CA2117468A1 - Ibv spike protein (2) - Google Patents

Abstract

A DNA molecule which codes substantially for a truncated Infectious Bronchitis Virus (IBV) spike protein polypeptide, said truncated IBV spike protein polypeptide being characterised in lacking the transmembrane and cytoplasmic anchor domains present in the native IBV spike protein.

Description

~i t i!
~o 93/17109 ~ PCT/GB93~00332 CA2 1 i 7468 IBV SPIKE PROTEIN (2) Backqro~d of the inventlon 1. Fi~ld of thQ inventiQn This invention relates to the spike protein of infectious brsnchitis virus (IBV) and to a recombinant DNA method of preparing lt. IBV is a virus which causes respiratory disease in the fowl, and is of particular importance in relation to poultry.

2. Pescris~iQn of the Drior art I8V is a virus of the type Coronaviridae. It has a single-stranded RNA genome, approximately 20 kb in length, of positive polarity, which specifies the production of three major structural proteins: nucleocapsid protein, membrane glycoprotein, and spike glycoprotein. The spike glycoprotein is so called because it is present in the teardrop-shaped surface projections or spikes protruding from the lipid membrane of the virus~ The spike protein is believed likely to be responsible for immunogen~city of the virus, partly by analogy with the spike prote~ns of other coronaviruses and partly by 7n v~tro neutralisation experiments, see, for example, D. Cav~nagh et al., Av~an Pathology 13, 573-583 (1984). Although the term "spike protein" is used to refer to the glycoproteinaceous material of the sp~ke, it has been characterised by D. Cavanagh, Journal.
General Virology 64, 1187-1191; 1787-1791; and 2577-2583 (1983) as comprising two or three copies each of two glycopolypept~des, Sl ~90,000 daltons) and S2 (84,000 daltons). The polypeptide components of the glycopolypept~des Sl and S2 have been estimated after enzymatic removal of oligosacchar1des to have a co~btned molecular weight of approx~mately 125,000 daltons. It appears that the spike protein is attached to the viral membrane by the S2 polypeptide. Thus, the protein comprises an extra cellular domain, a transmembrane domain and a cytoplasmic anchor domain.
European Patent Application Publication No. 218625A NRDC ~and equiva1ent US Patent S,032,520 and corresponding application in

3~ Japan) d7scloses the clon~ng of cDNA sequences cod~ng for the spike protein ~recursor as well as sequences coding specifically r~liG~,31~0332 CA2117468 1; ~ i ,9~

for the Sl and S2 polypept~des. Such a DNA mole~ule wh1ch codes for an IBV sp1ke prote1n will hereinafter be referred to as "spike DNA" for brev1ty. The d~sclosed splke DNA codes for the whole spike prote1n, i.e. all 3 domains.
Summary of the ~nven~ion It has now been found that it is unnecessary to clone the whole sp~ke cDNA dlsclosed in EP 21862~A 1n order to obta~n an ~mmunological response: a truncated "sp1ke DNA" cons~derably shorter ~n length may be cloned and expressed to produce a polypept~de that w111 generate an immunolog~cal response. Thus, the present ~nvent10n relates to a DNA molecule whlch codes substantially for a truncated IBV spike protein polypeptide. The truncated IBV sp~ke protein polypept~de produced as a resu1t of the clon~ng and expression of a DNA molecule of the present lnvent~on 1s characterised in lacking the transmembrane doma~n and cytoplasmic anchor reg10n of the nat1ve IBV S2 spike protein polypeptide, but otherwise encodtng the whole of the Sl and rema1nder of the S2 polypept1de except that the Sl polypeptide can lack up to lO amino ac~ds at the N-term~nal end and the truncated S2 polypeptide can lack up to 10 am1no ac~ds at ~ts truncated end.
A DNA molecule according to the 1nventlon ~s shown in the Sequence L~st1ng (SEQ ID N0: 1). Th~s DNA ~olecule was obta~ned as a result of research on ~he M41 stra1n o~ IBV, but ~b~-~ s expected that slmllarly truncated sptke pro~e~n of cDNA of other IBV serotypes and stra~ns such as Beaudette, M~2, ~l~2, Connect1cut 1solate A5968, Arkansas and Holland stralns H120, H5~, Ma5, D207, D212, D3128 and D3896, whether or not exh~b~t~ng a h~gh degree of homology w~th M41, wil1 express I~V sp1ke prote1n.
In referr~ng to a DNA molecule def~nëd as codlng substantially for a truncated IBV sp1ke prote~n ~t w111 be appreclated that it ~s 1ntended not to exclude DNA flanklng sequences, wh~ch may be, for example, cDNA to flank~ng sequences ~n the IBV RNA
genome (other than transmembrane sequences) or may be fore~gn sequences der1ved from other genes, such as leader sequences that may ass1st ~n dr1v1ng expresslon of the truncated polypept~de or may be a short sequence of plasm~d DNA. Also, ~t ~s not ~ntended that the DNA molecule should necessar11y code for ~, 1..., r ,~
_ . _ .. _ , . ...

