# Patent

## Title
Chimaeric protein

## Abstract

The present invention provides a chimaeric coronavirus S protein which is based on an S protein from a coronavirus strain with restricted tissue tropism, but which comprises at least part of the S2 subunit from a coronavirus strain with extended tissue tropism, such that a virus comprising the chimaeric S protein has extended tissue tropism. The present invention also provides a virus comprising such a chimaeric S protein.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. §371 National filing of International Application No. PCT/GB2010/001293, filed Jul. 5, 2010, incorporated herein by reference, which claims priority benefit of Great Britain patent application No. 0911794.6, filed Jul. 7, 2009.
FIELD OF THE INVENTION
The present invention relates to a chimaeric coronavirus S protein. In particular a chimaeric coronavirus S protein which, when used to produce a virus, causes the virus to have extended tissue tropism. The present invention also relates to nucleotide sequences encoding such a chimaeric protein; viral particles comprising such a chimaeric protein and their use in a vaccine to prevent and/or treat a disease.
BACKGROUND TO THE INVENTION
Infectious Bronchitis Virus (IBV)
Avian infectious bronchitis virus (IBV) is a highly infectious and contagious pathogen of domestic fowl that replicates primarily in the respiratory tract but also in epithelial cells of the gut, kidney and oviduct. IBV is a member of the Coronaviridae and genetically very similar coronaviruses cause disease in turkeys and pheasants.
Clinical signs of IB include sneezing, tracheal rales, nasal discharge and wheezing. Meat-type birds have reduced weight gain, whilst egg-laying birds lay fewer eggs. The respiratory infection predisposes chickens to secondary bacterial infections which can be fatal in chicks. The virus can also cause permanent damage to the oviduct, especially in chicks, leading to reduced egg production and quality; and kidney, sometimes leading to kidney disease which can be fatal.
Both live and attenuated vaccines are currently used in IB vaccination. To date, the most efficacious vaccines are live attenuated viruses empirically produced following blind repeated passages through embryonated eggs.
A problem with this approach is that, upon serial passaging, the immunogenicity of the virus decreases. It is necessary to achieve a balance between an acceptable degree of attenuation to make the virus safe, and an acceptable loss of immunogenicity such that the virus vaccine is still efficacious. This “balancing” of attenuation is a trial and error approach, rendering the outcome of the attenuation process uncertain.
Since attenuation by serial passage is effectively a random event, the resultant vaccine is ill-defined genetically as the molecular basis of the attenuation is unknown. Each batch of attenuated virus will be different, making it difficult to achieve consistency of the resulting vaccine and reproducibility of the protective/therapeutic effect in vivo.
A further disadvantage is that embryonated eggs are expensive and cannot be used as a prolonged source of virus.
Growth of virus on embryonated eggs is a cumbersome process as each egg must be sterilized, candled, inoculated with virus and incubated before harvesting small volumes of allantoic fluid from each egg and pooling before purification. The lack of reliable supplies of high quality eggs results in limitations in the amount of vaccine which may be produced, particularly in an emergency situation.
In addition to these logistic and supply problems, embryonated eggs have other limitations as a host system for vaccine production. For example, there are increasing concerns about the presence of adventitious viruses, particularly retroviruses in eggs, which would compromise the production of live, attenuated viral vaccines.
There is therefore a need for alternative IBV vaccines and methods for their production which do not suffer from the above mentioned drawbacks.
Coronaviruses
Coronaviruses are enveloped viruses that replicate in the cell cytoplasm and contain an unsegmented, single-stranded, positive sense RNA genome of 27 to 32 kb.
All coronavirus lipid envelopes contain at least three membrane proteins: the spike glycoprotein (S), integral membrane protein (M), and small membrane protein (E). The coronavirus S protein is a type I glycoprotein which oligomerizes in the endoplasmic reticulum and is assembled into virion membranes through non-covalent interactions with the membrane protein. Following incorporation into coronavirus particles, the S protein is responsible for binding to the target cell receptor and fusion of the viral and cellular membranes. The S glycoprotein consists of four domains: a signal sequence that is cleaved during synthesis; the ectodomain, which is present on the outside of the virion particle; the transmembrane region responsible for anchoring the S protein into the lipid bilayer of the virion particle; and the cytoplasmic tail.
The IBV S protein (1,162 amino acids) is cleaved into two subunits, S1 (535 amino acids; 90 kDa) comprising the N-terminal half of the S-protein, and S2 (627 amino acids; 84 kDa) comprising the C-terminal half of the S protein.
The S2 protein subunit associates non-covalently with the S1 subunit and contains the transmembrane and C-terminal cytoplasmic tail domains.
The S1 subunit has been widely reported to comprise the receptor-binding activity of the S protein.
For example, it has been shown for the serogroup I coronavirus, human coronavirus HCoV-229E, that of three variants having truncation in the N-terminal domain of S1, two were unable to bind the receptor, implicating the region between amino acids 417 and 547 as important for receptor binding (Bonavia et al (2003) J. Virol 77. 2530-2538).
The first 330 amino acids of the 769-residue S1 subunit of the mouse hepatitis virus (MHV) S protein are sufficient to bind the MHV receptor (Kubo et al (1994) J. Virol. 68:5403-5410). Similarly an 193-amino acid fragment of the SARS S protein (residues 318-510) binds to the receptor and blocks S-protein mediated infection (Wong et al (2004) J. Biol. Chem. 279:3197-3201).
It is also reported that amino acids 1-510 of the SARS-CoV S glycoprotein represent a domain containing the receptor binding site (amino acids 270-510) analogous to the S1 subunit of other coronavirus S glycoproteins (Babcock et al (2004) J. Virol. 4552-4560).
The S protein is a determinant of the cell tropism of the virus (Casias et al (2003) J. Virol. 77:9084-9089). It has been shown that amino acid substitutions in the N-terminal region of the S1 protein are associated with the extended host range of a virus variant of murine hepatitis virus (MHV) (Thackray and Holmes (2004) Virology 324:510-524). Moreover, it is generally thought that species specificity of infection is due to the specificity of the virus-receptor interaction (Compton et al (1992) J. Virol. 7420-7428; Gagneten et al (1995) J. Virol. 69:889-895).
It has, to date, thus been widely assumed that cell tropism is a property of the S1 domain of the S protein of coronaviruses.
SUMMARY OF ASPECTS OF THE INVENTION
The present inventors have surprisingly shown that cell tropism of coronaviruses is determined by the S2 protein, and that substitution of the S2 protein with all or part of another coronavirus can alter (extend or reduce) the cell tropism of the virus, depending on the cell tropism of the virus from which the S2 protein was derived.
This means that immunogenic virus vaccines which are unable to grow on cell lines can be induced to do so by substitution of all or part of their S2 proteins.
Thus, in a first aspect, the present invention provides a chimaeric coronavirus S protein which is based on an S protein from a coronavirus strain with restricted tissue tropism, but which comprises at least part of the S2 subunit from a coronavirus strain with extended tissue tropism, such that a virus comprising the chimaeric S protein has extended tissue tropism.
The chimaeric S protein may comprise all or part of the S2 subunit from the coronavirus strain with extended tissue tropism.
For example, a chimaeric infectious bronchitis virus (IBV) S protein may comprise a portion of the S2 sequence which comprises the sequence XBBXBX (SEQ ID NO: 9) in the part of the S2 protein corresponding to between residues 686 and 691 of the sequence given as SEQ ID No. 1, where B is a basic residue and X is any amino acid.
The sequence XBBXBX (SEQ ID NO: 9) may, for example, be the sequence SRRKRS (SEQ ID NO: 10) or SRRRRS (SEQ ID NO: 11). The chimaeric S protein may comprise the sequence SRRKRSLIE (SEQ ID NO: 12) or SRRRRSVIE (SEQ ID NO: 13) in the part of the S2 protein corresponding to between residues 686 and 694 of the sequence given as SEQ ID No. 1.
The coronavirus may, for example be: Infectious bronchitis virus (IBV); Canine coronavirus (CCoV); Feline coronavirus (FeCoV); Human coronavirus 229E (HCoV-229E); Porcine epidemic diarrhoea virus (PEDV); Transmissible gastroenteritis virus (TGEV); Human Coronavirus NL63 (NL or New Haven); Bovine coronavirus (BCoV); Canine respiratory coronavirus (CRCoV); Human coronavirus OC43 (HCoV-OC43); Mouse hepatitis virus (MHV); Porcine haemagglutinating encephalomyelitis virus (HEV); Rat coronavirus (RCV); Turkey coronavirus (TCoV); HCoV-HKU1; Severe acute respiratory syndrome coronavirus (SARS-CoV); or Turkey coronavirus (Bluecomb disease virus).
Where the coronavirus is IBV, the IBV strain with extended tissue tropism may be IBV Beaudette.
In a second aspect, the present invention provides a nucleotide sequence capable of encoding a chimaeric S protein according to the first aspect of the invention.
The invention also provides plasmid comprising a nucleotide sequence according to the second aspect of the invention.
In a third aspect, the present invention provides a viral particle comprising a chimaeric S protein according to the first aspect of the invention, and/or a nucleotide sequence according to the second aspect of the invention.
The viral particle may be a recombinant vaccinia virus (rVV) or a coronavirus.
The viral particle may be capable of growing on a cell line such as Vero cells.
The infection of Vero cells by a viral particle according to the third aspect of the invention may be blocked by soluble heparin.
In a fourth aspect, the present invention provides a method for making a viral particle according to the third aspect of the invention a method for making a viral particle by:

    
    
        (i) transfecting a plasmid as described in the previous section into a host cell;
        (ii) infecting the host cell with a recombining virus comprising the genome of the coronavirus strain with restricted tissue tropism, minus at least part of the S2 subunit;
        (iii) allowing homologous recombination to occur between the S gene sequences in the plasmid and the corresponding sequences in the recombining virus genome to produce a chimaeric S gene; and
        (iv) selecting for recombining virus comprising the chimaeric S gene.
    
    


The recombining virus may be vaccinia virus.
In order to generate a recombinant coronavirus particle, the virus DNA from step (iv) may be used to generate coronavirus RNA in situ using a reverse genetics system.
The above method therefore optionally includes the step:

    
    
        (v) recovering recombinant coronavirus comprising the chimaeric S gene from the DNA from the recombining virus from step (iv).
    
    


In a fifth aspect, the present invention provides a cell capable of producing a viral particle according to the fourth aspect of the invention. The cell may, for example, be a cell, such as a primary chick kidney cell, capable of producing recombinant virus using a reverse genetics system, or a cell infected with a viral particle according to the fourth aspect of the invention.
The cell infected with a viral particle according to the fourth aspect of the invention may be derivable from a cell line, such as a Vero cell.
In a sixth aspect, the present invention provides a vaccine comprising a viral particle of the fourth aspect of the invention.
Further aspects of the invention provide:

    
    
        (i) a method for treating and/or preventing a disease in a subject which comprises the step of administering a vaccine according to the sixth aspect of the invention to the subject;
        (ii) a vaccine according to the sixth aspect of the invention for treating and/or preventing a disease in a subject;
        (iii) the use of a viral particle according to the fourth aspect of the invention in the manufacture of a vaccine for treating and/or preventing a disease in a subject.
        (iv) a method for producing a vaccine according to the sixth aspect of the invention, which comprises the step of infecting Vero cells with a viral particle according to the fourth aspect of the invention;
        (v) a method for altering the cell tropism of a coronavirus which comprises the step of substitution of at least a part of the S2 protein with the S2 protein, or corresponding part thereof, from a different strain; and
        (vi) a cell culture comprising a cell or a population of cells according to the fifth aspect of the invention.
    
    


The extended cell tropism conferred on the virus by the presence of the chimaeric gene means that virus stock for vaccine production can be produced by growing on cell lines, rather than embryonated eggs or primary cells.
The use of cell lines such as Vero cell has many advantages:

    
    
        (i) it has been previously validated for growth of viruses and diagnostic purposes;
        (ii) the cells (and therefore virus) can be grown in suspension, rather than flat beds; and
        (iii) it is possible to achieve consistent yields.
    
