Proteolytic targeting of influenza viruses, methods for preparation and uses thereof

Abstract

The present application provides proteolytic targeting of influenza viruses, methods for their preparation and use. [Means] The proteolytic target influenza virus comprises a hydrolysis target M1 protein, and a TEVp recognition site and a proteolytic target molecule recognized by the ubiquitin-proteasome system are sequentially inserted into the C-terminus of the hydrolysis target M1 protein, and the proteolytic target molecule recognized by the ubiquitin-proteasome system comprises the amino acid sequence shown in SEQ ID No. 1-353. The hydrolysis target M1 protein can be recognized and hydrolyzed by the ubiquitin-proteasome system, which reduces the viral replication ability and ensures high safety. The proteolytic target influenza virus of the present application has a wide variety of subtypes, which plays an important role in the research and development of influenza virus vaccines.

Description

[Technical Field]

[0001] The present application is in the field of biotechnology, and specifically relates to proteolytic targeting of influenza viruses, methods for their preparation and use. [Background technology]

[0002] Influenza viruses are classified into three types: A, B, and C. Among these, influenza A virus outbreaks are the most frequent and widespread. The influenza A virus genome consists of eight independent single-stranded RNA fragments encoding 10 proteins, including hemagglutinin protein (HA), matrix protein (M), neuraminidase (NA), nucleocapsid protein (NP), nonstructural protein (NS), and three polymerases: PB1, PB2, and PA. The matrix proteins M1 and M2, and the nonstructural proteins NS1 and NEP, play important roles in maintaining the virion morphology and pathogenicity of the virus.

[0003] Influenza A viruses can seasonally infect humans and birds, and large-scale epidemics can cause extremely high morbidity and mortality, severely threatening human health. Currently, vaccination is the primary means of preventing influenza and controlling its spread.

[0004] CN102899294A discloses an H1N1 swine influenza virus vaccine strain and its use. The H1N1 swine influenza virus vaccine strain is an inactivated vaccine for preventing swine influenza, which provides good protection to pigs challenged with the same highly virulent strain, has good immunogenicity, and can effectively prevent H1N1 swine influenza in either a single vaccine or a combined vaccine.

[0005] CN106075424A discloses an avian influenza virus vaccine, the antigen of which is an inactivated H9 subtype avian influenza virus, and the HA and EID50 of the new virulent strain of the H9 subtype avian influenza virus FJ11 strain used in the avian influenza virus vaccine have high titers, good immunogenicity, and can protect against attacks by H9 subtype avian influenza viruses isolated in various regions. The vaccine is safe, and analysis of the characteristic shape, safety test, and efficacy test data in shelf-life tests shows that all indicators are stable and effective compared to similar products, and the inactivated H9 subtype avian influenza vaccine rapidly produces antibodies.

[0006] However, inactivated vaccines may destroy or alter effective antigenic components during the inactivation process, potentially affecting their immune efficacy. Furthermore, the immune effect of inactivated vaccines is short-lasting, requiring multiple doses for reinforcement, and generally high cost. Furthermore, the influenza virus vaccines can only protect against a small number of virus subtypes. Therefore, developing a safe, controllable, targeted, and subtype-rich influenza virus library plays an important role in the research and development of influenza virus vaccines. Summary of the Invention [Problem to be solved by the invention]

[0007] The present application provides a proteolytically targeted influenza virus, its preparation method, and use. The proteolytically targeted influenza virus contains a hydrolysis-targeted M1 protein, which can be recognized by the ubiquitin-proteasome system and cleaved by tobacco etch virus protease (TEVp). It can be mass-produced in a cell line overexpressing TEVp. In contrast, in a normal cell line, the ubiquitin-proteasome system recognizes the proteolytically targeted molecule fused to the viral protein and degrades the viral protein, thereby weakening or even completely eliminating the viral replication ability. The resulting proteolytically targeted influenza virus is highly safe. The proteolytically targeted influenza virus can be used to prevent influenza as a live or attenuated vaccine. It can also be used as an oncolytic virus for tumor treatment. The proteolytically targeted influenza virus of the present application has a wide variety of subtypes, making it an important tool for the research and development of influenza virus vaccines. [Means for solving the problem]

[0008] In aspect 1, the present application provides: 1. A proteolytically targeted influenza virus comprising a hydrolysis-targeted M1 protein, a TEVp recognition site and a proteolytic target molecule recognized by the ubiquitin-proteasome system are sequentially inserted into the C-terminus of the hydrolysis target M1 protein, and the proteolytic target molecule recognized by the ubiquitin-proteasome system comprises an amino acid sequence represented by SEQ ID Nos. 1 to 353; Proteolytic targeting of influenza viruses is provided.

[0009] In this application, the design principle of the proteolytic target virus is as follows:

[0010] (1) Proteolytic target molecules introduced into specific sites of viral proteins can be recognized by the ubiquitin-proteasome system in normal host cells, degrading the corresponding viral proteins and inactivating the virus.