~ 93~171Q~ PCT/GB93/00332 _ ~ _ amino acids extending right up to the 5'- term~nus or 3'-truncated end. It may be possible to obtain expression of the truncated sp~ke protein lacking say, up to 5 or eYen 10 of the amino acids (30 nucleotides) at either end.
The invent~on also includes a vector containing the above defined DNA molecule, including a cloning vector sllch as a plasmid or phage or expression vector, preferably a pox virus vector, and a host contalning the vector. Mammalian cells contain~ng the above-defined DNA molecule, whether as naked DNA
or contained in a vector, are also included. Further, the invention includes isolated biosynthetic truncated spike protein polypeptide and ~ts expression from mammalian cells.
Brief descriDtion of the drawinas Figures 1-17 show plasmid constructs of use in the prepara~ion of DNA molecules of the present invention.
DescriDtion of the preferred embodiments SEQ ID NO: 1 shows the complete nucleotide sequence of a cDNA
molecule of the invention obtained from IBV genomic RNA M41 strain. The IBV RNA of other strains is believed to be fairly s~milar to that of M41, and therefore oligonucleotides derived from DNA of the present invention can be used as primers for sequencing RNA of other serotypes thus enabling truneated cDNA
for all or virtually all other serotypes to be prepared usin~-~methods described hereinafter. Obviously, those serotypes in wh kh the ent~re IBV spike protein cDNA has a h~gh degree of nucleotide sequence homology with IBV M41 stra~n are slightly pre~erred, as giving a w~der choice of potent~al oligonucleot~des.
The vectors included in the invention are clon~ng and expression vectors. The DNA molecule of the present invention is conv~niently multiplied by insertion in a prokaryotic vector, for example pBR322, and cloning in an appropriate host such as a bacterial host, especially E. coli. Alternatively, using appropriate dlfferent vectors it could be multiplied in (say~
8acillus species, or a yeast. For expression, mammalian cells can be transfectod by the calcium phosphate precip~tat~on method or transformed by a viral vector. Viral vectors include WO 93/17109 PCI/GB93/003~2 retroviruses and poxviruses such as fowlpox virus or vaccinia virus.
A DNA molecule of the present invention may be prepared by first obtaining full length IBV spike DNA in a suitable plasmid.
European Patent 218625A NRDC predicts the probable transmembrane domain of the spike protein and indieates the region of DNA
coding for it. A suitable endonuclease restriction site near the beginning of the DNA sequence coding for the transmembrane domain, can ~hen be ident~fied. Uslng the des~red endonuclease, the IBV spike DNA may be cleaved and the truncated DNA molecule coding for the extracellular domain, introduced into a viral vector as described below. Care is needed to ensure either that the chosen restriction site is a unique one in the spike DNA. or that a cloning procedure such as described in the Example is devised to compensate. In the Example, a two-step cloning ~ proeess was used to overcome a second Styl site in the M41 spike - DNA molecule. Alternatively, once it is known where the sequencecoding for the transmembrane domain begins, the truncat1On can be brought about uslng Polymerase Chain Reaction (PCR) cloning or by using ollgonucl20tide site-dlrected mutagenesis. In the latter method, a stop codon is ~nserted at the desired positlon.
The truncated IBV sp~ke DNA can be introduced into the viral vector as ~ollows. The DNA is inserted into a plasmid containi~
an appropriate non-essential region of poxvirus DNA, such as the thymldine kinase gene of vacc1nia virus or ~nto any su~table non-essent~al region of fowlpox virus, e.g. as described in European Patent 353851A, so that the insert interrupts the NER
sequence. A poxvirus promoter, e.g. the vaccinia virus p7.5K
promoter, which is usable in vaccinia virus or avipoxviruses. or a fowlpox virus promoter as described in our prior patent appllcations publication Nos. W089/03879, W090104638 and W091/02072, is also introduced into the NER sequence in such a pos~tion that it will operate on the inserted truncated spike DNA
sequence. When an intergenic NER is used a "marker" gene w~th its own promoter e.g. the lac Z gene will be inser~ed along with the sequense coding for the truncated spike protein. When the SU13STITUTE SHE~ET

~o 93/17109 PCT/GB93/00332 poxvirus and the plasmid recombinant DNA are co-transfected into a mammalian cel1, homologous recombination takes place between the poxvirus NER, such as TK in vacclnia virus, or a said non-essential region of fowlpox virus and the same gene or region S present in the plasmid. Since the truncated IBV spike DNA has thereby interrupted the poxvirus gene, viruses lacking the gene expression product, such as TK, are selected. If the NER used is an intergenic region, viruses expressing the truncated spike protein ~11 be identified by the co-expression of the "marker"
gene e.g. blue plaques colonies if lac Z ~s the marker gene.
Once such a recombinant virus vector has been thus constructed it can be used to introduce the truncated IBV spike DNA directly into the desired host cells without the need for any separate step of transfec~ing plasmid recsmbinant DNA into the cells.
In order to improve the expression of the truncated spike protein it may be preferable to replace part or all of the untranslated leader sequence upstream of the spike gene. The leader sequence is the region between the TAATTATT of the promoter sequence and the ATG initiation codon of the gene. By replacing part or all of the native FPV IBV leader sequence wlth leader sequences derived from other related vlruses such as poxviruses it may be poss1ble to initiate stronger translation in FPV.
Such leader sequences could be deriYed from: (1) part or all of the sequences found downstream of other poxvlral promoters e~g. the vaccinia v1rus p7.5 pro0oter (li) part or all of the leader sequences from foreign gsnes that have been shown to be well expressed in sells infected by the appropriate recombinant poxviruses or (~ synthetlc sequences shown to promote efficient translation in poxvirus-infected cells. The replacement of appropriate sequences can be accomplished using PCR cloning or by inserting synthetic oligonucleotides. The choice of leader sequence to be used and the method of inser~ion is well within the ability of skilled man. The Example 2 hereinafter illustrates how the procedure could be performed.

SUBSTITUTE SHEET

W O 93~17109 PCT/GB93/00 The invention therefore further relates to a vector wherein containing part or all of a sequence found downstream of a poxvirus promoter, not being the poxvirus promoter of use in the vector, between the promoter and the IBV DNA Molecule.
With a view ultimately to obtaining expression of the recombinant virus in ViYo, the preferred poxvirus is fowlpox virus. It may be that the inserted truncated IBV DNA contains a sequence, which, in the fowlpox vector, leads to premature termination of transcription. In this case, the truncated sp~ke DNA would have to be mod~fied slightly by one or two nucleotides, thereby to allow transcription to proceed along the full length of the gene.
The vector can be introduced into any appropriate host by any method known in recombinant DNA technology. Hosts include E. coli, Bacillus 5pp, animal cells such as avian or mammalian cells and yeasts. The method of introduction can be transformed by a plasmid or cosmid vector, or infection by a phage or viral vector etc. as known in recombinant DNA technology.
The following Examples illustrate the invention. All temperatures are in C.

I. Prepara~ion ~f a full lenqth IBV s~ike prot~n cDNA from M41 ~tra~n Example 2 of European Patent Application Publicat~on No. 218625 (NRDC) describes the preparation of cDNA coding for the spike protein precursor of IBV strain M41. It describes therein the preparat~on of plasmids pMB276 and pMB2~ containing the entire M41 sp~ke protein cDNA sequence.
An initial step in the preparation of a DNA molecule encoding a truncated IBV spike protein was to join pMB276 and pMB250 to produce a full length clone of the IBV M41 spike gene.
1. Joinlnq pMB276 and pMB250 a~ a shared Ndel site to produce a ~ull lenath çLone of the IBV M41 s~ike aene ~in DMB374) Plasmids pMB276 and pMB250 were digested w~th Ndel (20 units) in 50mM tris-HCl pH 8.0, lOmM MgC12, SOmM NaCl, final volume 20~1.

