Mr Lion
https://aim4truth.org/2020/03/14/cat-report-333/
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: Can Anyone Look Up This Patent Information?
: The patent # is: U.S. patent: 10 130 701
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: Thanks To All Who Replied: ------------------------
: ( 1 of 1 )
: 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
: Sperry Journal of Virology, 2005, vol. 79, No. 6, pp.
: 3391-3400. cited by examiner .
: Altschul et al., Basic local alignment search tool. J. Mol.
: Biol. 215: 403-10 (1990). cited by applicant .
: Ammayapppan et al.,
: Identification of sequence changes responsible for the
: attenuation of avian infectious bronchitis virus strain
: Arkansas DPI, Arch. Virol., 154(3):495-9 (2009). cited by
: applicant .
: Anonymous: "EM_STD:KF377577", Oct. 30, 2013. cited
: by applicant .
: Armesto et al., A recombinant avian infectious bronchitis
: virus expressing a heterologous spike gene belonging to the
: 4/91 serotype, PLoS One, 6(8):e24352 (2011). cited by
: applicant .
: Armesto et al.,
: The replicase gene of avian coronavirus infectious bronchitis
: virus is a determinant of pathogenicity, PLoS One,
: 4(10):e7384 (2009). cited by applicant .
: Armesto et al.,
: Transient dominant selection for the modification and
: generation of recombinant infectious bronchitis
: coronaviruses, Methods Mol. Biol., 454:255-73 (2008). cited
: by applicant .
: Ausubel et al.,
: Short Protocols in Molecular Biology, 4th edition, Chapter 18
: (1999). cited by applicant .
: Britton et al.,
: Generation of a recombinant avian coronavirus infectious
: bronchitis virus using transient dominant selection, J.
: Virol. Methods, 123(2):203-11 (2005). cited by applicant .
: Britton et al.,
: Modification of the avian coronavirus infectious bronchitis
: virus for vaccine development, Bioeng. Bugs., 3(2):114-9
: (2012). cited by applicant .
: Casais et al.,
: Recombinant avian infectious bronchitis virus expressing a
: heterologous spike gene demonstrates that the spike protein
: is a determinant of cell tropism, J. Virol., 77(16):9084-9
: (2003). cited by applicant .
: Casais et al.,
: Reverse genetics system for the avian coronavirus infectious
: bronchitis virus, J. Virol., 75(24):12359-69 (2001). cited
: by applicant .
: Devereux et al.,
: A comprehensive set of sequence analysis programms for the
: VAX. Nucl. Acids Res.12: 387-95 (1984). cited by applicant
: .
: Cavanagh et al.,
: Manipulation of the infectious bronchitis coronavirus genome
: for vaccine development and analysis of the accessory
: proteins, Vaccine, 25(30):5558-62 (2007). cited by
: applicant .
: International Preliminary Report on Patentability,
: International Application No. PCT/GB2015/052124, dated Jan.
: 24, 2017. cited by applicant .
: International Search Report and Written Opinion, International
: Application No. PCT/GB2015/052124, dated Oct. 9, 2015.
: cited by applicant .
: Larkin et al.,
: Clustal W and Clustal X version 2.0, Bioinformatics,
: 23(21):2947-8 (2007). cited by applicant .
: Menachery et al.,
: Attenuation and restoration of severe acute respiratory
: syndrome coronavirus mutant lacking 2'-o-methyltransferase
: activity, J. Virol., 88(8):4251-64 (2014). cited by
: applicant .
: Tatusova et al., BLAST 2 Sequences, a new tool for comparing
: protein and nucleotide sequences, FEMS Microbiol. Lett.,
: 174(2):247-50 (1999). cited by applicant .
: Wang et al.,
: Attenuation of porcine reproductive and respiratory syndrome
: virus strain MN184 using chimeric construction with vaccine
: sequence, Virology, 371(2):418-29 (2008). cited by
: applicant .
: Wei et al.,
: Development and characterization of a recombinant infectious
: bronchitis virus expressing the ectodomain region of S1
: gene of H120 strain, Appl. Microbiol. Biotechnol.,
: 98(4):1727-35 (2014). cited by applicant.
: 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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