    





DESCRIPTION OF THE FIGURES
FIG. 1—Schematic diagram of the IBV S protein. The functional S protein is glycosylated and is present as a homotrimer in the virion membrane. The trimeric form of the S1 subunits constitutes the receptor binding domain.
FIG. 2—Schematic diagram showing the S proteins from Beaudette (Beau-R) and M41 and the chimaeric S proteins BeauR-M1B2(S), BeauR-B1M2(S), Beau-S-M41-Hep and M41-S-Beau-Hep. The transmembrane (TM) domain and cytoplasmic (cyto) tail domain of all chimaeric S proteins are derived from the Beaudette S protein. The position of the heparan sulphate-binding site in the S2 subunit is indicted as HSBS.
FIG. 3—Generation of plasmids containing chimaeric S glycoprotein genes. The S1 and S2 subunits of Beau-R and M41 were amplified from two plasmids, pGPT-M41S and pGPT-IBV-StuI-BamHI, by PCR using primers located in the replicase gene, across the S1/S2 junction and gene 3. Overlapping PCR was used to combine the subunits, forming the chimaeric S genes, which were then cut with NsiI (restriction site located in the replicase gene) and BspEI (restriction site located in gene 3). The receiver plasmid, pGPT-IBV-StuI-BamHI, was also cut with NsiI and BspEI and the Beaudette S gene was removed by gel extraction. The chimaeric S genes were ligated into the remaining pGPT-IBV-StuI-BamHI backbone containing the E. coli guanine xanthine phosphoribosyltransferase (gpt) gene.
FIG. 4—Diagrams of pGPT-IBV-StuI-BamHI and pGPT-M41S.
The plasmids pGPT-IBV-StuI-BamHI and pGPT-M41S were used to amplify the Beau-R and M41 S1 and S2 subunits by PCR. The replicase gene and gene 3 surrounding the S genes are derived from Beaudette. The C-terminus of the M41 S gene in pGPT-M41 S has been exchanged for the Beau-R S gene C-terminus because this is the area that interacts with the M protein. The C-terminal ends of the M41 and Beau-R S proteins are very different to each other so to enable interaction of the chimaeric S protein with the other structural proteins from Beau-R, it was decided to keep the Beau-R S gene C-terminus. The Beau-R S gene was removed from pGPT-IBV-StuI-BamHI and the chimaeric S genes inserted in its place to create the plasmids pGPT-S1M41S2Beau and pGPT-S1BeauS2M41.
FIG. 5—The IBV reverse genetics system.
A plasmid vector containing gpt is constructed from the chimaeric S gene and parts of the neighbouring genes in the IBV genome. The plasmid is transfected into Vero cells infected with rVV containing the IBV genome minus the S gene. Three rounds of plaque purification in the presence of gpt selection components and three rounds in absence are carried out to select for rVV containing the chimaeric S gene. Large virus stocks are prepared in BHK-21 cells from which virus is purified and DNA extracted. Recombinant IBV cDNA within the VV genome and a plasmid expressing the IBV N protein are transfected into CK cells infected with rFPV-T7. The supernatant is filtered and used for passage on CK cells and rIBV with the chimaeric S gene is recovered.
FIG. 6—Infected Vero cells 48 hours post-infection with various IBVs.
FIG. 7—Confocal microscopy of rIBV growth on Vero cells. Vero cells were infected with rIBVs and were fixed 24 hours post-infection and immunolabelled with mouse anti-dsRNA, secondary antibody AlexaFluor 488 goat anti-mouse (green, Invitrogen) to detect IBV infected cells. Nuclei of all the cells were labelled with DAPI (blue).
FIG. 8—Growth kinetics of rIBVs on Vero cells. Vero cells were infected with Beau-R, M41-CK, BeauR-M41(S), BeauR-M1B2(S), BeauR-B1M2(S), Beau-S-M41-Hep and M41-S-Beau-Hep at a multiplicity of infection of 0.1. Supernatant was harvested at 1, 12, 24, 48 and 72 hours post-infection and titrated on CK cells. Three replicates were performed and the averages taken. Error bars indicate standard error of the mean.
FIG. 9—Plaque reduction assay on Vero cells of IBV pre-treated with increasing amounts of soluble heparin.
FIG. 10—Bar chart comparing the effect of soluble heparin on the percentage of IBV-infected Vero cells. IBV strains Beau-R and BeauR-M1B2(S) were incubated with either PBS (−) or soluble heparin, 15 mg/ml (+) for 30 minutes at room temperature prior to infecting Vero cells. Cells were fixed 24 hours post-infection then immunolabelled with mouse α-dsRNA, secondary antibody goat α-mouse IgG (green, Invitrogen) and nuclei were stained blue with DAPI. Ten fields of vision per sample were analysed by confocal microscopy at ×40 magnification and the percentage of infected cells was calculated.
FIG. 11—Alignment between Beaudette (Beau-R), M41, Beaudette having the heparan-sulphate-binding site modified to the corresponding M41 sequence (Beau-S-M41-Hep) and M41 having the Beaudette heparan-sulphate-binding site (M41-S-Beau-Hep)
FIG. 12—Comparison of Beaudette and M 41 a) S protein sequences, and b) S2 region.
FIG. 13—Amino acid differences between M41 and Beaudette S proteins.
FIG. 14—Amino acid differences between M41 and Beaudette S2 regions.



DETAILED DESCRIPTION
Coronavirus
Coronavirus is a genus of animal virus belonging to the family Coronaviridae. Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and a helical symmetry. The genomic size of coronaviruses ranges from approximately 27 to 32 kilobases, which is the longest size for any known RNA virus.
Coronaviruses primarily infect the upper respiratory and gastrointestinal tract of mammals and birds. Four to five different currently known strains of coronaviruses infect humans. The most publicized human coronavirus, SARS-CoV which causes SARS, has a unique pathogenesis because it causes both upper and lower respiratory tract infections and can also cause gastroenteritis. Coronaviruses are believed to cause a significant percentage of all common colds in human adults. Coronaviruses also cause a range of diseases in farm animals and domesticated pets, some of which can be serious and are a threat to the farming industry. Economically significant coronaviruses of farm animals include infectious bronchitis virus (IBV) which mainly causes respiratory disease in chickens and seriously affects the poultry industry worldwide; porcine coronavirus (transmissible gastroenteritis, TGE) and bovine coronavirus, which both result in diarrhoea in young animals. Feline coronavirus has two forms, feline enteric coronavirus is a pathogen of minor clinical significance, but spontaneous mutation of this virus can result in feline infectious peritonitis (FIP), a disease associated with high mortality. There are also two types of canine coronavirus (CCoV), one that causes mild gastrointestinal disease and one that has been found to cause respiratory disease. Mouse hepatitis virus (MHV) is a coronavirus that causes an epidemic murine illness with high mortality, especially among colonies of laboratory mice. Coronaviruses are divided into three groups, as shown below:
Group 1