[0011] (2) Proteolytic target molecules introduced into specific sites of viral proteins can be inhibited in specific virus production systems or selectively cleaved by the linker chain to separate from the viral protein, thereby preventing or reducing the degradation of the viral protein by the ubiquitin-proteasome system.

[0012] (3) The proteolytic target molecule introduced into a specific site of the viral protein cannot be inhibited in normal host cells, or the linker linking the proteolytic target molecule and the viral protein cannot be cleaved in normal host cells. Therefore, the prepared virus can be recognized and degraded by the ubiquitin-proteasome system in host cells of animals and humans, reducing its replication ability and even completely losing its ability to replicate and reproduce, thereby increasing the safety of the virus.

[0013] In this application, 353 conditionally cleavable proteolytic target molecules are introduced into the C-terminus of the hydrolysis target M1 protein, and the ubiquitin-proteasome system present in normal cells of humans and animals can recognize the proteolytic target molecules expressed in fusion with viral proteins, allowing the viral M1 protein to be degraded by the ubiquitin-proteasome system, resulting in degradation of the viral protein.The hydrolysis target influenza virus containing the hydrolysis target M1 protein has weakened replication in animals and humans, and is therefore unable to replicate and reproduce, increasing the safety of the virus, and the resulting hydrolysis target influenza virus can be prepared into a live influenza virus vaccine.

[0014] The amino acid sequence of the proteolytic target molecule is as follows: Among them, SEQ ID Nos. 1 to 4 and 146 to 167 correspond to Keap1-Cul3 E3 ubiquitin ligase, SEQ ID Nos. 5 to 7 correspond to SOCS2 E3 ubiquitin ligase, SEQ ID No. 8 corresponds to SOCS3 E3 ubiquitin ligase, SEQ ID No. 9 corresponds to SOCS6 E3 ubiquitin ligase, SEQ ID No. 10 corresponds to KLHL-12 E3 ubiquitin ligase, SEQ ID No. 11 corresponds to C-terminal Degrons-Cullin-RING E3 ubiquitin ligase, SEQ ID Nos. 12 to 17 correspond to KLHDC3 E3 ubiquitin ligase, SEQ ID Nos. 18 to 26 correspond to KLHDC2 E3 ubiquitin ligase, and SEQ ID No. 27 corresponds to APPBP2. SEQ ID Nos. 28-31, 53-60, and 168-174 correspond to SPOP E3 ubiquitin ligase, SEQ ID Nos. 32-34 and 175-176 correspond to KLHL3 E3 ubiquitin ligase, SEQ ID Nos. 35-38 correspond to KLHL20 E3 ubiquitin ligase, SEQ ID Nos. 39-40 correspond to SPSB E3 ubiquitin ligase, SEQ ID Nos. 41-43 correspond to FBXO31 E3 ubiquitin ligase, SEQ ID Nos. 44-46, 126-130, and 311-326 correspond to β-TrCP1 E3 ubiquitin ligase, and SEQ ID Nos. 311-326 correspond to β-TrCP1 E3 ubiquitin ligase. No. 47~50, SEQ ID No. 114~120, SEQ ID No. 303~305 are SCF FBW7SEQ ID Nos. 51-52 correspond to ITCH E3 ubiquitin ligase, SEQ ID Nos. 61-64 and 177-182 correspond to MDM2 E3 ubiquitin ligase, SEQ ID Nos. 65-67 correspond to CRBN E3 ubiquitin ligase, SEQ ID Nos. 68-75 and 183-206 correspond to SEQ ID No. 183 E3 ubiquitin ligase, SEQ ID Nos. 76-85 and 207-260 correspond to APC / C-Dbox E3 ubiquitin ligase, and SEQ ID Nos. 86-90, 136-140, 261-278 and 279 correspond to APC / C-Dbox E3 ubiquitin ligase. SEQ ID Nos. 331 to 345 correspond to APC / C-KENbox E3 ubiquitin ligase, SEQ ID Nos. 91 to 95 and 294 to 298 correspond to COP1 E3 ubiquitin ligase, SEQ ID Nos. 96 to 100 and 299 to 300 correspond to CRL4_CDT2_1 E3 ubiquitin ligase, SEQ ID Nos. 101 to 105 and 301 to 302 correspond to N-end UBR-box E3 ubiquitin ligase, SEQ ID Nos. 106 to 113 correspond to VHL E3 ubiquitin ligase, and SEQ ID Nos. 121 to 123 correspond to SCF Skp2-Cks1 SEQ ID Nos. 124-125 and 306-310 correspond to E3 ubiquitin ligases. TIR1 SEQ ID Nos. 131 to 135 and 327 to 330 correspond to SIAH-1 E3 ubiquitin ligase, SEQ ID Nos. 141 to 145 and 346 to 347 correspond to APC / C-ABBA E3 ubiquitin ligase, and SEQ ID Nos. 279 to 293 correspond to APC / C-TPR1 E3 ubiquitin ligase.

