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Illlll llllllll Ill lllll lllll lllll lllll lllll United States Patent(10)Bickerton et al.(45)(54)CORONAVIRUS(71)Applicant: THE PIRBRIGHT INSTITUTE,Pirbright, Woking (GB)(72)Inventors: Erica Bickerton, Woking (GB); SarahKeep, Woking (GB); Paul Britton,Woking (GB)(73)Assignee: THE PIRBRIGHT INSTITUTE,Woking, Pirbright (GB)( *)Notice:(21)Appl. No.:15/328,179(22)PCT Filed:Jul. 23, 2015(86)PCT No.:PCT/GB2015/052124§ 371 (c)(l),(2) Date:Jan.23, 2017(87)OTHER PUBLICATIONSSubject to any disclaimer, the term ofthispatent is extended or adjusted under 35U.S.C. 154(b) by 0 days.PCT Pub. No.: W02016/012793PCT Pub. Date: Jan. 28, 2016(65)Prior Publication DataUS 2017/0216427 Al(30)(51)(52)( 58)(56)Aug. 3, 2017Foreign Application Priority DataJul. 23, 2014(GB) . 1413020.7Int. Cl.A61K 391215(2006.01)(2006.01)C12N 7100C12N 9112(2006.01)A61K 39100(2006.01)U.S. Cl.A61K 391215 (2013.01); C12N 7100CPC(2013.01); C12N 91127 (2013.01); C12Y207107048 (2013.01); A61K 203915254(2013.01); A61K 2039154 (2013.01); Cl2N2770120021 (2013.01); Cl2N 2770120022(2013.01); Cl2N 2770120034 (2013.01); Cl2N2770120051 (2013.01); Cl2N 2770120062(2013.01)Field of Classification SearchCPC . A61K 39/215See application file for complete search history.7,452,542 B2 * 11/2008 Denison .C07K 14/005424/221.1FOREIGN PATENT DOCUMENTSW0-2004/092360W0-2005/049814W0-2007 2011Sperry Journal of Virology, 2005, vol. 79, No. 6, pp. 3391-3400.*Altschul et al., Basic local alignment search tool. J Mo!. Biol. 215:403-10 (1990).Ammayapppan et al., Identification of sequence changes responsible for the attenuation of avian infectious bronchitis virus strainArkansas DPI, Arch. Virol., 154(3):495-9 (2009).Anonymous: "EM STD:KF377577", Oct. 30, 2013.Armesto et al., A recombinant avian infectious bronchitis virusexpressing a heterologous spike gene belonging to the 4/91 serotype,PLoS One, 6(8):e24352 (2011).Armesto et al., The replicase gene of avian coronavirus infectiousbronchitis virus is a determinant of pathogenicity, PLoS One,4(10):e7384 (2009).Armesto et al., Transient dominant selection for the modificationand generation of recombinant infectious bronchitis coronaviruses,Methods Mo!. Biol., 454:255-73 (2008).Ausubel et al., Short Protocols in Molecular Biology, 4th edition,Chapter 18 ( 1999).Britton et al., Generation of a recombinant avian coronavirusinfectious bronchitis virus using transient dominant selection, J.Virol. Methods, 123(2):203-11 (2005).Britton et al., Modification of the avian coronavirus infectiousbronchitis virus for vaccine development, Bioeng. Bugs., 3(2): 114-9(2012).Casais et al., Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein isa determinant of cell tropism, J. Virol., 77(16):9084-9 (2003).Casais et al., Reverse genetics system for the avian coronavirusinfectious bronchitis virus, J. Virol., 75(24):12359-69 (2001).Devereux et al., A comprehensive set of sequence analysis programmsfor the VAX. Nucl. Acids Res.12: 387-95 (1984).Cavanagh et al., Manipulation of the infectious bronchitis coronavirusgenome for vaccine development and analysis of the accessoryproteins, Vaccine, 25(30):5558-62 (2007).International Preliminary Report on Patentability, International Application No. PCT/GB2015/052124, dated Jan. 24, 2017.International Search Report and Written Opinion, InternationalApplication No. PCT/GB2015/052124, dated Oct. 9, 2015.Larkin et al., Clustal Wand Clustal X version 2.0, Bioinformatics,23(21):2947-8 (2007).Menachery et al., Attenuation and restoration of severe acuterespiratory syndrome coronavirus mutant lacking 2' -omethyltransferase activity, J. Virol., 88(8):4251-64 (2014).Tatusova et al., BLAST 2 Sequences, a new tool for comparingprotein and nucleotide sequences, FEMS Microbiol. Lett., 174(2):24750 (1999).(Continued)Primary Examiner - Bao Q Li(74) Attorney, Agent, or Firm - Marshall, Gerstein &Borun LLP(57)References CitedU.S. PATENT DOCUMENTSWOWOWOWOPatent No.:US 10,130,701 B2Date of Patent:Nov. 20, 2018ABSTRACTThe present invention provides a live, attenuated coronavirus comprising a variant replicase gene encoding polyproteins comprising a mutation in one or more of non-structuralprotein(s) (nsp)-10, nsp-14, nsp-15 or nsp-16. The coronavirus may be used as a vaccine for treating and/or preventinga disease, such as infectious bronchitis, in a subject.25 Claims, 15 Drawing SheetsSpecification includes a Sequence Listing.