    
    
        Canine coronavirus (CCoV)
        Feline coronavirus (FeCoV)
        Human coronavirus 229E (HCoV-229E)
        Porcine epidemic diarrhoea virus (PEDV)
        Transmissible gastroenteritis virus (TGEV)
        Human Coronavirus NL63 (NL or New Haven)
    
    


Group 2

    
    
        Bovine coronavirus (BCoV)
        Canine respiratory coronavirus (CRCoV)—Common in SE Asia and Micronesia
        Human coronavirus OC43 (HCoV-OC43)
        Mouse hepatitis virus (MHV)
        Porcine haemagglutinating encephalomyelitis virus (HEV)
        Rat coronavirus (RCV). Rat Coronavirus is quite prevalent in Eastern Australia where, as of March/April 2008, it has been found among native and feral rodents colonies.
        Turkey coronavirus (TCoV)
        (No common name as of yet) (HCoV-HKU1)
        Severe acute respiratory syndrome coronavirus (SARS-CoV)
    
    


Group 3

    
    
        Infectious bronchitis virus (1BV)
        Turkey coronavirus (Bluecomb disease virus)
    
    


The virus of the present invention may be a group 1 coronavirus such as TGEV; a group 2 coronavirus such as MHV; or a group 3 coronavirus such as IBV.
IBV
Avian infectious bronchitis (IB) is an acute and highly contagious respiratory disease of chickens which causes significant economic losses. The disease is characterized by respiratory signs including gasping, coughing, sneezing, tracheal rales, and nasal discharge. In young chickens, severe respiratory distress may occur. In layers, respiratory distress, nephritis, decrease in egg production, and loss of internal egg quality and egg shell quality are common.
In broilers, coughing and rattling are common clinical signs, rapidly spreading in all the birds of the premises. Morbidity is 100% in non vaccinated flocks. Mortality varies depending on age, virus strain, and secondary infections but may be up to 60% in non-vaccinated flocks.
The first IBV serotype to be identified was Massachusetts, but in the United States several serotypes, including Arkansas and Delaware, are currently circulating, in addition to the originally identified Massachusetts type.
The IBV strain Beaudette was derived following at least 150 passages in chick embryos. IBV Beaudette is no longer pathogenic for adult birds but rapidly kills embryos.
FIG. 12 and Table 1 shows the amino acid differences between IBV Beaudette and M41.
H120 is a commercial live IBV Massachusetts serotype vaccine strain, attenuated by approximately 120 passages in embryonated chicken eggs. H52 is another Massachusetts strain, and represents an earlier and slightly more pathogenic passage virus (passage 52) during the development of H120. Vaccines based on H120 and H52 are commonly used.
S-Protein
The coronavirus S protein comprises a large, heavily glycosylated ectodomain that can be cleaved during biosynthesis into two subunits (S1 and S2) by a furin-like enzyme in the Golgi apparatus. Not all coronaviruses are cleaved, yet even without cleavage the basic subunit structure of the S protein is conserved. S1 comprises the receptor binding domain (Li et al (2005) Science 309:1864-1868) and S2 comprises the fusion domain. The S protein of IBV is fully cleaved at the S1/S2 boundary, especially in chicken embryo systems.
The S2 domain contains five domains or functional regions, as shown in FIG. 1: two domains, HR1 and HR2 form helical structures resulting in the stalk structure of the protein; a transmembrane domain responsible for anchoring the protein to the virion membrane; a cysteine-rich cytoplasmic domain responsible for interacting with other virus structural proteins and a fifth domain, the fusion peptide, responsible for virus-cell fusion or cell-to-cell fusion.
The amino acid differences between M41 and Beaudette S proteins and S2 regions are shown in FIGS. 13 and 14, respectively.
Tissue Tropism
Coronaviruses show strong species and tissue tropism. Likewise, clinical isolates of IBV show distinct tropism both in vivo and in cell culture.
The M41 strain has been adapted for growth on primary chick kidney (CK) cells and is restricted to infection of primary chicken cells, and so needs to be grown on embryonated eggs or CK cells.
The Beaudette strain, on the other hand is known to be able to infect a range of cells in culture, including Vero and baby hamster kidney (BHK) cells.
A coronavirus with restricted tissue tropism is able to infect a smaller number of cell types than a coronavirus with extended tissue tropism.
A coronavirus with restricted tissue tropism, may, for example, be restricted to infection of primary cells, whereas a coronavirus with extended tissue tropism may (in addition to being able to infect primary cells) be able to infect one or more cell lines.
A coronavirus with extended tissue tropism may, for example, have the capacity to infect Vero cells.
The Vero cell lineage was isolated in 1962 from kidney epithelial cells extracted from an African green monkey (Cercopithecus aethiops). Vero cells are used for many experimental and clinical purposes, including acting as host cells for growing virus.
The Vero cell lineage is continuous in that it can be replicated through many cycles of division and not become senescent.
The Vero cell lineage has been licensed for use in the manufacture of vaccines and is currently used for the production of polio and rabies vaccines.
The strain with restricted tissue tropism may be immunogenic and capable of inducing a protective or therapeutic immune response in vivo. The strain with restricted tissue tropism may be, for example, a strain currently used for vaccine production. For IBV, this includes strains such as: H52, H120, Ma5, 4/91, D41, D274 and W93. The strain with restricted tissue tropism may be or be derived from an isolate “from the field” such as BJ1, BJ2, or BJ3 (Li and Yang (2001) Avian Pathol 30:535-541).
For IBV, the strain with extended tissue tropism may, for example, be IBV Beaudette, so that the chimaeric protein of the invention comprises all or part of the IBV Beaudette S2 protein.
Cell tropism may be established experimentally by simply challenging a given cell type with infection by a virus. The cytopathic effect (cpe) and the degree of formation of syncytia may then be analysed after a certain number of passages, as described in the Examples. Change in morphology of the infected cells may be analysed using microscopy.
Chimaeric Protein
The present invention relates to a chimaeric coronavirus spike protein (S protein) which is based on an S protein from a coronavirus strain with restricted tissue tropism, but which comprises at least part of the S2 subunit from a coronavirus strain with extended tissue tropism.
The term “based on” indicates that at least the S1 domain is derived or derivable from the strain with restricted tissue tropism. The chimaeric protein may also comprise a part of the S2 domain from the strain with restricted tissue tropism. For example, the transmembrane and/or cytoplasmic domains may be derived or derivable from the strain with restricted tissue tropism.
The chimaeric protein may comprise all or a part of the S2 subunit from the coronavirus strain with extended tissue tropism.
The chimaeric protein may comprise the heparan sulphate binding site located within the S2 subunit of, for example, IBV Beaudette.
A chimaeric infectious bronchitis virus (IBV) S protein may, for example, comprise the sequences XBBXBX (SEQ ID NO: 9) in the part of the S2 protein corresponding to between residues 686 and 691 of the sequence given as SEQ ID No. 1 (FIG. 12A BEAU-CK sequence), where B is a basic residue and X is any amino acid.
For example, the protein may comprise the sequence SRRKRS (SEQ ID NO: 10) or SRRRRS (SEQ ID NO: 11) in the part of the S2 protein corresponding to between residues 686 and 691 of the sequence given as SEQ ID No. 1.
For example, the protein may comprise the sequence SRRKRSLIE (SEQ ID NO: 12) or SRRRRSVIE (SEQ ID NO: 13) in the part of the S2 protein corresponding to between residues 686 and 694 of the sequence given as SEQ ID No. 1.
The chimaeric protein may comprise substantially all of the portion of S2 sequence which is N-terminal to the HR1 domain from the strain with extended tissue tropism. The chimaeric protein may comprise the portion of S2 sequence which is N-terminal to the HR1 domain, together with the HR1 domain from the strain with extended tissue tropism. The chimaeric protein may comprise the portion of S2 sequence which is N-terminal to the HR1 domain, the HR1 domain and the portion of sequence between the HR1 and HR2 domains from the strain with extended tissue tropism. The chimaeric protein may comprise the portion of S2 sequence which is N-terminal to the HR1 domain, the HR1 domain, the portion of sequence between the HR1 and HR2 domains and the HR2 domain from the strain with extended tissue tropism.
The chimaeric protein may comprise substantially all of the S2 subunit from the coronavirus strain with extended tissue tropism.
The chimaeric protein may comprise one or more of the nineteen amino acid changes of the S2 region shown in Table 1 and FIG. 14. In particular the chimaeric protein may comprise an arginine residue at position 687, and a lysine residue at position 689. The chimaeric protein may also comprise a leucine residue at position 692.
The term “substantially all” means that the chimaeric protein comprises at least 95% of the corresponding portion of the wild-type sequence. The missing amino acids may be positioned anywhere within the sequence and may be grouped together (so that a small section of sequence is missing) or separate.
Where a portion of the chimaeric protein is derivable from an S protein for a given strain, that portion may have the same amino acid sequence as the corresponding portion of the wild-type sequence, or it may have one or more amino acid substitutions, additions or deletions compared to the wild-type sequence as long as the original function of that portion of the sequence is retained.
The term “wild type” is used to mean a polypeptide having a primary amino acid sequence which is identical with the native protein (i.e., the viral protein).
A mutant sequence may arise naturally, or may be created artificially (for example by site-directed mutagenesis). The mutant may have at least 70, 80, 90 or 95% sequence identity with the corresponding portion of the wild type sequence. The mutant may have less than 20, 10, or 5 mutations over the corresponding portion of the wild-type sequence.
Identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % identity between two or more sequences. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).
The sequence may have one or more deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent molecule. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the activity is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:



















