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The patent # is: U.S. patent: 10 130 701
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United States Patent 10,130,701
Bickerton , et al. November 20, 2018
Coronavirus
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.
Inventors: Bickerton; Erica (Woking, GB), Keep; Sarah
(Woking, GB), Britton; Paul (Woking, GB)
Applicant:
Name City State Country Type
THE PIRBRIGHT INSTITUTE
Pirbright, Woking
N/A
GB
Assignee: THE PIRBRIGHT INSTITUTE (Woking, Pirbright, GB)
Family ID: 51494985
Appl. No.: 15/328,179
Filed: July 23, 2015
PCT Filed: July 23, 2015
PCT No.: PCT/GB2015/052124
371(c)(1),(2),(4) Date: January 23, 2017
PCT Pub. No.: WO2016/012793
PCT Pub. Date: January 28, 2016
Prior Publication Data
Document Identifier Publication Date
US 20170216427 A1 Aug 3, 2017
Foreign Application Priority Data
Jul 23, 2014 [GB] 1413020.7
Current U.S. Class: 1/1
Current CPC Class: C12N 7/00 (20130101); C12N 9/127 (20130101); C07K 14/005 (20130101); A61K 39/215 (20130101); C12Y 207/07048 (20130101); C12N 2770/20062 (20130101); C12N 2770/20022 (20130101); C12N 2770/20034 (20130101); C12N 2770/20051 (20130101); A61K 2039/70 (20130101); C12N 2770/20021 (20130101); A61K 2039/5254 (20130101); A61K 2039/54 (20130101); C12N 2770/20071 (20130101)
Current International Class: A61K 39/215 (20060101); C12N 9/12 (20060101); C12N 7/00 (20060101); A61K 39/00 (20060101)
References Cited [Referenced By]
U.S. Patent Documents
7452542 November 2008 Denison
Foreign Patent Documents
WO-2004/092360 Oct 2004 WO
WO-2005/049814 Jun 2005 WO
WO-2007/078203 Jul 2007 WO
WO-2011/004146 Jan 2011 WO
Other References
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Primary Examiner: Li; Bao Q
Attorney, Agent or Firm: Marshall, Gerstein & Borun LLP
Claims
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.
Description
FIELD OF THE INVENTION
The present invention relates to an attenuated coronavirus comprising a variant replicase gene, which causes the virus to have reduced pathogenicity. The present invention also relates to the use of such a coronavirus in a vaccine to prevent and/or treat a disease.
BACKGROUND TO THE INVENTION
Avian infectious bronchitis virus (IBV), the aetiological agent of infectious bronchitis (IB), 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 Order Nidovirales, Family Coronaviridae, Subfamily Corona virinae and Genus Gammacoronavirus;
genetically very similar coronaviruses cause disease in turkeys, guinea fowl 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 and produce poor quality 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.
IBV has been reported to be responsible for more economic loss to the poultry industry than any other infectious disease.
Although live attenuated vaccines and inactivated vaccines are universally used in the control of IBV, the protection gained by use of vaccination can be lost either due to vaccine breakdown or the introduction of a new IBV serotype that is not related to the vaccine used, posing a risk to the poultry industry.
Further, there is a need in the industry to develop vaccines which are suitable for use in ovo, in order to improve the efficiency and cost-effectiveness of vaccination programmes.
A major challenge associated with in ovo vaccination is that the virus must be capable of replicating in the presence of maternally-derived antibodies against the virus, without being pathogenic to the embryo.
Current IBV vaccines are derived following multiple passage in embryonated eggs, this results in viruses with reduced pathogenicity for chickens, so that they can be used as live attenuated vaccines.
However such viruses almost always show an increased virulence to embryos and therefore cannot be used for in ova vaccination as they cause reduced hatchability. A 70% reduction in hatchability is seen in some cases.
Attenuation following multiple passage in embryonated eggs also suffers from other disadvantages.
It is an empirical method, as attenuation of the viruses is random and will differ every time the virus is passaged, so passage of the same virus through a different series of eggs for attenuation purposes will lead to a different set of mutations leading to attenuation.
There are also efficacy problems associated with the process: some mutations will affect the replication of the virus and some of the mutations may make the virus too attenuated. Mutations can also occur in the S gene which may also affect immunogenicity so that the desired immune response is affected and the potential vaccine may not protect against the required serotype.
In addition there are problems associated with reversion to virulence and stability of vaccines.