US 10,130, 701 B2Page 2(56)References CitedOTHER PUBLICATIONSWang et al., Attenuation of porcine reproductive and respiratorysyndrome virus strain MN184 using chimeric construction withvaccine sequence, Virology, 371(2):418-29 (2008).Wei et al., Development and characterization of a recombinantinfectious bronchitis virus expressing the ectodomain region of S 1gene of Hl20 strain, Appl. Micro biol. Biotechnol., 98(4): 1727-35(2014).* cited by examiner
U.S. PatentUS 10,130,701 B2Sheet 1 of 15Nov. 20, 2018FIGURE iM41 growth curve8-"E6':Jc.0'014.2.IDi 2o.o """"'ll"' 'li""""" """'li"" """"""'ll"""""" -2040Time ahours6080
U.S. PatentUS 10,130,701 B2Sheet 2 of 15Nov. 20, 2018FIGURE 2!ill mock Beau-R M41-R6 M41-R 12ill M41-CK EP4Day 3Day 4100.80Ill70u:t:::Day 6Day 7Wheezing90-0IllDay 5IIfl!"'.-0:ii60II50I Id0Ill00.s:::fl!l:!.w0.ill mock I40302010II.IJ I--,-- ---0Day 3Day 4Day 5Day 6Day 7 Beau-R M41-R 6fil M41-R 12. M41-CK EP4
U.S. PatentNov. 20, 2018Sheet 3 of 15US 10,130,701 B2FIGURE 3Assessment of Ciliary Activity mock Beau-R M41-R 6 M41-R 12 M41-CK EP4
U.S. PatentNov. 20, 2018US 10,130,701 B2Sheet 4of15FIGURE 4wM41R-nsp10rep .,. .,-- ---M41R-nsp14,l5,16n:.'P
U.S. PatentNov. 20, 2018Sheet 6of15FIGURE 6US 10,130,701 B2
U.S. PatentNov. 20, 2018US 10,130,701 B2Sheet 7of15FIGURE 710090 ··:80' .,70i\;60i!!!Mock2"'1:!:.00 mt:i1il M41R-nsp 10, 15, 16 rep 8 :: I!NJ41R-nsp 10, 14, 15 mp 10 Ii30 1i1il t:0""'M41R-nsp 10, 14, 16 rep 4 40 : 50(I i!I! fv141R-nsp 10, 15 rep 1fili!1mNJ41-CK EP4IllD.:iy3Oay4Oay5Oay6Day7L. . . . . . . . . . . . . . . . . . . . . . . . . . . , , , , , , , , , , , , , , , , , , , , , , , ,. ,. ,.",""""""""""". . . . . . . .",,. . . . . . . ., . ., . . . . . . . . . . . . . . ., , , , . . . ., , . . . , , , , . . ,, ,, ,, ,, ,, n,, ,, ,, ,, ,,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, . """"'""""''""'""'"""""''···. . ,.,. ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,. . ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, . .:
U.S. PatentNov. 20, 2018US 10,130,701 B2Sheet 8of15100 ·;80:;;:;0 U. -.m60· (1)Cf)l! 40cwewa.20Q mockEmKJ M41 R-nspc::J M41R-nsp M41R-nspGm M41R-nsp-era10, 15 rep 110, 14, 16 rep 410, 15, 16 rep 8'10, 14, 15 rep 10M41-K6!\.1141-CK EP4
U.S. PatentUS 10,130,701 B2Sheet 9of15Nov. 20, 2018FIGURE 9A 8-- -·" '"M41-RM41-CKM41-K 6B·.-. f-·.8M41R-nsp10 repM41R-nsp14, 15. 16 repM41 R nsp10 15 rep (1)M41 R-nsp10.15.16 rep {8)M41 R-nsp10.14.15 rep (10)fV14i R-nsp10.14:16 rep (4)6ouaTime (hours)noo
U.S. PatentNov. 20, 20180.·"ll""'C LLSheet 10 of 15US 10,130,701 B2