SUBSTITU I E SHEET

YYO 93/1710g P~T/GB93/00332 The digested DNA was then phenol-extracted with an equal volume of TE-saturated phenol, ether extracted twice with an equal volume o~ water-saturated ether, then ethanol- precip~tated. The precip~tated DNA was resuspended in 15~1 water. Then 2.5~1 of each digest were ligated together in a total volume of 10~1 in 50mM tris~HCl pH 7.5, lOmM MgC12, lOmM ~TT, lmM ATP, 1 unit T4 DNA ligase at 4C overn~ght. Ligated DNA, in 1~1 of the ligation mix, was transformed into competent E. col1 DH5 and transformed bacteria were selected on agar plates containing tetracycline.
Transformant colonies were grown in L broth plus tetracycline and DNA was isolated therefrom using a standard procedure described by Holmes and Quigley (1981), Analytical Biochemistry 114: 193-197. Following digestion of the isolated DNA with Ndel and agarose gel electrophoresis, i~ was apparent that, of 48 clones screened, one Cno. 17) had ~nher~ted the desired fragments from the parental plasmids, viz. a fragment of circa 6kbp from pM3276, Fig. 1 and a fragment of about 4kbp from pMB 250, F~g. 2.
The desired recombinant plasmid would also have a fragment, following Pstl d1gestion, equivalent to the length of pBR322 (~stl sites flank the M41 spike cDNA). Analysis of clone 17 : showed that ~t d~d not have a pBR322-s~zed Pstl fragment, :~ indicat7ng that the two Ndel fragmen~s had tlgated together in the wrong relative orientaff 0n. Clone 17 DNA was therefor~
digested with Ndel and religated (using procedures -described above) to allow isolation of recomblnan~s with the two Ndel fragments in the correct or3entat~on. Analys~s of Pstl-digested DNA from a number of clones showed that about 50X had religated to glve the correct or3entat30n. One of these clones was saved, as pMB374, Fig. 3.
2. Clonlna _the IBV M41 sPike Qene under cQntrJ2L~ hp vacc1nia v1rus ~7.5 Dromoter to make DGSS2 The IBV M41 spike protein gene was cut out of pMB374 by digest10n of the plasmid with Tthlll 1, see Fig. 3, in lOmM
trls-Hcl pH 7.4, lOmM MgC12~ 50mM NaCl, lOmM ~-mercaptoethanol, at 65C ln a final volumæ o~ 20~1. The DNA was made blunt-ended SUE~STITIJTE SHEET

by the addition of 0.025mM dATP, dCTP, dGTP, dTTP and 5 units of Klenow polymerase, followed by ineubation for lh at room temperature. The digestion products were electrophore~ed on an agarose gel using standard procedures as described by Maniatis et al., (1982) in "Molecu1ar cloning: a laboratory manual" ~Cold Spring Harbor Laboratory) and a Skb fragment, containing the sp~ke gene was purified using "Geneclean II" (Blo lOl~ as per supplier's ~nstructions. The purified DNA was then cloned into the ~m~l site of pGS20 (from Dr. G. L. Smith, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OXl 3RE, as described in Mackett, Smith & Moss (l984), J. V~rol. 49, 857-B64) to make pGSS2, see Fig. 4.
II. TruncatiQn o~ M41 spike gene It was desired to truncate the spike protein gene so that the lS protein would not carry a transmembrane segment and a cytoplasmic domain. This could be conveniently achieved by cutting the gene ; at the ~y1 site (position 4384 in pGSS2). As there is another StYl s1te within the gene, a two step process was devised. This involved the transfer of the spike prote~n gene sequences (wlthout the p7.5 promoter~ to pUCl9 (Yanlsch-Perron, Vie1ra &
Messing~ 1985, Gene 33, 103-ll9). Finally the truncated spike gene was transferred to the fowlpoxvirus expression plasmid, pEFL29. These steps are descr1bed below. .
1. Transfer of the ~yl fra~ment within the spike qene frQm D~SSZ to pU~l9 to make pUC/M4lStv pGSS2 was digested with Stvl, the DNA was made blunt-ended with Klenow polymerase and the 1.95kb fragment (2430-43843 was ; recovered and pur~f~ed. This fragment was ligated into pUCl9 di gested wi th ~m~l- Recombi nants carrying the inserted fragment were isolated and the orientation of the inserted fragment was checked by digestion of their plasmid DNA with Mlul and BamHl.
The required recombinant had a small Mlul/BamHl fragment of 480 bp (and not 1480bp) and was given the title pUC/M41Sty, Fig. 5 (note that ligation of the blunt-ended Styl fragment into the ~m~l site restores the Stvl sites but not the Smal site~.

- SUBSTITUl-E SWEEi~l' ` CA2117468 ~0 93/17109 PCr/GB93/00332 2. Cloninq ~he N-terminal part of the sDike qene from DG~S2 intQ pUC/M41 S~y ~o g~ve pU~/M41 Bam-Stv (conta1ninq spike se~u~nces ~ mm the N-terminus to the C-terminal ~tyl site) Plasmids pGSS2, Fig. 4, and pUC/M41Sty, Fig. 5, were both d~gested with BamHl and Afl2 and fragments of 2.2kb and 3.8kb, respectlvely, were recovered. The purified fra~ments were liyated together and recombtnants were isolated. The required recombinant (tltled pUCJM41 Bam-Sty3, Fig. 6, had the 0.85kb BamHllAfl2 fragment of pUC/M41Sty replaced by a 2.2kb fragment from pGSS2, Fig. 6.
3. Tr~n~fer of ~he truncated s~ik~ qene from pUC/M41 Bam-~y into the f~wlDoxvirus exDression vectQr. ~EFL29. to aive uEFS17 The entire truncated spike gene sequences were cut out of pUC/M41 Bam-Sty using ~mHl and E~oRl. Followlng repair of the ends of the DNA w~th Klenow polymerase, the 3.3kb fragment was isola~ed. purified and blunt-end liga~ed ~nto pEFL29, F~g. 7, digested with ~m~l- Recomb1nants were screened by digestion with BamHl/B~12 to check that the sp~ke gene insert was in the correct or~entation relat~ve to the p7.5 promoter in pEFL29. Correct recombinants were titled pEFSl7, Fig. 8. (The derivation of pEFL2~ ~s descr7bed below).

4. Derivatlon of pEFL29 _~
A DNA fragment containing the fowlpoxvirus 4b- promoter dr1vlng a l~Z reporter gene was cut sut of plasmid pNM~b30 (see the releYant fowlpox virus promoter patent spec~f~cat~on (W089l03879), page 35, Table 2~ using ~QRl and ~L~l. The fragment was end-repaired and ~as then blunt-end ligated ~nto the end-repaired B~12 s~te of a plasmid containiny part of the terminal ~mHl fragment of fowlpoxvirus (p83ME, described in Boursnell et al., l990, J. Gen. Virol. 71, 621-628) to create plasmid pEFLlO.
The vacc~nia virus p7.5 promoter was then introduced, on a 300bp çQRl (end-repaired) DNA fragment from pGSZ0 (see above), into the ~ç~l site of pEFLlO. A recombinant with the 5UBSTITUTE SHE~T

w o 93~17109 PCT/GBs3/003.~?~

p7.5 promoter in such an orientation that transcript~on from it is initiated in the opposite direction to that from the fowlpoxvirus 4b promoter, identified by restriction analysis using BamHl, was titled pEFL29.