[0015] SEQ ID No. 1:LDPETGEYL, SEQ ID No. 2:LDPETGEFL, SEQ ID No.3:LDEETGEFL、 SEQ ID No.4:AFFAQLQLDEETGEFL、 SEQ ID No.5:PVPDYTSIHIV、 SEQ ID No.6:NIDFYAQVSDI、 SEQ ID No.7:ASFEYTILDPS、 SEQ ID No.8:MPPPGAPSFPSPPTEPSSEVPEQPSAQPLPGSPPRR、 SEQ ID No.9:NGNNYVYIDPT、 SEQ ID No.10:PGAPPGRDLA、 SEQ ID No.11:YYCFG、 SEQ ID No.12:YKKVGTMAAG、 SEQ ID No.13:RWGRRG、 SEQ ID No.14:RPCQRG、 SEQ ID No.15:RAPRQRSRDG、 SEQ ID No.16:SWRLTGFSGMKG、 SEQ ID No.17:RGPSSGG、 SEQ ID No.18:PPPMAGG、 SEQ ID No.19:RGPSSGG、 SEQ ID No.20:EAIGLLGG、 SEQ ID No.21:HLRGSPPPMAGG、 SEQ ID No.22:RGSPPPMAGG、 SEQ ID No.23:SPPPMAGG、 SEQ ID No.24:PPMAGG、 SEQ ID No.25:PMAGG、 SEQ ID No.26:RRGPSSGG、 SEQ ID No.27:VLIRVTYCGL、 SEQ ID No.28:KADTTTPTT、 SEQ ID No.29:PEQDCAVTSGE、 SEQ ID No.30:DEVTSTTSSS、 SEQ ID No.31:KAASADSTTEGTPAD、 SEQ ID No.32:EPEEPEADQHQ、 SEQ ID No.33:GPPSVFPPEPEEPEADQHQ、 SEQ ID No.34:ECEETEVDQHV、 SEQ ID No.35:LPDLV、 SEQ ID No.36:LPDMV、 SEQ ID No.37:LGLPDLVAKYN、 SEQ ID No.38:LGLPDMVAKHN、 SEQ ID No.39:ELNNNL、 SEQ ID No.40:DINNNN、 SEQ ID No.41:PTDVRDVDI、 SEQ ID No.42:PTDVRDIDL、 SEQ ID No.43:PTDVTAIHL、 SEQ ID No.44:KKERLLDDRHDSGLDS、 SEQ ID No.45:KAWQQQSYLDSGIHS、 SEQ ID No.46:LDSGIHS、 SEQ ID No.47:SPLPSGLLTPPQSG、 SEQ ID No.48:CSLIPTPDKE、 SEQ ID No.49:FELLPTPPLS、 SEQ ID No.50:PPTPPGSH、 SEQ ID No.51:TPEAPPCYMDVI、 SEQ ID No.52:KFMPPPTYTEVD、 SEQ ID No.53:EVSIIQGADSTT、 SEQ ID No.54:MVADSTTKID、 SEQ ID No.55:ADSTT、 SEQ ID No.56:EVSIIQGADSTT、 SEQ ID No.57:ASSST、 SEQ ID No.58:DEVTSTTSSS、 SEQ ID No.59:ASSSS、 SEQ ID No.60:PSSSS、 SEQ ID No.61:FSDLWKLL、 SEQ ID No.62:FEHLWSSL、 SEQ ID No.63:FEAQWAAL、 SEQ ID No.64:FQHIWDFL、 SEQ ID No.65:LVDKKSGEK、 SEQ ID No.66:FQCNQCGAS、 SEQ ID No.67:AINITNGEE、 SEQ ID No.68:RAVENQYSFY、 SEQ ID No.69:RAINNQYSFV、 SEQ ID No.70:GDYRR、 SEQ ID No.71:VDYRT、 SEQ ID No.72:GDYRE、 SEQ ID No.73:YDYRG、 SEQ ID No.74:RDYRD、 SEQ ID No.75:DDEGDSYSDNP、 SEQ ID No.76:RKRLPVT、 SEQ ID No.77:RMWLKIT、 SEQ ID No.78:RRTLKMI、 SEQ ID No.79:RSGLQLS、 SEQ ID No.80:RKALETL、 SEQ ID No.81:PRRTLKVIQ、 SEQ ID No.82:QRAALGNIS、 SEQ ID No.83:PRTALGDIG、 SEQ ID No.84:HRKHLQEIP、 SEQ ID No.85:SRKALGNVN、 SEQ ID No.86:NKENE、 SEQ ID No.87:DKENV、 SEQ ID No.88:DKENG、 SEQ ID No.89:EKENE、 SEQ ID No.90:KKENG、 SEQ ID No.91:EEIRRVPEF、 SEQ ID No.92:DEQFVPDF、 SEQ ID No.93:SDQIVPEY、 SEQ ID No.94:EEHFLVPDL、 SEQ ID No.95:EEPQTVPEM、 SEQ ID No.96:TPITDYFPKRKKI、 SEQ ID No.97:RRVTDFFARRRP、 SEQ ID No.98:MRVTDFFSQSKRG、 SEQ ID No.99:RKLTDFYPVRRS、 SEQ ID No.100:PLISDFFAKRKRS、 SEQ ID No.101:AKDAS、 SEQ ID No.102:RRFSP、 SEQ ID No.103:MREGSA、 SEQ ID No.104:VDNNTN、 SEQ ID No.105:MCGGA、 SEQ ID No.106:LAPFVDTYDMMQM、 SEQ ID No.107:IAPVNTKATIRL、 SEQ ID No.108:LAPYISMDDDFQL、 SEQ ID No.109:LAPAAGDTIISLDF、 SEQ ID No.110:LAPYIPMDDDDFQL、 SEQ ID No.111:LAPTPGDAIISLDF、 SEQ ID No.112:LAPYIPMDGEDFQL、 SEQ ID No.113:LAPVAPHSPFLL、 SEQ ID No.114: LLPTPPLS、 SEQ ID