e 00Fig. 10 (cont.} Nsp14 **20100**40120**60140**80 160M41 Nsp14:Mod Nsp14: z0 N'"o*180*200*220*240M41 Nsp14 'I ,,,, '. , . ,,.,,. 0 ",,,,.Jft.L-«' ! 'iJK! ,. WC . "'w.JB ih :- , Y' ,., ,.mt: '.- ,Mod Nsp14 VV ,r LCBS.OC'il'itW'ifW3(;f Ll'll1IftW'fJK!filK1*Plk5rJZ5M!IFNfiifttJAY fl#'A'i%Jff*260*280*300*320M41 Nsp14 fl!IW"2 '.t N nlli !iVfirutAWlASADA.Im!MZLJ %:WffAF P§if:fil!iT iUiIME niSSC!f'il iJT liMod Nsp14 DI i'Sfffe dD f'-ifiG1flffHlA5JaJffii!MI'.illlU::iAX lgzr i'Jk'fi:DLTY.PffIAN.EniYJNSEC.R.YLQFJ!'i "M41 Nsp14 :.,,Mod Nsp14:*340*360*380*400N0.QO1J1 .('D('D.0Ul460480*420440***M41 Nsp14 f.SMFlfL .51,'tffe :PKEfilMS.DJ EFE"i!lS.SlNJETIQ.LDGM efSLlfE ITKCNIGG.WlC:EiMod Nsp14; lh'W Wf* JGGSJ,,,f'IJt SDN lfm:SJJGW-IDIQWifX?if D.LVS:tltl'mIXZ:Xm? .llIGB'll 500520*M41 Nsp14 H:DriA!Yb'''tJ!'SYWQ V- iEWFWifrmli'NFH{LWtf:fWSk!AMod Nsp14: fl'Af Afifill'WSY.tfA.A1P;f if#!' i"fID',,,BSWS.,*drJl'"w"' """'"" "'"" N
e 00 Fig. 10 (cont.)Nsp15 I - · - · · -.20**40M41 Nsp15tMod Nsp15:*100*120**60140**80160M41 Nsp15 N!1Wl1ID'J Tl !'Eti MWfilKZV:l'!D!EN%J;.rvr:.Y!J!Htit'iD tlSf'f 1if 1feflliff%r f'tfmYSiVE! fl!ill4fB,3IFfMod Nsp15: l.IWDWWUHLY.Rm'M"t010A%JJW1E.WJGiiL'' 1 D1JRYGDYQS ImtWef:S7 !51'JEIBSNELV'Qf JIPl*180M41 Nsp15 tMod Nsp15:*200*220*z0 N'"oN0.QO2401J1 .('D('D.*260*280*300*320M41 Nsp15 l ffeJNMUM:ffM! Sr Wt 4 i"' l"&lV"C W f UD.r:li f! i'G1.r4t;SW#'ef1VlS!§fffm!Mod Nsp15 .!m::HJJWJi iMlttlS1' "' l'l!, 1 !110-l'W!JlS!DYSS'!N0.Ul*M41 Nsp15: E'Mlr mr.rw.rtrtr ·Mod Nsp15t DGXIff:fI'C:ii?.@IGdrJl'"w"""" """" """" N
e 00 Fig. 10 (cont.) Nsp16*20*40*60*80M41 Nsp16: S.1&1'1XtG"i 1 i&r 2 1:Wlf:t lGctAtPSG.'1 l1&7i1Hltt2Xti 1'l.g;VF t GSirn'G"iAm*'l twlQllfil'MMEEC.NI,," YW'ifG1ALFSG1 iT ,J tkflP"tlNMR.'MffWB'b.G.S. \WefltFGMod Nsp16: S C?z0 N'"o*100*120*140*180KFfl!llI&tHfir:m w. lI.AmIGNWlfi:tLSSF ,M41 Nsp16; TM"'',&Z F:EtWr1.lf?IDfilff'? lSVA1refS"efLBMod Nsp16: P.M! EEG!:t!.VtM iV!:frffeS l&lbS l!S.tifi!mFa.5W l.!% r' flfi L SW!i : ·*180*200. .,*220*240IN0.QO1J1 .('D('D.(.H*260*280*300M41 Nsp16· - s .Sl %j; -. iLP !F"v'or@' 'l§fflv4., 002 . ".·'l ;i;.Ss: CLMod Nsp16 Qis: ti'Sim.l !Jt .:r: MV'N :rtf!efENL!R'C t;. W SWWS.DSW#e 0.UldrJl'"w"' """'"" "'"" N
U.S. PatentUS 10,130,701 B2Sheet 14 of 15Nov. 20, 2018Fig. 11ALOO ······I I.II 11 ····I···Ii. �····.II IIII ······l···I··········e'· , .I .············I·····I II II II. I I I III IIIIII . II. .l jLW � "'til.00 · ·····!.;It.: G.8f: . '.' .· , . ············· ·········l SMo ;k M41R·n"Sp 10, 14 n p ':' M4Hl·nsp 10, 16 ""P :'M41·! o.4{; . .G.W ·0(} (li:)J L.].Dciy4(k y:.!);,y6D.;Jv:1Fig. 118II11 I11IIII IIIIII1111111111III IIII. . 11IIIII I1111 111111II111111111111 111111IIII11111111II . ll. Mocklit M41R-nsp :lU, l.4 rep:;:, M41R·mp 10, 16 repi M 1l.·!