5. Isolation of recombinant fowlpoxvirus exDressina the truncated IBV M41 spike gene Chick embryo f~broblasts (CEFs), at 80% confluence, were infected with the Duphar "Poxine" strain of fowlpoxvirus at a ~ultiplic~ty of infection (m.o.i.) of 1. At 4h post-infection, pEFS17 DNA (10~9 per 25cm2 flask) was introduced to the cells using the 'Lipofectin' method ~8RL) under manufacturer~s instructions. Five days post-infection, when there was complete cytopathic effect, the cells were harvested. Virus, released from the cells by freeze/thawing three times, was used at various dilutions to infect CEFs which were then overlaid w1th agarose to allow plaques to form. When plaques were vis1ble the plates were overlaid with X-gal agarose. Two days la~er, blue plaques were p~cked and virus was released by freeze/thawing. The virus was titrated again, overlaid with X-gal agarose and blue plaques were picked again. This procedure was repeated three more times.
Finally two plaques (fpl74Pxllll and fpl74Px1121) were chosen for further characterisation.
III. Characterisat~on of the fowlpoxvirus/EFS17 recombinant ~1!~
Z~ 1. (a) Plaque hybridisation analysls These viruses were propagated and plaques on CEFs were onee more obtai ned . The agarose overlay was removed and a nitrocellulose f~lter was applied to the cell sheet. A piece of 3MM filter paper, soaked in 20X SSC, was applied to the nitrocellu1Ose filter. The nitrocellulose filter was then removed and baked at 80C in a vacuum oven. The filters were then probed with a 32P-radiolabelled probe speci~ic ~or the IBV
M41 spike protein gene, to verify that the recombinan~ fowlpox viruses carried the IBV M41 spike protein gene.

SUBSTITUTE SH8~ET

CA2i i 7468 YVO 93/17109 pcT/GBs3/oo332 2. (b) Radio-immunoprecipitation assay (RIPA) CEFs were infected with the fpEFSl7 recombinant viruses (or with a control 'poxine'/lacZ recombinant virus or mock-infected) at a m.o.i. of 10. At 24h post-infection the tissue culture medium was replaced with methionine-free medium to 'starve' the cells (i.e. to deplete the cells of their intracellular methion~ne pool~ for lh. The cells were then labelled with 35S-methionine (lOO~Ci) for 3h. Then they were harvested, washed and lysed ~n RIPA buffer (the RIPA procedures used are described in detail in "Antibodies: a laboratory manual", Harlow and Lane ~1988), Cold Spring Harbor Laboratory, New York). A polyclonal serum raised in rabbits against purified IBV M41 spike protein was added to the clarified extracts and immune complexes were precipitated with protein-A/Sepharose.
The protein-A/Sepharose was washed thrice with RIPA buffer, then resuspended in SDS-PAGE sample buffer and boiled for 3 min. The samples were then applied to a 5-lOZ gradient SDS-PAGE gel and electrophoresed. The gel was fixed and exposed by fluorography.
RIPA analysls sho~ed that cells infected with the fpEFSl7 recomb~nant v~ruses, but not those infected ~th control 'pox~ne'/lacZ recombinant nor uninfected cells, synthesised a new prote~n (apparent molecular we~ght about 160K). The band appeared 'fuzzy', characteristic of an extensively glycosylate~.
prote~n sueh as the spike protein. When the infected eells were 'starved' and labelled in the presence of tunicamycin, an ~nhibitor of N-11nked glycosylation, a fa~nt band was seen at about 120K (the predicted size of the unmodified primary translation product) but most of the new product appeared as two closely migrating b~nds of 90-9SK, suggesff ng that the 30 unglycosylated protein was unstable and was being cleaved by protease activity.

SUBSTITUTE SHEET

WO 93/17109 PCl/GB93/003 The Example below describes the replacement of the untranslated IBV spike sequences with sequences derived from part of the leader downstream of the p7.5 promoter, by cloning S synthetic oligonucleotides between the BamHI site in the leader and a Spel site near the 5' end of the I~V spike coding sequence. The complete leader is then cloned upstream of the truncated IBV spike gene from pEFS 17 to give pEFS 20.
In summary the 83 base pair BamHI-Spel fragment (SEQ ID N0 3) 10in pEFS17 i 5 replaced with a synthetic leader based on p7.5 (SEQ ID N0 4) using the oligonucleotides MAS-H7 and MAS-H8 (SEQ ID 5 and 6 respectively).
1) Replacinq non-translated leader from IBV in_pGSS2 with leader sequences from the va~cinia virus D7.5 promoter 15Plasmid pGSS2 (Fig. 4) was digested with BamHI (1059) and Spel (3358), and fragments of lOkb and 2.2kb (Fig. 9 were recovered. To anneal synthetic oligonucleotides MAS-H7 and MAS-H8, 50 pmol of each were mixed in 10~1 water. They were then bolled for 3 minutes and allowed to cool slowly to room temperature. The annealed oligonucleotide duplex (0.2 to 5 pmol) was then ligated to the 10 kb BamHI-Spel fragment from pGSS2.
The required recombinant, pGSS3 (~ig. 10), had retained the BamHI
and Spel sites but had deleted a 2.2 kb Spel fragment relative t~
pGSS2.
2) Replasina the ~eleted 2.2 kb S~el fraament from vGSS2 into DGS~3 ~o make p~SS4 The 2.2 kb Spel fragment from pGSS2 (Fig 11) was ligated into pGSS3 11nearised w~th Spel. The presence and orientation of the inserted Spel fragment in the resultant recombinant, pGSS4 ~Fig. 12), was verified by digestion ~ith Spel, Aflll, Mlul or BamHI/Sall.