It is important that new and safer vaccines are developed for the control of IBV. Thus there is a need for IBV vaccines which are not associated with these issues, in particular vaccines which may be used for in ovo vaccination.
SUMMARY OF ASPECTS OF THE INVENTION
The present inventors have used a reverse genetics approach in order to rationally attenuate IBV. This approach is much more controllable than random attenuation following multiple passages in embryonated eggs because the position of each mutation is known and its effect on the virus, i.e. the reason for attenuation, can be derived.
Using their reverse genetics approach, the present inventors have identified various mutations which cause the virus to have reduced levels of pathogenicity.
The levels of pathogenicity may be reduced such that when the virus is administered to an embryonated egg, it is capable of replicating without being pathogenic to the embryo.
Such viruses may be suitable for in ovo vaccination, which is a significant advantage and has improvement over attenuated IBV vaccines produced following multiple passage in embryonated eggs.
Thus in a first aspect, 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 variant replicase gene may encode a protein comprising one or more amino acid mutations selected from the list of: Pro to Leu at position 85 of SEQ ID NO: 6, Val to Leu at position 393 of SEQ ID NO: 7; Leu to Ile at position 183 of SEQ ID NO: 8; Val to Ile at position 209 of SEQ ID NO: 9.
The replicase gene may encode a protein comprising the amino acid mutation Pro to Leu at position 85 of SEQ ID NO: 6.
The replicase gene may encode a protein comprising the amino acid mutations Val to Leu at position 393 of SEQ ID NO: 7; Leu to Ile at position 183 of SEQ ID NO: 8; and Val to Ile at position 209 of SEQ ID NO: 9.
The replicase gene may encodes a protein comprising the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6; Val to Leu at position 393 of SEQ ID NO:7; Leu to Ile at position 183 of SEQ ID NO:8; and Val to Ile at position 209 of SEQ ID NO: 9.
The replicase gene may comprise one or more nucleotide substitutions selected from the list of:
C to T at nucleotide position 12137;
G to C at nucleotide position 18114;
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.
The coronavirus may be an infectious bronchitis virus (IBV).
The coronavirus may be IBV M41.
The coronavirus may comprise an S protein at least part of which is from an IBV serotype other than M41.
For example, the S1 subunit or the entire S protein may be from an IBV serotype other than M41.
The coronavirus according to the first aspect of the invention has reduced pathogenicity compared to a coronavirus expressing a corresponding wild-type replicase, such that when the virus is administered to an embryonated egg, it is capable of replicating without being pathogenic to the embryo.
In a second aspect, the present invention provides a variant replicase gene as defined in connection with the first aspect of the invention.
In a third aspect, the present invention provides a protein encoded by a variant coronavirus replicase gene according to the second aspect of the invention.
In a fourth aspect, the present invention provides a plasmid comprising a replicase gene according to the second aspect of the invention.
In a fifth aspect, the present invention provides a method for making the coronavirus according to the first aspect of the invention which comprises the following steps: (i) transfecting a plasmid according to the fourth aspect of the invention 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.
The recombining virus may be a vaccinia virus.
The method may also include the step: (v) recovering recombinant coronavirus comprising the modified replicase gene from the DNA from the recombining virus from step (iv).
In a sixth aspect, the present invention provides a cell capable of producing a coronavirus according to the first aspect of the invention.
In a seventh aspect, the present invention provides a vaccine comprising a coronavirus according to the first aspect of the invention and a pharmaceutically acceptable carrier.
In an eighth aspect, the present invention provides a method for treating and/or preventing a disease in a subject which comprises the step of administering a vaccine according to the seventh aspect of the invention to the subject.
Further aspects of the invention provide: the vaccine according to the seventh aspect of the invention for use in treating and/or preventing a disease in a subject. use of a coronavirus according to the first aspect of the invention in the manufacture of a vaccine for treating and/or preventing a disease in a subject.
The disease may be infectious bronchitis (IB).
The method of administration of the vaccine may be selected from the group consisting of; eye drop administration, intranasal administration, drinking water administration, post-hatch injection and in ovo injection.
Vaccination may be by in ova vaccination.
The present invention also provides a method for producing a vaccine according to the seventh aspect of the invention, which comprises the step of infecting a cell according to the sixth aspect of the invention with a coronavirus according to the first aspect of the invention.
More data here:
http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&co1=AND&d=PTXT&s1=10130701.PN.&OS=PN/10130701&RS=PN/10130701
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