U.S. PatentNov. 20, 2018Sheet 15 of 15US 10,130,701 B2Fig. 11CfilM41R·nw 10, 16n?p""'M41·K
US 10,130,701 B212CORONAVIRUSIt is important that new and safer vaccines are developedfor the control of IBV. Thus there is a need for IBV vaccineswhich are not associated with these issues, in particularvaccines which may be used for in ovo vaccination.FIELD OF THE INVENTIONThe present invention relates to an attenuated coronaviruscomprising a variant replicase gene, which causes the virusto have reduced pathogenicity. The present invention alsorelates to the use of such a coronavirus in a vaccine toprevent and/or treat a disease.SUMMARY OF ASPECTS OF THE INVENTION10BACKGROUND TO THE INVENTIONAvian infectious bronchitis virus (IBV), the aetiologicalagent of infectious bronchitis (IB), is a highly infectious andcontagious pathogen of domestic fowl that replicates primarily in the respiratory tract but also in epithelial cells ofthe gut, kidney and oviduct. IBV is a member of the OrderNidovirales, Family Coronaviridae, Subfamily Corona virinae and Genus Gammacoronavirus; genetically very similarcoronaviruses cause disease in turkeys, guinea fowl andpheasants.Clinical signs of IB include sneezing, tracheal rales, nasaldischarge and wheezing. Meat-type birds have reducedweight gain, whilst egg-laying birds lay fewer eggs andproduce poor quality eggs. The respiratory infection predisposes chickens to secondary bacterial infections which canbe fatal in chicks. The virus can also cause permanentdamage to the oviduct, especially in chicks, leading toreduced egg production and quality; and kidney, sometimesleading 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 infectiousdisease. Although live attenuated vaccines and inactivatedvaccines are universally used in the control of IBV, theprotection gained by use of vaccination can be lost either dueto vaccine breakdown or the introduction of a new IBVserotype that is not related to the vaccine used, posing a riskto the poultry industry.Further, there is a need in the industry to develop vaccineswhich are suitable for use in ovo, in order to improve theefficiency and cost-effectiveness of vaccination programmes. A major challenge associated with in ovo vaccination is that the virus must be capable of replicating in thepresence of maternally-derived antibodies against the virus,without being pathogenic to the embryo. Current IBV vaccines are derived following multiple passage in embryonatedeggs, this results in viruses with reduced pathogenicity forchickens, so that they can be used as live attenuated vaccines. However such viruses almost always show anincreased virulence to embryos and therefore cannot be usedfor in ova vaccination as they cause reduced hatchability. A70% reduction in hatchability is seen in some cases.Attenuation following multiple passage in embryonatedeggs also suffers from other disadvantages. It is an empiricalmethod, as attenuation of the viruses is random and willdiffer every time the virus is passaged, so passage of thesame virus through a different series of eggs for attenuationpurposes will lead to a different set of mutations leading toattenuation. There are also efficacy problems associated withthe process: some mutations will affect the replication of thevirus and some of the mutations may make the virus tooat
US 2017/0216427 Al Aug. 3, 2017 (30) Foreign Application Priority Data Jul. 23, 2014 (GB) . 1413020.7 (51) Int. Cl. A61K 391215 . (10) Patent No.: US