~A2 1 1 ~4 68 ~0 93/17109 PCT/GB93/00332 3) Cloninq_~he IBV s~ike qene with the new leader from pGSS4 into the eX~ressiQn vector. pEFL29 Plasmid pGSS4 was digested with BamHI and EcoRI, repaired with Klenow polymerase then a 4.~ kb fragment ~Fig. l3) was recovered and ligated into pEFL29 (Fig. 7) digested with Smal.
The presence and orientation of the spike gene insert in the desired recombinant, pEFSl9 (Fig. 14), was checked by digestion with BamHl, EcoRI, Styl or BamHI/Styl.
4) Comb~ninq the new leader and 5'-terminus of the I~V spike qene (fr~m DEF~l9) with the ~-terminus nf the truncated sDike qene (from pEF~17) Plasmids pEFS17 and pEFSl9 were digested with Ncol and BglII
then 3 kb (Fig. l5) and ll.8 kb (Fig. 16) fragments, respectively, were recovered and ligated together. The required lS recombinant pEFS20 (Fig. 17) was checked by digestion with Kpnl, BamHI, Styl and BamHI/Styl.
Recomb~nant fowlpox viruses were derived, using pEFS20, and analysed as described above in Example l.III for pEFSl7.

5LIBE;TITUTE SHEET

WO93/17109 PCT/GB93/00~
_ ",,~ _ SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: British, Technology Group Ltd (ii) TITLE OF INVENTION: IBV Spike Protein (2) (iii) NUMBER OF SEQUENCES~ 6 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: British Technology Group Ltd (B) STREET: l0l Newington Causeway (C) CITY: London (E) COUNTRY: U.K.
(F) ZIP: SEl 6BU
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy ~isk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #l.0, Version ~l.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B~ FI~ING DATE:
(C) CLASSIFICATION:
(vii~ PRIOR ~PPLICATION DATA:
(A) APPLICATION NUMBER: GB 9203509.6 ~B) FILING DATE: l9-FEB-1992 tviii) ATTORNEY/AGENT INFORMATION:
t~) NAM~: Percy, R K
(C3 REFERENCE/DOCKET NUMBER: 135324 ~
(ix) TELECO~MUNICATION INFORMATION:
(A) TELEPHONE: 017 403 6666 (B) TELEFAX: 071 403 7568 (2~ INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LEN~TH: 3281 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: cDNA
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Infectious bronchitis virus (B) STRAI~: M4l SUBSTITU I t SHEET

93~17109 PCT/GB~3/00332 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: l..3281 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATG TTG GTA ACA CCT CTT TTA CTA GTG ACT CTT TTG TGT GTA CTA TGT

Met Leu Val Thr Pro Leu Leu Leu Val Thr Leu Leu Cys Val Leu Cys l 5 lO 15 AGT GCT GCT TTG T~T CAC AGT AGT TCT TAC GTT TAC TAC TAC CAA AGT

Ser Ala Ala Leu Tyr A~p Ser Ser Ser Tyr Val Tyr Tyr Tyr Gln Ser GCC TTT AGA C~A CCT AAT GGT TGG CAT TTA CAC GGG GGT GCT TAT GCG

Ala Phe Arg Pro Pro Asn Gly Trp His Leu His Gly Gly Ala Tyr Ala GTA GTT AAT ATT TCT AGC GAA TCT AAT AAT GCA GGC TCT TCA CCT GGG

Val Val Asn Ile S~r Ser Glu Ser Asn Asn Ala Gly Ser Ser Pro Gly TGT ATT GTT GGT ACT ATT CAT GGT GGT CGT GTT GTT AAT GCT TCT T

Cys Ile Val Gly Thr Ile His Gly Gly Arg Val Val ~sn Al~ Ser Ser

6~ 70 75 ~9 ATA GCT ATG ACG GCA CCG TCA TCA GGT ATG GCT TGG TCT AGC AGT CAG

Ile Ala Met Thr Ala Pro Ser Ser Gly Met Ala Trp Ser Ser Ser Gln go 95 TTT TGT ACT GCA CAC TGT AAC TTT TCA GAT ACT ACA GTG TTT GTT ACA

Phe Cys Thr Ala His Cys Asn Phe Ser Asp Thr Thr Val Phe Val Thr lO0 105 lt0 CAT TGT TAT AAA TAT GAT GGG TGT CCT ATA ACT GGC ATG CGT CAA AAG
3~4 CA~ I 1 7468 W093/17l09 PCT/GB93/00~

His Cys Tyr Lys Tyr Asp Gly Cys Pr~ Ile Thr Gly Met Arg Gln Lys 115 lZ0 125 AAT TTT TTA CGT GTT TCT GCT ATG AAA AAT GGC CAG CTT TTC TAT AAT

Asn Phe Leu Arg Val Ser Ala Met Lys Asn Gly Gln Leu Phe Tyr Asn TTA ACA GTT AGT ~TA GCT AAG TAC CCT ACT TTT AAA TCA TTT CAG TGT
48~
Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Phe Lys Ser Phe Gln Cys GTT AAT AAT TTA ACA TCC GTA TAT TTA AAT GGT GAT CTT GTT TAC ACC

Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Tyr Thr TCT AAT GAG ACC ACA GAT GTT ACA TCT GCA GGT GTT TAT TTT AAA GCT

Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala 1~0 185 190 GGT GGA CCT ATA ACT TAT AAA GTT ATG AGA G~A GTT AAA GCC CTG GCT

Gly Gly P~o Ile Thr Tyr Lys Val Met Arg Glu Val Lys Ala Leu A~
195 20~ 205 TAT TTT GTT AAT GGT ACT GCA CAA GAT GTT ATT TTG TGT GAT GGA TCA

Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys Asp Gly Ser CCT AGA GGC TTG TTA GCA TGC CAG TAT AAT ACT GGC AAT TTT TCA GAT

Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp ~GC TTT TAT CCT TTT ATT AAT AGT AGT TTA GTT AAG CAG AAG TTT ATT
76~
Gly Phe Tyr Pro Phe Ile Asn Ser Ser Leu Val Lys Gln Lys Phe Ile ~;UE~STITUTE SHEET

CA2i 1 7468 .~O 93/17l09 PCr/GE~93/00332 GTC TAT CGT GAA AAT AGT GTT AAT ACT ACT TTT ACG TTA CAC AAT TTC

Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Phe Thr Leu His Asn Phe ACT TTT CAT AAT GAG ACT GGC GC:C AAC CCT AAT CCT AGT GGT GTT CAG

Thr Phe His Asn Glu Thr Gly Ala Asn Pro Asn Pro Ser Gly Val Gln AAT ATT CA~ ACT TAC CAA ACA CAA ACA GCT CAG AGT GGT TAT TAT AAT
gl2 Asn ïle. Gln Thr Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn TTT AAT TTT TCC TTT CTG AGT AGT TTT GTT TAT AAG GAG TCT AAT TTT

Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Glu Ser Asn Phe 3~5 310 315 320 ATG TAT GGA TCT TAT CAC CC}~ AGT TGT AAT TTT AGA CTA GAA ACT ATT
10~8 Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr I le 325 330 335_.<, AAT A~T GGC TTG TGG TTT AAT TCA CTT TCA GTT TCA ATT GCT TAC GGT