ALIPHATIC
Non-polar
G A P






I L V





Polar - uncharged
C S T M






N Q





Polar - charged
D E






K R




AROMATIC

H F W Y










The present invention also provides a method for altering the cell tropism of a coronavirus which comprises the step of substitution of at least part of the S2 protein with the S2 protein or corresponding part thereof, from a different strain.
Portions of sequence may be exchanged by methods known in the art, such as by excision and ligation.
By “corresponding part” it is meant that, when the sequence of the S protein of the strain with restricted tissue tropism is aligned with the sequence of the S protein with extended tissue tropism (as shown, for example in FIGS. 12A and 12B) the part of one sequence which aligned with the other.
An alignment between S proteins of different strains is straightforward because coronaviruses share a common domain structure and, between strains, should have a relatively high level of sequence identity. Alignment software may be used such as the BLASTTM package described above.
Nucleotide Sequence
The present invention also provides a nucleotide sequence capable of encoding the chimaeric protein.
The nucleotide sequence may be natural, synthetic or recombinant. It may be double or single stranded, it may be DNA or RNA or combinations thereof. It may, for example, be cDNA, PCR product, genomic sequence or mRNA.
The nucleotide sequence may be codon optimised for production in the host/host cell of choice.
It may be isolated, or as part of a plasmid, virus or host cell.
Plasmid
A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. They are usually circular and double-stranded.
Plasmids, or vectors (as they are sometimes known), may be used to express a protein in a host cell. For example a bacterial host cell may be transfected with a plasmid capable of encoding a particular protein, in order to express that protein. The term also includes yeast artificial chromosomes and bacterial artificial chromosomes which are capable of accommodating longer portions of DNA.
The plasmid of the present invention comprises a nucleotide sequence capable of encoding the chimaeric S gene. It may also comprise one or more additional coronavirus nucleotide sequence(s), or nucleotide sequence(s) capable of encoding one or more other coronavirus proteins such as the replicase gene and/or gene 3.
The plasmid may also comprise a resistance marker, such as the guanine xanthine phosphoribosyltransferase gene (gpt) from Escherichia coli, which confers resistance to mycophenolic acid (MPA) in the presence of xanthine and hypoxanthine and is controlled by the vaccinia virus P7.5 early/late promoter.
Viral Particle
The present invention also relates to a viral particle with a chimaeric S gene. The viral particle may be a recombinant vaccinia virus (rVV) or a coronavirus.
The viral particle may be recombinant.
The viral particle may be made using a reverse genetics system, such as a vaccinia-virus based reverse genetics system.
In this respect, the present invention also provides a method for making a viral particle by:

    
    
        (i) transfecting a plasmid as described in the previous section into a host cell;
        (ii) infecting the host cell with a recombining virus comprising the genome of the coronavirus strain with restricted tissue tropism, minus at least part of the S2 subunit;
        (iii) allowing homologous recombination to occur between the S gene sequences in the plasmid and the corresponding sequences in the recombining virus genome to produce a chimaeric S gene;
        (iv) selecting for recombining virus comprising the chimaeric S gene.
    
