Chimaeric protein
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.
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.