Asn Asn Gly Leu Trp Phe Asn Sor Leu Ser Val Ser Ile Ala Tyr ~;ly CCT CTT CAA GGT GGT TGC AAG CAA TCT GTC TTT AGT GGT AGA GCA ACT

Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Gly Arg Ala Thr TGT TGT TAT GCT TAT TCA TAT GGA GGT CCT TCG CTG TGT AAA GGT GTT

Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Ser Leu Cys Lys Gly Val 37~ 375 380 SUBSTITUTE SHEET

~;A~ i /46~

WO 93/171Og ~5 PCI~GB93/003?^`

TAT TCA GGT GAG TTA GAT CTT AAT TTT GAA TGT GGA CTG TTA GTT TAT

Tyr Ser Gly Glu Leu Asp Leu Asn Phe Glu Cys Gly Leu Leu V~l Tyr 38~ 390 395 40~

GTT ACT AAG AGC GGT GGC TCT CGT ATA CAA ACA GCC ACT GAA CCG CCA

Val Thr Lys Ser Gly Gly Ser Arg Ile Gln Thr Ala Thr Glu Pro Pro GTT ATA ACT CGA CAC AAT TAT ~AT AAT ATT ACT TTA AAT ACT TGT GTT
I296 .
Val Ile Thr Arg His Asn Tyr Asn Asn Ile Thr Leu Asn Thr Cys Val . 420 425 430 GAT TAT AAT ATA TAT GGC AGA ACT GGC CAA GGT TTT ATT ACT AAT GTA

Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr Asn Val ACC ~AC TCA GCT GTT AGT TAT AAT TAT CTA GCA GAC GCA GGT TTG GCT
13g2 Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Ala ~sp Ala Gly Leu Ala ATT ~T~ GAT ACA TCT GGT TCC ATA GAC ATC TTT GTT GTA CAA GGT GAA~

Ile Leu Asp Thr Ser Gly Ser Ile Asp Ile Phe Val Val `Gln Gly Glu 465 470 475 ~80 TAT GGT CTT ACT TAT TAT AAG GTT AAC CCT TGC GAA GAT GTC AAC CAG

Tyr ~ly Leu Thr Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gln CAG TTT GT;~ GTT TCT GGT GGT ~ TTA GTA GGT ATT CTT ACT TCA CGT
153~
Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg ~AT GAG ACT GGT TCT CAG CTT CTT GAG AAC CAG TTT TAC ATT AAA ATC

CA27 i 7468 ~v~93~17109 ~q_ PCTJGB93/00332 Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn Gln Phe Tyr Ile Lys Ile ACT AAT GGA ACA CGT CGT TTT AGA CGT TCT ATT ACT GAA AAT GTT GCA

Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser Ile Thr Glu Asn Val Ala AAT TGC CCT TAT GTT AGT TAT GGT AAG TTT TGT ATA AAA CCT GAT GGT

Asn Cys Pro Tyr Val Ser Tyr Gly Lys PhP Cys Ile Lys Pro Asp Gly TCA ATT GCC ACA ATA GTA CCA AAA CAA TTG GAA CAG TTT GTG GCA CCT

Ser Ile Ala Thr Ile Val Pro Lys Gln Leu Glu Gln Phe Val Ala Pro 565 ~70 575 TTA CTT AAT GTT ACT GAA AAT GTG CTC ATA ~CT AAC AGT TTT AAT TTA

Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu 5~0 585 590 ACT GTT ACA GAT GA& TAC ATA CAA ACG CGT ATG GAT AAG GTC CAA ATT
1~24 Thr Yal Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln ~e 595 600 60~ ~

AAT TGT CTG CAG TAT GTT TGT GGC AAT TCT CTG ~AT TGT AGA GAT TTG

Asn Cy5 Leu Gln Tyr Val Cys Gly A~n Ser Leu Asp Cy5 Arg Asp Leu 610 6lS 620 TTT CAA CAA TAT GGG CCT GTT TGT GAC AAC ATA TTG TCT GTA GTA AAT

Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn 6~5 630 635 640 AGT ATT GGT CAA AAA GAA GAT ATG GAA CTT TTG AAT TTC TAT TCT TCT

Ser Ile Gly Gln Lys Glu Asp Met Glu Leu Leu Asn Phe Tyr Ser Ser ~:IJR~:TITII~ ~ F F T

WOg3/l7l09 PCr/GB93/00 _ 2O-645 65~ 655 ACT AAA CCG GCT GGT TTT AAT ACA CCA TTT CTT AGT AAT GTT AGC ACT

Thr Lys Pro Ala Gly Phe Asn Thr Pro Ph Leu Ser Asn Val Ser Thr GGT GAG TTT AAT ATT TCT CTT CTG TTA ACA ACT CCT AGT AGT CCT AGA

Gly Glu Phe Asn Ile Ser Leu Leu Leu Thr Thr Pro Ser Ser Pro Arg 675 6~0 685 AGG CGT TCT TTT ATT GAA GAC CTT CTA TTT ACA AGC GTT GAA TCT GTT

Arg Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Yal Glu Ser Val 690 ~95 700 GGA TTA CCA ACA GAT GAC GCA TAC AAA AAT TGC ACT GCA GGA CCT TTA

Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu 705 710 715 ~20 GGT TTT CTT AAG GAC CTT GCG T&T GCT CGT GAA TAT AAT GGT TTG CTT

Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu 725 730 735 ~, GTG TTG CCT CCC ATT ATA ACA GCA GAA ATG CAA ACT TTG TAT ACT AGT

Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Leu Tyr Thr Ser 740 745 75~

TCT CTA GTA GCT TCT ATG GCT TTT GGT GGT ATT ACT GCA GCT GGT GCT
~304 Ser Leu Val Ala Ser Met Ala Phe Çly Gly Ile Thr Ala Ala Gly Ala ATA CCT TTT GCC ACA CAA CTG CAG GCT AGA ATT AAT CAC TTG GGT ATT
235~
Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile 770 77~ 780 SUBSTITUTE 5HEE~T

CA-2 i i 74h8 ~'~93/17109 PCT/GB93~00332 ACC CAG TCA CTT TT~ TTG AAG AAT CAA GAA AAA ATT GCT GCT TCC TTT

Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe 785 ?90 795 800 AAT AAG GCC ATT GGT CGT ATG CAG GAA GGT TTT AGA AGT ACA TCT CTA