    


The genome of the coronavirus strain with restricted tissue tropism may lack the part of the S2 protein corresponding to the part provided by the plasmid, so that a chimaeric protein is formed.
The recombining virus is one suitable to allow homologous recombination between its genome and the plasmid. The vaccinia virus is particularly suitable as homologous recombination is routinely used to insert and delete sequences for the vaccinia virus genome.
The above method optionally includes the steps:

    
    
        (v) recovery of recombinant coronavirus comprising the chimaeric S gene from the DNA from the recombining virus from step (iv).
    
    


Methods for recovering recombinant coronavirus, such as recombinant IBV, are known in the art (See Britton et at (2005) see page 24).
For example, the DNA from the recombining virus from step (iv) may be inserted into a plasmid and used to transfect cells which express cytoplasmic T7 RNA polymerase. (The cells may, for example be pre-infected with a fowlpox virus expressing T7 RNA polymerase). Recombinant coronavirus may then be isolated, for example, from the growth medium.
When the plasmid is inserted into the vaccinia virus genome, an unstable intermediate is formed. Recombinants comprising the plasmid may be selected for e.g. using a resistance marker on the plasmid.
Positive recombinants may then be verified to contain the chimaeric S gene by, for example, PCR and sequencing.
Large stocks of the recombining virus including the chimaeric S gene (e.g. recombinant vaccinia virus, rVV) may be grown up and the DNA extracted in order to carry out step (v).
Suitable reverse genetics systems are known in the art (Casais et al (2001) J. Virol 75:12359-12369; Casais et al (2003) J. Virol. 77:9084-9089; Britton et al (2005) J. Virological Methods 123:203-211; Armesto et al (2008) Methods in Molecular Biology 454:255-273).
Cell
The viral particle may be used to infect a cell.
Since the viral particle comprising the chimaeric S gene has extended tissue tropism, the cell may be derivable from or a part of a cell line.
The cell may, for example, be a baby hamster kidney cell (e.g. BHK-21) or a Vero cell.
The cell may be used to produce the viral particle.
Thus the present invention also provides a method for producing a viral particle which comprises the following steps:

    
    
        (i) infection of a cell with a viral particle according to the sixth aspect of the invention;
        (ii) allowing the virus to replicate in the cell; and
        (iii) harvesting the progeny virus.
    
    


