Coronavirus – United States Patent 10,130,701 Bickerton , et al. November 20, 2018

ABSTRACT: The present invention provides a live, attenuated coronavirus comprising a variant replicase gene encoding polyproteins comprising a mutation in one or more of non-structural protein(s) (nsp)-10, nsp-14, nsp-15 or nsp-16. The coronavirus may be used as a vaccine for treating and/or preventing a disease, such as infectious bronchitis, in a subject.

http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=/netahtml/PTO/srchnum.htm&r=1&f=G&l=50&s1=10130701.PN.&OS=PN/10130701&RS=PN/10130701

The invention claimed is:

1. A live, attenuated coronavirus comprising a variant replicase gene encoding polyproteins comprising a mutation in one or both of non-structural protein(s) nsp-10 and nsp-14, wherein the variant replicase gene encodes a protein comprising an amino acid mutation of Pro to Leu at the position corresponding to position 85 of SEQ ID NO: 6, and/or wherein the variant replicase gene encodes a protein comprising an amino acid mutation of Val to Leu at the position corresponding to position 393 of SEQ ID NO: 7.

2. The coronavirus according to claim 1 wherein the variant replicase gene encodes a protein comprising one or more amino acid mutations selected from: an amino acid mutation of Leu to Ile at the position corresponding to position 183 of SEQ ID NO: 8; and an amino acid mutation of Val to Ile at the position corresponding to position 209 of SEQ ID NO: 9.

3. The coronavirus according to claim 1 wherein the replicase gene encodes a protein comprising the amino acid mutations Val to Leu at the position corresponding to position 393 of SEQ ID NO: 7; Leu to Ile at the position corresponding to position 183 of SEQ ID NO: 8; and Val to Ile at the position corresponding to position 209 of SEQ ID NO: 9.

4. The coronavirus according to claim 1 wherein the replicase gene encodes a protein comprising the amino acid mutations Pro to Leu at the position corresponding to position 85 of SEQ ID NO: 6; Val to Leu at the position corresponding to position 393 of SEQ ID NO: 7; Leu to Ile at the position corresponding to position 183 of SEQ ID NO: 8; and Val to Ile at the position corresponding to position 209 of SEQ ID NO: 9.

5. The coronavirus according to claim 1 wherein the replicase gene comprises at least one nucleotide substitutions selected from: C to Tat nucleotide position 12137; and G to C at nucleotide position 18114; compared to the sequence shown as SEQ ID NO: 1; and optionally, comprises one or more nucleotide substitutions selected from T to A at nucleotide position 19047; and G to A at nucleotide position 20139; compared to the sequence shown as SEQ ID NO: 1.

6. The coronavirus according to claim 1 which is an infectious bronchitis virus (IBV).

7. The coronavirus according to claim 1 which is IBV M41.

8. The coronavirus according to claim 7, which comprises an S protein at least, part of which is from an IBV serotype other than M41.

9. The coronavirus according to claim 8, wherein the S1 subunit is from an IBV serotype other than M41.

10. The coronavirus according to claim 8, wherein the S protein is from an IBV serotype other than M41.

11. The coronavirus according to claim 1 which has reduced pathogenicity compared to a coronavirus expressing a corresponding wild-type replicase, wherein the virus is capable of replicating without being pathogenic to the embryo when administered to an embryonated egg.

12. A variant replicase gene as defined in claim 1.

13. A protein encoded by a variant coronavirus replicase gene according to claim 12.

14. A plasmid comprising a replicase gene according to claim 12.

15. A method for making the coronavirus according to claim 1 which comprises the following steps: (i) transfecting a plasmid according to claim 14 into a host cell; (ii) infecting the host cell with a recombining virus comprising the genome of a coronavirus strain with a replicase gene; (iii) allowing homologous recombination to occur between the replicase gene sequences in the plasmid and the corresponding sequences in the recombining virus genome to produce a modified replicase gene; and (iv) selecting for recombining virus comprising the modified replicase gene.

16. The method according to claim 15, wherein the recombining virus is a vaccinia virus.

17. The method according to claim 15 which also includes the step: (v) recovering recombinant coronavirus comprising the modified replicase gene from the DNA from the recombining virus from step (iv).

18. A cell capable of producing a coronavirus according to claim 1.

19. A vaccine comprising a coronavirus according to claim 1 and a pharmaceutically acceptable carrier.

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

21. The method of claim 20, wherein the disease is infectious bronchitis (IB).

22. The method according to claim 20 wherein the method of administration is selected from the group consisting of; eye drop administration, intranasal administration, drinking water administration, post-hatch injection and in ovo injection.

23. The method according to claim 21 wherein the administration is in ovo vaccination.

24. A method for producing a vaccine according to claim 19, which comprises the step of infecting a cell according to claim 18 with a coronavirus according to claim 1.

25. The coronavirus according to claim 1, further comprising a mutation in one or both of nsp-15 and nsp-16.

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