Asn Lys Ala Ile Gly Arg Met Gln Glu Gly Phe Arg Ser Thr Ser Leu GCA TTA CAA CAA ATT CAA GAT GTT GTT AAT AAG CAG AGT GCT ATT CTT
24g6 Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ala Ile Leu ACT GAG ACT ATG GCA TCA CTT AAT AAA AAT TTT GGT GCT ATT TCT TCT

Thr Glu Thr Met ~la Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser Ser GTG ATT CAA G~A ATC TAC CAG CAA CTT GAC GCC ATA CAA GCA AAT GCT
25~2 Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Ala 8~0 855 860 CAA GTG GAT CGT CTT ATA ACT GGT AGA TTG TCA TCA CTT TCT GTT T~

Gln Val A~p Arg ~eu Ile Thr Gly Arg Leu Ser S~r Leu Ser Val Leu 8~5 870 ~75 880 GCA TCT GCT AAG CAG GCG GAG CAT ATT AGA GTG TCA CA~ CAG CGT GAG

Ala 5er ~la ~ys Gln Ala Glu His Ile Arg Val Ser Gln Gln Arg Glu 885 890 ~95 TTA GCT ACT CAG AAA ATT AAT GAG TGT GTT AAG TCA CAG TCT ATT AGG

Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Ile Arg 9~0 905 9lO

TAC TCC TTT TGT GGT AAT G~A CGA CAT GTT CTA ACC ATA CCG CAA AAT
27~4 SUBSTITIJTE SHEET

C A 2 i ~ 7 4 ~
WO93~7109 -~2- PCT/GB93/00~

Tyr `Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn GCA CCT AAT GGT ATA GTG TTT ATA CAC TTT TCT TAT ACT CCA GAT AGT
2~32 Ala Pro Asn Gly Ile Val Phe Ile His Phe Ser Tyr Thr Pro Asp Ser TTT GTT AAT GTT ACT GCA ATA GTG GGT TTT TGT GTA ~AG CCA GCT AAT
28~0 Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Lys Pro Ala Asn GCT AGT CAG TAT GCA ATA GTA CCC GCT AAT GGT AGG GGT ATT TTT ATA

Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn ~ly Arg Gly Ile Phe Ile CAA GTT AAT GGT P~GT TAC TAC ATC ACA GCA CGA GAT ATG TAT ATG CCA
29~6 Gln Val ~sn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro AGA GCT ATT ACT GCA GGA GAT ATA GTT ACG CTT ACT TCT TGT CAA GCA

Arg Ala Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln Ala~
9~5 1000 1005 -AAT TAT GTA AGT GTA AAT AAG ACC GTC ATT ACT ACA TTC GTA GAC AAT

Asn Tyr Val Ser Val Asn Lys Thr Val Ile Thr Thr Phe Val Asp Asn GAT GAT TTT GAT TTT AP~T GAC GAA TTG TCA AAA TGG TGG AAT GAC ACT

Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Lys Trp Trp Asn Asp Thr 1~25 1030 1035 1040 AAG CAT GAC~ CTA CCA GAC TTT GAC AAA TTC AAT TAC ACA GTA CCT ATA

Ly5 His Glu Leu Pro Asp Phe Asp Lys Phe Asn Tyr Thr Val Pro Ile 1045 1050 10~5 SUE~STITLJTE SHEET

C~2i 1 7468 `V093/17109 PCT/GB93/00332 -~3-CTT GAC ATT GAT AGT GAA ATT GAT CGT ATT CAA GGC GTT ATA CAG GGT

Leu Asp Ile Asp Ser Glu Ile Asp Arg Ile Gln Gly Val Ile Gln Gly CTT AAT GAC TCT TTA ATA GAC CTT GAA AAA CTT TCA ATA CTC AAA ACT

Leu Asn Asp Ser Leu Ile Asp ~u Glu Lys Leu Ser Ile Leu Lys Thr TAT ATT A~G TGG CCA AG

Tyr Ile Lys Trp Pro (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: lO93 amino acids (B) TYPE: amino acid ~D) TOPOLOGY: linear ( ii ) MOLECIJLE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Leu Val Thr Pro Leu Leu Leu Val Thr Leu Leu Cys Val Leu Cys Ser Ala Ala Leu Tyr Asp Ser Ser Ser Tyr Val Tyr Tyr Tyr Gln Ser Ala Phe Arg Pro Pro Asn Gly Trp His Leu His Gly &ly Ala Tyr Ala Val Val Asn Ile Ser Ser Glu Ser Asn Asn Ala Gly Ser Ser Pro Gly Cys Ile Val Gly Thr Ile His Gly Gly Arg Val Val Asn Ala Ser Ser Ile Ala Met Thr Ala Pro Ser Ser Gly Met Ala Trp Sar Ser Ser Gln Phe Cys Thr Ala His Cys Asn Phe Ser Asp Thr Thr Val Phe Val Thr lOO . 105 llO

SIJBSTITUT~ SHEET

CA2i 1 7468 WO93/17l09 PCT/GB93/003.

His Cys Tyr Lys Tyr Asp Gly Cys Pro Ile Thr Gly Met Arg Gln Lys Asn Phe Leu ~rg Val Ser Ala Met ~ys Asn Gly Gln Leu Phe Tyr Asn Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Phe Lys Ser Phe Gln Cys ~al Asn Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Tyr Thr Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Glu Val Lys Ala Leu Ala ; Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys ~sp Gly Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp Gly Phe Tyr Pro Phe Ile Asn Ser Ser Leu Val Lys Gln Lys Phe Ile Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Phe Thr Leu His Asn Phe Thr Phe His Asn GlU Thr Gly Ala Asn Pro Asn Pro Ser Gly Val Gln Asn Ile Gln Thr Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Glu Ser Asn Phe 3~5 310 315 320 Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Ile Ala Tyr Gly SUE~STITUTE 5HEET

CA2i 1 7468 ~093/17109 PCT/GB93/00332 _2S-Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Gly Arg Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Ser Leu Cys Lys Gly Val Tyr Ser Gly Glu Leu Asp Leu Asn Phe Glu Cys Gly Leu Leu Val Tyr Val Thr Lys Ser Gly Gly Ser Arg Ile Gln Thr Ala Thr Glu Pro Pro .~.