No.115:PGTPPSS、 SEQ ID No.116:LTPPPS、 SEQ ID No.117:ITPFTS、 SEQ ID No.118:PGETPPLS、 SEQ ID No.119:FPPSPPSS、 SEQ ID No.120:SLIPTPDK、 SEQ ID No.121: SVEQTPKK、 SEQ ID No.122:AVEQTPRK、 SEQ ID No.123:SVEQTPRK、 SEQ ID No.124:QIVGWPPVRSNRK、 SEQ ID No.125:QVVGWPPVCSYRK、 SEQ ID No.126:DSGIES、 SEQ ID No.127:DSGKGS、 SEQ ID No.128:DSGIIT、 SEQ ID No.129:DSGHES、 SEQ ID No.130:DSGNES、 SEQ ID No.131:PPVAQVMPG、 SEQ ID No.132:IPTACVRPT、 SEQ ID No.133:IPCAAVSPN、 SEQ ID No.134:APTAVVLPH、 SEQ ID No.135:RPVAMVRPT、 SEQ ID No.136:NKENC、 SEQ ID No.137:DKENV、 SEQ ID No.138:EKENH、 SEQ ID No.139:AKEND、 SEQ ID No.140:DKENG、 SEQ ID No.141:FDIYMD、 SEQ ID No.142:FHVFED、 SEQ ID No.143:ITVFDE、 SEQ ID No.144:FTPYVE、 SEQ ID No.145:FSIFDE、 SEQ ID No.146:NQETGE、 SEQ ID No.147:SEE、 SEQ ID No.148:SEEN、 SEQ ID No.149:DNETGE、 SEQ ID No.150:MONEY、 SEQ ID No.151:DHDTGE、 SEQ ID No.152:DPSTGE、 SEQ ID No.153:DPSTGE、 SEQ ID No.154:DGETGE、 SEQ ID No.155:SPETGE、 SEQ ID No.156:DEETGE、 SEQ ID No.157:DEETGE、 SEQ ID No.158:DEETGE、 SEQ ID No.159:NVESGE、 SEQ ID No.160:QDIDLGV、 SEQ ID No.161:QDIDLGV、 SEQ ID No.162:DPSTGE、 SEQ ID No.163:DGETGE、 SEQ ID No.164:SPETGE、 SEQ ID No.165:DEETGE、 SEQ ID No.166:NVESGE、 SEQ ID No.167:QDIDLGV、 SEQ ID No.168:ADSST、 SEQ ID No.169:ADSST、 SEQ ID No.170:PSSTS、 SEQ ID No.171:PSSTS、 SEQ ID No.172:VSSST、 SEQ ID No.173:VSSTS、 SEQ ID No.174:VTSTT、 SEQ ID No.175:LQLDEETGEFL、 SEQ ID No.176:ECEETEVDQHV、 SEQ ID No.177:FEHLWSSL、 SEQ ID No.178:FSDLWKLL、 SEQ ID No.179:FEAQWAAL、 SEQ ID No.180:FEAQWAAL、 SEQ ID No.181:FQHIWDFL、 SEQ ID No.182: FSDLWKLL、 SEQ ID No.183: LLQPNNYQFC、 SEQ ID No.184:DYR、 SEQ ID No.185:RAVENQYSFY、 SEQ ID No.186:SHVENDYIDNPS、 SEQ ID No.187:LIEDNEYTARQG、 SEQ ID No.188:SIFNSLYTTLSD、 SEQ ID No.189:SIFDNLYTTLSD、 SEQ ID No.190:VSFNPYEPELA、 SEQ ID No.191:LLQPNNYQFC、 SEQ ID No.192:INGNNYVYIDP、 SEQ ID No.193:STNHIYSNLAN、 SEQ ID No.194:IPFLDYRTYAV、 SEQ ID No.195:NESVDYRATFP、 SEQ ID No.196:YSGSDYRSGLA、 SEQ ID No.197:ADYRE、 SEQ ID No.198:IPFLDYRTYAM、 SEQ ID No.199:RDYRD、 SEQ ID No.200:IRNTNEYTEGPT、 SEQ ID No.201:DSFLQRYSSDP、 SEQ ID No.202:KGDGGLYSSLPP、 SEQ ID No.203:TLNSDGYTPEPA、 SEQ ID No.204:RDNVYYYDEEG、 SEQ ID No.205:KEDPIYDEPEG、 SEQ ID No.206:AEDSTYDEYEN、 SEQ ID No.207:RLCLPQV、 SEQ ID No.208:RNSLRQT、 SEQ ID No.209:RAPLSTL、 SEQ ID No.210:RKALETL、 SEQ ID No.211:RMKLPSP、 SEQ ID No.212:RNFLTEQ、 SEQ ID No.213:RTPLSIV、 SEQ ID No.214:RYGLHPD、 SEQ ID No.215:RSAFEDL、 SEQ ID No.216:RHFLDRV、 SEQ ID No.217:RHCLPTL、 SEQ ID No.218:RKKLVLE、 SEQ ID No.219:RLPLRAV、 SEQ ID No.220:RSPLLEV、 SEQ ID No.221:RKALGTV、 SEQ ID No.222:RKALGTV、 SEQ ID No.223:RGSLEGA、 SEQ ID No.224:RSPLFIF、 SEQ ID No.225:RSSLLSR、 SEQ ID No.226:RSTLAEL、 SEQ ID No.227:RCQLAKK、 SEQ ID No.228:KTPLKEG、 SEQ ID No.229:RGTLKRQ、 SEQ ID No.230:RAALAVL、 SEQ ID No.231:RDELGGG、 SEQ ID No.232:RFALSQL、 SEQ ID No.233:RPELQDA、 SEQ ID No.234:RKFLSLA、 SEQ ID No.235:RLPLRAQ、 SEQ ID No.236:RRPLILK、 SEQ ID No.237:RRRLPLP、 SEQ ID No.238:RLPLPVQ、 SEQ ID No.239:RKSLSQK、 SEQ ID No.240:HLSLKDI、 SEQ ID No.241:RLPLVPE、 SEQ ID No.242:RARLCSS、 SEQ ID No.243:RVRLSWA、 SEQ ID No.244:RRFLENC、 SEQ ID No.245:RLPLAQV、 SEQ ID No.246:RSPLTIL、 SEQ ID No.247:RAALDRL、 SEQ ID No.248:RNLLHDN、 SEQ ID No.249:RKRLGDD、 SEQ ID No.250:RRGLILA、 SEQ ID No.251:RENLQRK、 SEQ ID No.252:RQPLSEA、 SEQ ID No.253:RASLNPR、 SEQ ID No.254:RMKLKKM、 SEQ ID No.255:RTPLASV、 SEQ ID No.256:RTQLETK、 SEQ ID No.257:RRSLSIS、 SEQ ID No.258:QRTVLGVIG、 SEQ ID No.259:NRKPLTVLN、 SEQ ID No.260:SRTPLTTLN、 SEQ ID No.261:NKENC、 SEQ ID No.262:EKENH、 SEQ ID No.263:EKENQ、 SEQ ID No.264:AKEND、 SEQ ID No.265:DKENG、 SEQ ID No.266:NKENK、 SEQ ID No.267:QKENG、 SEQ ID No.268:NKENL、 SEQ ID No.269:EKENL、 SEQ ID No.270:SKENI、 SEQ ID No.271:DKENR、 SEQ ID No.272:EKENC、 SEQ ID No.273:QKENR、 SEQ ID No.274:YKENK、 SEQ ID No.275:LKENS、 SEQ ID No.276:LKENS、 SEQ ID No.277:DKENQ、 SEQ ID No.278:QKENV、 SEQ ID No.279: QYLIR、 SEQ ID No.280: NKRIR、 SEQ ID No.281:FNQIR、 SEQ ID No.282:VNHIR、 SEQ ID No.283:RTTIR、 SEQ ID No.284:RTQIR、 SEQ ID No.285:KGNLR、 SEQ ID No.286:YKGIR、 SEQ ID No.287:LMTMR、 SEQ ID No.288:VNRIR、 SEQ ID No.289:VNRIR、 SEQ ID No.290:FTRIR、 SEQ ID No.291:YRSIR、 SEQ ID No.292:ILGMR、 SEQ ID No.293:HQGIR、 SEQ ID No.294:DEQFVPDY、 SEQ ID No.295:EELLMVPDM、 SEQ ID No.296:EEHFLVPDL、 SEQ ID No.297:EQTLQEVPTGL、 SEQ ID No.298:SLLDVQRVPSF、 SEQ ID No.299:TSMTDFYHSKRRL、 SEQ ID No.300:NDITNYYKVKRRP、 SEQ ID No.301:MCKGL、 SEQ ID No.302:MCRTL、 SEQ ID No.303:PPTPPPE、 SEQ ID No.304:PTPPLS、 SEQ ID No.305:PFLTPSPE、 SEQ ID No.306:QVVGWPPVRSYRK、 SEQ ID No.307:QIVGWPPVRSYRK、 SEQ ID No.308:QVVGWPPVRNYRK、 SEQ ID No.309:QVVGWPPIGLHRM、 SEQ ID No.310:PVVGWPPVRSSRR、 SEQ ID No.311:DSGRGS、 SEQ ID No.312:DSGNYS、 SEQ ID No.313:DSGICMS、 SEQ ID No.314:DSGVETS、 SEQ ID No.315:DSGLDS、 SEQ ID No.316:DSGIHS、 SEQ ID No.317:DSGIDS、 SEQ ID No.318:DSGIDS、 SEQ ID No.319:DSGLPS、 SEQ ID No.320:DSGLGS、 SEQ ID No.321:DSGRGDS、 SEQ ID No.322:DSGYNT、 SEQ ID No.323:DSGQGS、 SEQ ID No.324:DSGIGT、 SEQ ID No.325:DSGYSS、 SEQ ID No.326:SEGSDDSGL、 SEQ ID No.327:RPTAAVTPI、 SEQ ID No.328:PPRALVLPH、 SEQ ID No.329:KPAAVVAPI、 SEQ ID No.330:KPTAYVRPM、 SEQ ID No.331:NKENE、 SEQ ID No.332:EKENQ、 SEQ ID No.333:NKENK、 SEQ ID No.334:QKENG、 SEQ ID No.335:NKENL、 SEQ ID No.336:EKENL、 SEQ ID No.337:SKENI、 SEQ ID No.338:DKENR、 SEQ ID No. 339: EKENC, SEQ ID No. 340: QKENR, SEQ ID No. 341: YKENK, SEQ ID No. 342:LKENS, SEQ ID No. 343:LKENS, SEQ ID No. 344:DKENQ, SEQ ID No. 345:QKENV, SEQ ID No. 346:FTIHVD, SEQ ID No. 347:FMLYEE, SEQ ID No.348:IARRNSLTRFLEKRKDRV, SEQ ID No.349:QARKASLARFLEKRKERV, SEQ ID No.350:QARKASLARFLEKRKERL, SEQ ID No.351:IARRASLHRFLEKRKDRV, SEQ ID No. 352:LARKASLARFLEKRKERV, or SEQ ID No.353:IARRASLHRFLEKRKDRI.

