«2 United States Patent Bickerton et al. USO10130701B2 (10) Patent No.: (4s) Date of Patent: US 10,130,701 B2 Nov. 20, 2018 (4) CORONAVIRUS. (71) Applicant: THE PIRBRIGHT INSTITUTE, Pirbeight, Woking (GB) (72) Inventors: Erica Bickerton, Woking (GB): Sarah Keep, Woking (GB): Paul Britton, Woking (GB) (73) Assignee: THE PIRBRIGHT INSTITUTE, Woking, Pirbright (GB) (*) Notice: Subject to any dislaimer, the term of this patent is extended or adjusted under 35 USC. 154) by 0 days. (21) Appl. Nos 15028,179 (22) PCT led: Jul 23, 2018 (86) PCTNo: —— PCTIGR2018/082124 5.371 (XD, (2) Date! Jan. 23, 2017 (87) PCT Pub. Nos 02016012793 PCT Pub, Date: Jan. 28, 2016 ws) Prior Publication Data US 201710216427 AL Aug 3, 2017 G0) Foreign Application Priority Data Jul 23, 2014 (GB) 14130207, (1) Inch. AGIK 397215 (2005.01) CRN 700 (2006.01) CRN 912 (2006.01) AGIK 39/00 (2006.01) (2) USC. cre AGIK 39/215 (2013.01): CI2N 700 (2013.01): CBN 94127 (201301), CRY 20707048 (2013.01); ABIK 2039/5254 (2013.01); 461K 203954 (201301), C12N 277020021 (2013.01); C12N 2770/20022 (2013.01); C12N 277020034 (2013.01); C12N 277020051 (2013.01); C12N 2770720062 (201301) (58) Fleld of Classification Seareh cee AGIK 39/215 See application fle for complete search history. 66) References Cited USS, PATENT DOCUMENTS. 782.542 B2* 11/2008 Denison OTK 14005 42211 FOREIGN PATENT DOCUMENTS. wo-2004 090360 A? 10.2004 Wor2o0soisi4 A? 6200s WO Woomrorsys aL 42007 WO Wo201LODHI4e AL 12011 (OTHER PUBLICATIONS Sperry Journal of Volo: 2005, vol. 79, No.6, sp. 3391-3400. Altschul eta Basic loca alignment search tool. J Mol Bio. 215 405-10 (990). Ammayapppan eta, Metifction of sence changes respon- ible forthe aftenuaion of avian infectious bronchitis vius sain Arkansts DP, Arch, Virol, 154034959 (2009), Anonymous: "EM STD KES77S77, Ost. 30, 2013, ‘Amesio etal. A recombinant avian infectious bonis vis ‘xqstingieeologous spike gene belonging othe 491 serotype, Phas One, 6024382 (2011). Amesto eta. The replicase gene of avian coronavis infectious Iwonchitis virus is a deteminant of pathogenicity, PLoS On, 107384 2000), Amio etal, Transient dominant selection for the mediation nd gencrtion of recombinant infectious bronchitis coronaire ‘Methods Mol iol, 454258-79 O08), Ausibel etal, Short Protocols in Molecular Biology, 4th edition, Chaper 18 1999), Bitton cal, Genertion of = recombinant avian coronavinss Infectious bronchitis virus using tansieat dominant seletion, J Vise. Methods, 123(2}203-11 (2008). Dita @ a, Modieation of the avian coronavins infectious bronchitis vis for vaccine development, Bioeng Bugs, 32) 1149 Gor (Casi al; Rocombiaant avian infetious bronchi Vins expos ‘heterologous spike gene demonsvates thal the spike protein is 2 termina of eal tropism, J. Vio. 77(16)90849 (2003). Casais etal, Reverse genetics system forthe avian coronas {infectious bronchitis vis, J. Vr, 7524) 1235969 (2001). Devers etal comprehensive set of sequence nays programms Ir the VAX. Nach elds Ret 1: 387.98 (1984) ‘Cavanagh cal, Manipulation of the infstious onc coronavins genome for vaccine development and analysis of the accessory Potsins, Vcine, 2500):5888-62 Q007), Imernationa Preliminary Report on PatatabiiyInratonal Api cation Nov PCT/GIB2018092124 dated fn. 24, 2017 International Seach Report and Writes Opinion, Intemational Application No. PCT'GH20181052124, dated Oxt9, 2018, Latkin etal, Clustl W and Clasal X version 20, Bioiformatics, 2301)2947-8 (07, ‘Menachery cal, Atemation and restoration of severe acute fespiratory syndrome coronavirus mutaat lacking 2-0 smetileasterase stv, J Virol 88(8)4251-64 Q014), Tatusova etal, BLAST 2 Sequences,» pe fol for comparing protein and aclotde sequences, FEMS Microbiol Let, 742) 247 S019) (Continved) Primary Examiner — Bao Q Li (14) Attorney, Agent, ar Firm — Marshall, Gerstein & Boru LLP on ABSTRACT ‘The present invention provides a live, attenuated coronavi- replicase gene encoding polypro- {eins comprising a mutation ia one of more of noa-strctiral protein(s) (nsp)-10, nsp-14, nsp-15 or nsp-l6, The eoron- Virus may be used a a veeine for weating andor preventing a disease, such as infectious broneitis, in a subject. 25 Claims, 15 Drawing Sheets Specification includes a Sequence Listing.