Val Ile Thr Arg His Asn Tyr Asn Asn Ile Thr Leu Asn Thr Cys Val Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr Asn Val Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala .
:i:le Leu A5p Thr Ser Gly Ser Ile Asp Ile Phe Val Val Gln Gly Glu Tyr Gly Leu Thr Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg 500 505 51~
-Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn ~;ln Phe Tyr Ile I,ys Ile Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser Ile Thr Glu Asn Val Ala Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile ~ys Pro Asp ~ly er Ile Ala Thr Ile Val Pro Lys ~ln Leu Glu Gln Phe Yal Ala Pro eu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Ph~ Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Yal Gln Ile SIJE~8TITUTE SHEET

~2~-Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Leu Asp Cys Arg Asp Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn Ser Ile Gly Gln Lys Glu Asp Met Glu Leu Leu Asn Phe Tyr Ser Ser Thr Lys Pro Ala Gly Phe Asn Thr Pro Phe Leu Ser Asn Val Ser Thr Gly Glu Phe Asn Ile Ser Leu Leu Leu Thr Thr Pro Ser Ser Pro Arg Arg Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu Met GIn Thr Leu Tyr Thr Ser . ,-~
Ser Leu Val Ala Ser Met Ala Phe Gly Gly Ile Thr Ala Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu ~ly Ile Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe Asn Lys Ala Ile Gly Arg Met Gln Glu Gly Phe Arg Ser Thr Ser Leu Ala Leu G1n Gln Ile Gln ~sp Val Val Asn Lys Gln Ser Ala Ile Leu 8~0 825 830 Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser SPr 835 ~40 845 CA2i`1 7468 `~'~ 93/17109 PCT/GB93/0033.

Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Ala 8~0 855 860 Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu Ala Ser Ala Lys Gln Ala Glu His Ile Arg Val Ser Gln Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Il~ Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn Ala Pro Asn ~ly Ile Val Phe Ile His Phe Ser Tyr Thr Pro Asp Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Lys Pro Ala Asn 945 950 955 g6~
Ala Ser Gln Tyr Ala ~le Val Pro Ala Asn Gly Arg Gly Ile Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro Arg Ala Ile Thr Ala ~ly Asp Ile Val Thr Leu Thr Ser Cys Gln A

Asn Tyr Val Ser Val Asn Lys Thr Val Ile Thr Thr Phe Val Asp Asn 1010 1015 102~

Asp Asp ~he Asp Phe Asn Asp Glu Leu Ser Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Lys Phe Asn Tyr Thx Val Pro Ile Leu Asp Ile Asp Ser Glu Ile Asp Arg Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Lys Leu Ser Ile Leu Lys Thr .

SUBSTlTlJTE SHEET

CA 2 i 1 74 68 W093/l7l09 PCT/GB93/003~-Tyr Ile Lys Trp Pro 10~0 (2) INFORMATION FQR SEQ ID NO:3:
(i~ SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 83 base pairs (B) TYPE: nucleic acid (C~ STRANDEDNESS: single ~D) TOPOLOGY: linear (ii3 MOLECULE TYPE: cDNA
(vi~ ORIGINAL SOURCE:
(A~ ORGANICM: Infectious bronchitis virus (B) STRAIN: M41 (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 7..57 ~D) OTHER INFORMATION: /function= "IBV LEADER SEQUENCE"
(ix) FEATURE:
(A) NAME/KEY: misc feature (B) LOCATION: 58..83 (D) OTHER INFORMATION: /function= "IBV SPIKE CODING
SEQUENCE"

(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:3:
GGATCCCCGA TCCCCTAGTCTTTAATTTAA TTAAGTGTGG TAAGTTACTG GTAAGAGATG

TTGGTAACAC CTCTTTTACT AGT

(2) INFORMATION FOR SEQ ID NO:4:
(i) SE~UENCE CHARACTERISTICS:
(A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(~i) ORIGINAL SOURCE:
(A) ORGANISM: Infectious bronchitis virus (B) STRAIN: M41 ~ix~ FEAT~RE:
~A) NAME/KEY: misc_feature (B) LOCATION: 7..20 (D) OTHER INFORMATION: /function= "VACCINIA P7.5 LEADER
SEQ~JENCE"
(ix) FEATURE:

SUBSTITUTE SHEET

~'~ 93~17109 ~ ; PCI/GB93J00332 (A) NAME/KEY: misc _ feat~e (B) LOCATION: 2l. . 46 (D) OTHER INFORMATION: /function= "IBV SPIKE CODING
SEQUENCE"

(xi) SEQUENCE r)ESCRIPTION: SEQ ID NO: 4:
GGATCCAATC: AATAGCAATC ATGTTGGTAA CACCTCTTTT ACTAGT

2 ) INFORMATION FOPc SEQ ID NO: 5:
( i ) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 4 O base pairs (B) TYPE: nucleic acid t C3 ST~A~DEDNESS: single ( D ~ TOPOLOGY: l inear MC~LECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTIO~I: SEQ ID NO:5:
GATCCAATCA ATAGCAATCA TGTTGGTAP,C ACCTCTTTTA

( 2 ) INFORMATION FOR S~Q ID NO: 6:
( i 3 SEQUENOE CHARACThRI STICS:
(A) LENGTH: 4 0 base pairs (B) TYPE:: nucleic acid (C~ STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESC~IPTION: SEQ ID NO:6:
CTAGTAAAAG AGGTGTT~CC AACATGATTG CTATTGATTG

~....
, SUBSTITU I ~ SHE~E~T

Claims (8)

- 30 -

1. A DNA molecule which codes substantially for a truncated Infectious Bronchitis Virus (IBV) spike protein polypeptide, said truncated IBV spike protein polypeptide being characterised in lacking the transmembrane and cytoplasmic anchor domains present in the native IBV S2 spike protein polypeptide, but otherwise encoding the whole of the S1 and remainder of the S2 polypeptide except that the S1 polypeptide can lack up to 10 amino acids at the N-terminal end and the truncated S2 polypeptide can lack up to 10 amino acids at its truncated end.

2. A DNA molecule according to claim 1, wherein said truncated IBV spike protein polypeptide is of IBV M41, M42, 6/82, H120, H52, Ma5, D207, D12, D3896, D3128 strains or serotypes or of Connecticut isolate A3968.

3. A vector carrying a DNA molecule claimed in claim 1 or 2.

4. A vector according to claim 3, further containing a poxvirus viral promoter sequence linked to an inserted sequence of the DNA
molecule.

5. A vector according to claim 4, further containing poxvirus sequence flanking the promoter and insert of the IBV DNA
molecule, said flanking sequence being effective for homologous recombination of the total vector insert.

6. A vector according to claim 3, 4 or 5, wherein the native leader sequence between the promoter and the IBV DNA molecule is partially or wholly replaced by part or all of a sequence found downstream of another poxvirus promoter.

7. A vector according to claim 4, 5 or 6 wherein the virus is fowlpox virus.
8. A vector according to any of claims 3 to 7 which is a prokaryotic cloning vector.
9. Animal cells containing a vector defined in any of claims 5 to 8.
10. A prokaryotic host incorporating a cloning vector defined in

claim 8.