[0016] Preferably, the TEVp recognition site comprises the amino acid sequence shown in SEQ ID No. 354.

[0017] SEQ ID No. 354:ENLYFQG.

[0018] Preferably, the C-terminus of the M1 protein and the TEVp recognition site are linked by a flexible linker 1.

[0019] Preferably, the flexible linker 1 comprises the amino acid sequence shown in SEQ ID No. 355.

[0020] SEQ ID No. 355:GSGG.

[0021] Preferably, the TEVp recognition site and the proteolytic target molecule are connected by a flexible linker 2.

[0022] Preferably, the flexible linker 2 comprises the amino acid sequence of GSG.

[0023] Preferably, the proteolytic target influenza virus is a type A, B or C virus.

[0024] Preferably, the influenza virus subtype is H1N1, H1N2, H1N3, H1N8, H1N9, H2N2, H2N3, H2N8, H3N1, H3N2, H3N8, H4N2, H4N4, H4N6, H4N8, H5N1, H5N2, H5N3, H5N6, H5N8, H5N9, H6N1, H6N2, H6N4, H6N5, H6N6, H6N8, H7N1, H7N2, H7N3, H7N 7, H7N8, H7N9, H8N4, H9N1, H9N2, H9N5, H9N8, H10N3, H10N4, H10N7, H10N8, H10N9, H11N2, H11N6, H11N9, H12N1, H12N3, H12N5, H13N6, H13N8, H14N5, H15N2, H15N8, H16N3, H17N10 or H18N11, or a combination of at least two of these.

[0025] In this application, the inventors have introduced the nucleotide sequence of a TEVp recognition site and a proteolysis target molecule into the genome of an influenza virus, so that in a cell system overexpressing TEVp, the influenza virus genome containing the proteolysis target molecule can replicate in conjunction with viral genome replication and can be expressed fused to a viral protein in conjunction with viral protein translation, thereby obtaining a virus site-specifically modified by the proteolysis target molecule, i.e., a proteolysis-targeting chimeric virus (PROTAC virus).

[0026] In normal cells, the ubiquitin-proteasome system recognizes the proteolytic target molecule fused with the viral M1 protein and degrades the viral protein, weakening the viral replication ability and eventually completely eliminating it, making PROTAC viruses highly safe. At the same time, because the PROTAC virus contains specific recognition sequences and sites for the ubiquitin-proteasome system, it can transform cold tumors into hot tumors and perform tumor lysis in tumor treatment.

[0027] In this application, the PROTAC virus can be efficiently replicated in a specific artificially modified cell line and produced on a large scale. The PROTAC virus can also be further modified, for example, by introducing immune enhancers into specific regions or specific amino acids of viral proteins to obtain viruses with improved performance or enhanced immunogenicity.

[0028] In the present application, the amino acid sequence structures of the flexible linker and TEVp recognition site (linking chain), and the flexible linker and proteolytic target molecule used may be applied to influenza B, or to other subtypes of influenza virus, or may be used to modify other types of viruses, such as HIV virus, novel coronavirus, hand, foot, and mouth virus, hepatitis D virus, or hepatitis E virus, or a combination of at least two of these viruses.

[0029] In a second aspect, the present application provides a nucleic acid molecule encoding the hydrolysis target M1 protein according to the first aspect.