The cell may be from or part of a cell line, such as a Vero cell. Viral particles may be harvested, for example from the supernatant by methods known in the art, and optionally purified.
The present invention also provides a cell capable of producing a recombinant viral particle according to the fourth aspect of the invention using a reverse genetics system. For example, the cell may comprise a recombining virus genome comprising a nucleotide sequence capable of encoding the chimaeric S gene.
The cell may be able to produce recombinant recombining virus (e.g. vaccinia virus) containing the chimaeric S gene. The cell may be a Vero cell.
Alternatively the cell may be capable of producing recombinant coronavirus by a reverse genetics system. The cell may express or be induced to express T7 polymerase in order to rescue the recombinant viral particle. The cell may be a CK cell.
Vaccine
The viral particle may be used to produce a vaccine.
The vaccine may by a live attenuated form of the viral particle.
The present invention also relates to a method for producing such a vaccine which comprises the step of infecting cells, for example Vero cells, with a viral particle comprising a chimaeric protein according to the first aspect of the invention.
Vaccination Method
The viral particle of the present invention may be used to treat and/or prevent a disease.
To “treat” means to administer the vaccine to a subject having an existing disease in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
To “prevent” means to administer the vaccine to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease (e.g. infection) or to reduce or prevent development of at least one symptom associated with the disease.
The disease may be any disease caused by a coronavirus, such as a respiratory disease and/or gastroenteritis in humans and hepatitis, gastroenteritis, encephalitis, or a respiratory disease in other animals.
The disease may be infectious bronchitis (IB); Porcine epidemic diarrhoea; Transmissible gastroenteritis; Mouse hepatitis virus; Porcine haemagglutinating encephalomyelitis; Severe acute respiratory syndrome (SARS); or Bluecomb disease.
The disease may be infectious bronchitis.
The vaccine may be administered to hatched chicks or chickens, for example by eye drop or intranasal administration. Although accurate, these methods can be expensive e.g. for large broiler flocks. Alternatives include spray inoculation of administration to drinking water but it can be difficult to ensure uniform vaccine application using such methods.
The vaccine may be provided in a form suitable for its administration, such as an eye-dropper for intra-ocular use.
The vaccine may be administered by the in ovo inoculation, for example by injection of embryonated eggs. In ovo vaccination has the advantage that is provides an early stage resistance to the disease. It also facilitates the administration of a uniform dose per subject, unlike spray inoculation and administration via drinking water.
The vaccine may be administered to any suitable compartment of the egg, including allantoic fluid, yolk sac, amnion, air cell or embryo. It may be administered below the shell (aircell) membrane and chorioallantoic membrane.
Usually the vaccine is injected into embryonated eggs during late stages of embryonic development, generally during the final quarter of the incubation period, such as 3-4 days prior to hatch. In chickens, the vaccine may be administered between day 15-19 of the 21-day incubation period, for example at day 17 or 18.
The process can be automated using a robotic injection process, such as those described in WO 2004/078203.
The vaccine may be administered together with one or more other vaccines, for example, vaccines for other diseases, such as Newcastle disease virus (NDV). The present invention also provides a vaccine composition comprising a vaccine according to the invention together with one or more other vaccine(s). The present invention also provides a kit comprising a vaccine according to the invention together with one or more other vaccine(s) for separate, sequential or simultaneous administration.
The vaccine or vaccine composition of the invention may be used to treat a human, animal or avian subject. For example, the subject may be a chick, chicken or mouse (such as a laboratory mouse, e.g. transgenic mouse).
Typically, a physician or veterinarian will determine the actual dosage which will be most suitable for an individual subject or group of subjects and it will vary with the age, weight and response of the particular subject(s).
The composition may optionally comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agents that may aid or increase the delivery or immunogenicity of the virus.
The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.
EXAMPLES
Example 1
Production of a Chimaeric S Genes Composed of (i) the IBV S1 Subunit from M41 and the S2 Subunit from Beaudette (M1B2); and (ii) the S1 Subunit from Beaudette and the S2 Subunit from M41 (B1M2)
An overlapping polymerase chain reaction (PCR) strategy was used to create two chimaeric S genes, one with the S1 subunit from M41 and the S2 subunit from Beaudette (M1B2), and the other with the S1 subunit from Beaudette and the S2 subunit from M41 (B1M2; see FIGS. 2 and 3). The S genes of Beau-R (a molecular clone of Beaudette) and M41 are contained within the plasmids pGPT-IBV-StuI-BamHI and pGPT-M41S respectively (see FIG. 4). Primers within the replicase gene and gene 3 were designed close to restriction sites already contained within the IBV genome to make it easier to recover the chimaeric S genes from the plasmids pGPT-S1M41S2Beau (M1B2) and pGPT-S1BeauS2M41 (B1M2). The S gene sequence is identical between Beau-R and M41 at the S1/S2 cleavage site so primers were also designed across this location. Around 500 nucleotides were left either side of the S gene to allow for easy manipulation.
The chimaeric S genes were cloned into plasmids containing the guanine xanthine phosphoribosyltransferase gene (gpt) from Escherichia coli, which confers resistance to mycophenolic acid (MPA) in the presence of xanthine and hypoxanthine and is controlled by the vaccinia virus P7.5 early/late promoter.
Example 2
Rescue of rIBVs Expressing the Chimaeric S Protein B1M2 or M1B2 and Establishing Whether they can Grow on Vero Cells
A vaccinia virus-based reverse genetics system was utilized to create recombinant IBVs (rIBVs) with a chimaeric S gene (see FIG. 5). The plasmids pGPT-S1M41S2Beau (M1B2) and pGPT-S1BeauS2M41 (B1M2) were transfected into Vero cells infected with a recombinant vaccinia virus (rVV) containing the full-length IBV cDNA genome minus the S gene. The plasmid was inserted into the vaccinia virus genome creating an unstable intermediate. Three rounds of plaque purification in the presence of mycophenolic acid, xanthine and hypoxanthine selected gpt-positive recombinants. GPT-negative recombinants were then selected by plaque purification three times in the absence of gpt selection medium.
BeauR-M1B2(S) and BeauR-B1M2(S) recombinants were screened by PCR and sequencing to determine that the chimaeric S gene sequence had been inserted successfully into the rVV genome. Large stocks of the rVV containing the IBV genome with the chimaeric S gene were grown in baby hamster kidney (BHK-21) cells until cytopathic effect (cpe) was observed. The vaccinia virus was purified and the DNA extracted.
Two isolates of rIBV BeauR-M1B2(S) and BeauR-B1M2(S), with the genomic background of Beau-R and the S1 or S2 subunit from M41, were recovered. CK cells infected with recombinant fowlpox virus expressing T7 RNA polymerase (rFPV-T7), were transfected with the rVV DNA and incubated at 37° C. Once  ̃60% cpe was observed, the culture medium was removed and filtered to remove any rFPV-T7. The culture medium containing the rIBV was passaged three times on CK cells. The total cellular RNA was isolated and analysed for the presence of IBV RNA by RT-PCR. The S gene was sequenced.
After three passages on CK cells, rIBV BeauR-M1B2(S) and BeauR-B1M2(S) were passaged three times on Vero cells. Photographs of rIBV BeauR-M1B2(S) and BeauR-B1M2(S)-infected Vero cells have been taken by brightfield microscopy (FIG. 6) and by confocal microscopy with immunofluorescence (FIG. 7). The recombinant viruses were titrated on CK cells and the growth kinetics on Vero cells investigated (FIG. 8).
BeauR-B1M2(S) formed no cpe on Vero cells and very few infected Vero cells were observed by confocal microscopy with indirect immunofluorescence. The growth curve indicates that BeauR-B1M2(S) is unable to replicate on Vero cells. BeauR-M1B2(S) caused extensive cpe on Vero cells after one passage and was further passaged on Vero cells to passage 7, forming syncytia from passage 5. The growth curve indicates that BeauR-M1B2(S) is able to replicate on Vero cells.
As shown in FIGS. 6-8, Vero cells do not support the growth of M41, BeauR-M41(S), the rIBV derived from Beaudette but expressing the M41 S protein, or
BeauR-B1M2(S), the morphology of the cells infected with these strains of IBV is similar to mock infected cells. However, the Vero cells infected with BeauR-M1B2(S) show the same morphology as those infected with Beaudette (Beau-R). These results indicate that the S2 subunit is involved in the infectivity of Vero cells.
Example 3
Soluble Heparin is Able to Block the Infection of Vero Cells Using rIBVs Beau-R and BeauR-M1B2(S)
Plaque reduction assays were carried out in which Beau-R and the two BeauR-M41(S1) isolates have been incubated in the presence of soluble heparin sodium salt at various concentrations 0-20 mg/ml before carrying out plaque assays on Vero cells. The number of plaques produced has been analysed to find out whether the presence of heparin has any effect on the ability of the viruses to grow on Vero cells (FIG. 9).
Beau-R and the two BeauR-M1B2(S) isolates have been incubated in the presence of heparin sodium salt at 15 mg/ml and in absence before infecting Vero cells. Confocal microscopy and indirect immunofluorescence was used to observe the effect of heparin on the number of infected cells present (FIG. 10). Ten fields of vision per sample were analysed by confocal microscopy at ×40 magnification and the percentage of infected cells was calculated.
FIG. 9 shows a plaque reduction assay on Vero cells of IBV pre-treated with increasing amounts of soluble heparin. The green line represents Beaudette and the blue and red lines represent the two rIBV expressing the chimaeric M1B2 S protein. FIG. 10 shows a bar chart comparing the effect of soluble heparin on the percentage of IBV-infected Vero cells, measured by confocal microscopy with indirect immunofluorescence. These results indicate that soluble heparin has the same effect, the blocking of infection, on the rIBVs as on Beaudette.
Example 4
Introduction of the Heparan Sulphate Binding Site into the S2 Subunit of the M41 S gene in BeauR-M41(S) and Replacement of the Heparan Sulphate Binding Site in the S Protein of Beau-R with the Corresponding Region from the M41 S Protein
Two plasmids have been designed, one with a section of the Beau-R S gene with the heparan sulphate binding site located with the S2 subunit swapped for the corresponding sequence of M41 (Beau-S-M41-Hep), and the other with a section of the M41 S gene with the heparan sulphate binding site from Beau-R (M41-S-Beau-Hep). Both plasmids contain the gpt gene for selection.
The reverse genetics system as described in example 2 and FIG. 5 has been used to create two chimaeric rIBVs, Beau-S-M41-Hep and M41-S-Beau-Hep (FIGS. 2 and 11). Two suitable recombinants of each were passaged three times on CK cells then their growth characteristics on Vero cells analysed by brightfield microscopy to assess whether the rIBVs cause cpe (FIG. 6), confocal microscopy of infected cells (FIG. 7) and growth kinetics (FIG. 8).
As shown in FIGS. 6-8, Vero cells do not support the growth of M41, BeauR-M41(S), BeauR-B1M2(S) or Beau-S-M41-Hep. The morphology of the cells infected with these strains of IBV is similar to mock infected cells and very few infected cells were observed by confocal microscopy with indirect immunofluorescence. However, the Vero cells infected with M41-S-Beau-Hep show the same morphology as those infected with Beaudette (Beau-R) and BeauR-M1B2(S). Growth curves on Vero cells showed that removing the heparan sulphate binding site from Beau-R removed its ability to grow on Vero cells and introducing the heparan sulphate binding site into the BeauR-M41(S) S protein allowed growth on Vero cells. These results indicate that the heparan sulphate binding site within the S2 subunit of Beaudette is involved in the infectivity of Vero cells.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, should be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or groups of elements or integers.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, virology or related fields are intended to be within the scope of the following claims.

## Claims

1. A chimaeric infectious bronchitis virus (IBV) S protein that comprises the S1 domain of an IBV strain with restricted tropism that is unable to grow on a cell line, and comprises at least part of the S2 domain from an IBV strain with extended tropism that is able to grow on said cell line, such that an IBV comprising the chimaeric S protein is able to grow on said cell line.

2. The chimaeric S protein according to claim 1, which comprises at least 95% of the sequence of the S2 domain of the IBV strain with extended tropism.

3. The chimaeric S protein according to claim 1, wherein said cell line is Vero.

4. The chimaeric S protein according to claim 1, which comprises the sequences XBBXBX (SEQ ID NO: 9) in the part of the S2 protein corresponding to residues 686 to 691 of SEQ ID NO: 1, where B is a basic residue and X is any amino acid.

5. The chimaeric S protein according to claim 4, which comprises the sequence SRRKRS (SEQ ID NO: 10) or SRRRRS (SEQ ID NO: 11) in the part of the S2 protein corresponding to residues 686 to 691 of SEQ ID NO: 1.

6. The chimaeric S protein according to claim 5, which comprises the sequence SRRKRSLIE (SEQ ID NO: 12) or SRRRRSVIE (SEQ ID NO: 13) in the part of the S2 protein corresponding to residues 686 to 694 of SEQ ID NO: 1.

7. The chimaeric S protein according to claim 1, wherein the IBV strain which is capable of growing on a cell line is IBV Beaudette.

8. An isolated nucleic acid comprising a nucleotide sequence encoding a chimaeric S protein, wherein the chimaeric S protein comprises the S1 domain of an IBV strain with restricted tropism that is unable to grow on a cell line, and comprises at least part of the S2 domain from an IBV strain with extended tropism that is able to grow on said cell line, such that an IBV comprising the chimaeric S protein is able to grow on said cell line.

9. A plasmid comprising a nucleic acid according to claim 8.

10. An infectious bronchitis virus comprising a chimaeric S protein and/or a nucleotide sequence that encodes the chimaeric S protein, wherein the chimeric S protein comprises the S1 domain of an IBV strain with restricted tropism that is unable to grow on a cell line, and comprises at least part of the $2 domain from an IBV strain with extended tropism that is able to grow on said cell line, such that an IBV comprising the chimaeric S protein is able to grow on said cell line.

11. An infectious bronchitis virus according to claim 10, which is able to grow on Vero cells.

12. An infectious bronchitis virus according to claim 11, whose infection of Vero cells is blocked by soluble heparin.

13. A method for making a recombining virus encoding an infectious bronchitis virus comprising a chimaeric S protein, comprising:
(i) transfecting a plasmid according to claim 9 into a host cell;
(ii) infecting the host cell with a recombining virus comprising a DNA sequence encoding the genome of the IBV strain with restricted tissue tropism, lacking at least part of the S2 subunit;
(iii) allowing homologous recombination to occur between the S gene sequences in the plasmid and the corresponding sequences in the recombining virus genome to produce a chimaeric S gene; and
(iv) selecting for recombining virus comprising the chimaeric S gene, wherein the chimaeric S gene comprises a nucleotide sequence that encodes a chimaeric S protein, wherein the chimaeric S protein comprises the S1 domain of an IBV strain with restricted tropism that is unable to grow on a cell line, and comprises at least part of the S2 domain from an IBV strain with extended tropism that is able to grow on said cell line, such that an IBV comprising the chimaeric S protein is able to grow on said cell line.

14. The method according to claim 13, wherein the recombining virus is a vaccinia virus.

15. The method according to claim 13 which further comprises:
(v) recovering recombinant IBV comprising the chimaeric S gene from the DNA from the recombining virus from step (iv).

16. An isolated cell transfected with a plasmid according to claim 9 or infected with the virus of claim 11.

17. A cell according to claim 16 that is a Vero cell.

18. A composition comprising an infectious bronchitis virus according to claim 10 and a pharmaceutically acceptable diluent, adjuvant, excipient, or carrier.

19. A vaccine comprising an infectious bronchitis virus according to claim 10.

20. A method for treating and/or preventing IBV in a subject which comprises the step of administering a vaccine according to claim 19 to the subject.

21. A method for producing a vaccine comprising infecting Vero cells with the infectious bronchitis virus according to claim 11 and producing a vaccine from virus grown in the infected Vero cells.