antibody was assembled from its constituent heavy and light chains (Wood et al., 1985) in vivo. We will attempt to co-express BTV capsid proteins in S. cereuisiue to investigate the requirements for interaction between proteins of BTV. This system would complement that established for recombinant baculoviruses in insect cells in which up to five structural proteins of BTV-10 have been co-expressed and assembled into double-shelled, virus-like particles (French et al., 1990; Loudon and Roy, 1991; Loudon et al.,1990 Apparently in this instance both bacteria and yeast expression systems are incapable of expressing this particular antigen in the correct conformational form. To this end we are cloning and expressing the VP2 antigens of bluetongue virus serotypes 1, 3 and 23 into baculovirus expression vectors to see if this antigen is as protective for the Australian BTV as for those overseas.

Acknowledgements We thank Ian G. Macreadie, C.S.I.R.O. Division of Biomolecular Engineering, Parkville, Victoria, Australia, for providing yeast strains and plasmid pYELC5 and for discussion, Alison Wade-Evans, AFRC Institute for Animal Health, Pirbright, UK, for providing the rabbit antiserum to a VP5 peptide of BTV-1 @A), Ian

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Pritchard, Christine Duch, Jacqueline Kattenbelt and Gary Crameri for excellent technical assistance and Linfa Wang for assistance with the Clone Manager and Plasmid Map Enhancer computer programs (Scientific and Educational Software, Philadelphia, PA). J.C.M. was supported by grant (CSlOlP) from the Australian Wool Corporation.

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