[0030] In a third aspect, the present application provides a recombinant vector comprising at least one copy of the nucleic acid molecule of the second aspect.

[0031] In a fourth aspect, the present application provides: (1) constructing an expression vector for preparing a proteolytic target influenza virus; and step (2) replacing a plasmid expressing the influenza virus M gene in the influenza virus rescue system with the expression vector obtained in step (1), and co-transfecting the vector into a cell line to obtain the proteolysis target influenza virus. A method for preparing a proteolytically targeted influenza virus according to embodiment 1 is provided.

[0032] This application provides 353 types of proteolytically targeted viruses, the C-terminus of the M1 protein of which contains a proteolytically targeted molecule that is recognized by the ubiquitin-proteasome system, the M1 protein and the proteolytically targeted molecule being linked by a TEVp recognition site that can be selectively cleaved, and the M1 protein and the TEVp recognition site, and the TEVp recognition site and the proteolytically targeted molecule being linked by two flexible molecules; the procedure for modifying viruses using this method is simple, and the resulting proteolytically targeted influenza viruses are safe, reliable, and highly immunogenic.

[0033] Preferably, in step (1), the expression vector comprises a coding sequence for the hydrolysis target M1 protein.

[0034] Preferably, in step (2), the cell line is an artificial cell line that overexpresses TEVp.

[0035] Preferably, in step (2), the influenza virus rescue system comprises a 12-plasmid rescue system of WSN influenza virus.

[0036] Preferably, the preparation method further comprises the step of replicating and producing on a large scale the proteolytic target influenza virus in an artificially modified cell line that overexpresses TEVp.

[0037] In the present application, the PROTAC virus can replicate only in cell lines that overexpress TEVp, and the dependency of the PROTAC virus on cell lines that overexpress TEVp can be utilized to mass-produce influenza virus in cell lines that overexpress TEVp.

[0038] As a preferred technical solution of the present application, the preparation method includes the following steps:

[0039] (1) An expression vector for preparing a protein hydrolysis target influenza virus is constructed, and a plasmid expressing the influenza virus M gene in the 12-plasmid rescue system of WSN influenza virus is modified using genetic engineering methods, and an insertion sequence is introduced into the C-terminus of the gene sequence of the M1 protein and before the stop codon to obtain an expression vector, which contains the coding sequence of the hydrolysis target M1 protein.

[0040] (2) The plasmid expressing the influenza virus M gene in the influenza virus rescue system is replaced with the expression vector obtained in step (1), and the replaced influenza virus rescue system is co-transfected with an artificial cell line overexpressing TEVp to obtain the proteolytic target influenza virus.

[0041] The proteolytically targeted virus is replicated in an artificially engineered cell line that overexpresses TEVp to produce the proteolytically targeted virus on a large scale.

[0042] In a fifth aspect, the present application provides an influenza virus vaccine comprising the proteolytically targeted influenza virus of the first aspect.

[0043] Preferably, the influenza virus vaccine is any one of an attenuated vaccine, a replication-defective live virus vaccine, or a replication-regulatable live virus vaccine.

[0044] In a sixth aspect, the present application provides a use of any one or a combination of at least two of the proteolysis targeted influenza virus according to aspect 1, the nucleic acid molecule according to aspect 2, the recombinant vector according to aspect 3, the method for preparing a proteolysis targeted influenza virus according to aspect 4, or the influenza virus vaccine according to aspect 5, in the preparation of a medicament for treating influenza and / or an oncolytic agent.

[0045] The numerical ranges referred to in this application include not only the recited point values ​​but also any point values ​​between the recited numerical ranges, and for reasons of space and clarity, this application does not exhaustively list specific point values ​​included in the ranges. [Effects of the Invention]

[0046] Compared to the prior art, the present invention has the following beneficial effects:

[0047] (1) This application utilizes the hydrolysis action of the ubiquitin-proteasome system and the cleavage action of TEVp to mass-produce proteolytically targeted influenza viruses in TEVp-overexpressing cell lines, while in normal cell lines, the ubiquitin-proteasome system recognizes the proteolytically targeted molecules fused with viral proteins and degrades the viral proteins, thereby weakening and even completely eliminating the replication ability of the virus. Therefore, the proteolytically targeted influenza viruses are highly safe and are attenuated, replication-defective, and replication-controllable live viruses.

[0048] (2) The method for preparing the proteolytically targeted influenza virus is simple, safe, and reliable. The proteolytically targeted influenza virus in this application is produced by a stable mammalian cell line, overcoming the drawbacks of conventional virus propagation using embryonated chicken eggs (virus propagation using embryonated chicken eggs is prone to cause adverse reactions such as human allergies). The obtained proteolytically targeted influenza virus has high immunogenicity and can be used in the preparation of vaccines and the development of drugs related to the treatment of viral infections, and has strong research and application value.

[0049] (3) The proteolytically targeted influenza virus may be further modified, for example, by introducing an immune enhancer into a specific region or specific amino acid of the viral protein to obtain a virus with improved performance and a proteolytically targeted influenza virus with enhanced immunogenicity.

[0050] (4) The proteolytic targeting influenza virus can activate the tumor microenvironment, transform cold tumors into hot tumors, and increase the efficacy of tumor treatment. [Brief explanation of the drawings]

[0051] [Figure 1] 1 is a schematic diagram of the preparation process of the proteolytic target influenza virus in Example 1. FIG. [Figure 2]Growth curves of proteolytically targeted influenza viruses in MDCK-TEVp (+TEVp) cells and normal MDCK (-TEVp) cells. Each E3 ubiquitin ligase was represented by a single toxin strain, and the data were displayed accordingly. Each toxin strain in the figure is renamed using its corresponding E3 ubiquitin ligase: M1-PTDKLHDC2 (M1-PTD22), M1-PTDKLHDC3 (M1-PTD43), M1-PTDAPPBP2 (M1-PTD71), M1-PTDKLHL20 (M1-PTD84), M1-PTDFBXO31 (M1-PTD91), M1-PTDβ-TrCP (M1-PTD94), M1-PTDSOCS2 (M1-PTD57), M1-PTDFEM1C (M1-PTD63), and M1-PTDITC. H(M1-PTD101), M1-PTDSPOP(M1-PTD109), M1-PTDMDM2(M1-PTD110), M1-PTDCRBN(M1-PTD116), M1-PTDAPC / C-ABBA (M1-PTD205), M1-PTDCBL (M1-PTD119), M1-PTDAPC / C-KENbox (M1-PTD138), M1-PTDCOP1 (M1-PTD148), M1-PTDN-end UBR-box(M1-PTD165), M1-PTDFBW7(M1-PTD174), M1-PTDSkp2-Cks1(M1-PTD183), M1-PTD SIAH-1 (M1-PTD194), M1-PTDAPC / C-TPR1 (M1-PTD143), M1-PTDAPC / C-Dbox (M1-PTD132). [Figure 3] Safety evaluation of proteolytically targeted influenza viruses in a mouse model (*** indicates significant differences compared to WT). Data are shown for representative strains: M1-PTDKLHDC2 (M1-PTD22), M1-PTDKLHDC3 (M1-PTD43), M1-PTDAPPBP2 (M1-PTD71), M1-PTDKLHL20 (M1-PTD84), M1-PTDFBXO31 (M1-PTD91), and M1-PTDβ-TrCP (M1-PTD94). [Figure 4]This is a verification of the mechanism of action of influenza viruses targeted by proteolysis. Each E3 ubiquitin ligase is selected as a representative toxin strain to display the data, so each toxin strain in the figure is renamed using the corresponding E3 ubiquitin ligase. [Figure 5] Evaluation of the immunogenicity of proteolytically targeted influenza virus in a mouse model, where (a) is the level of neutralizing (NT) and hemagglutination-inhibition (HI) antibodies, (b) is the level of anti-HA IgG and anti-NP IgG antibodies, (c) is the level of anti-NP IgA antibodies, (d) is the T cell immune response to influenza virus M1 antigen peptide (MGLIYNRM) in mouse lung tissue, (e) is the level of T cell immune response to influenza virus NP antigen peptide (ASNENMETME) (left) and M1 antigen peptide (MGLIYNRM) (right) in mouse spleen tissue, and (f) is the antigen presentation level of proteolytically targeted influenza virus in macrophage Raw264.7 cells. *, **, *** indicate significant differences compared to wild-type virus (WT), #, ##, and ### indicate significant differences compared to inactivated influenza vaccine (IIV), and +, ++, and +++ indicate significant differences compared to cold-adapted attenuated influenza vaccine (CAIV). Data are shown for representative viruses: M1-PTDKLHDC2 (M1-PTD22), M1-PTDKLHDC3 (M1-PTD43), M1-PTDAPPBP2 (M1-PTD71), M1-PTDKLHL20 (M1-PTD84), M1-PTDFBXO31 (M1-PTD91), and M1-PTDβ-TrCP (M1-PTD94). [Figure 6]The cross-immunoprotective effect of a proteolytic target influenza virus vaccine was evaluated in an adult (7-week-old) mouse model. (a) Wild-type WSN influenza virus titer in lung tissue 3 days after infection; (b) Mouse body weight and survival rate within 2 weeks after infection; (c) Wild-type X-31 influenza virus titer in lung tissue 3 days after infection; (b) Mouse body weight and survival rate within 2 weeks after infection. Data are shown for M1-PTDβ-TrCP (M1-PTD94). [Figure 7] (a) Evaluation of the immunoprotective effect of a proteolysate-targeted influenza virus vaccine in aged (15-month-old) mouse and ferret models, where (a) is the level of neutralizing (NT) antibodies, hemagglutination-inhibition (HI) antibodies, anti-HA IgG antibodies, and anti-NP IgG antibodies induced by the proteolysate-targeted influenza virus vaccine in aged mice; (b) is the level of T cell immune responses to influenza virus NP antigen peptide (ASNENMETME) induced by the proteolysate-targeted influenza virus vaccine in the lung tissue (left panel) and spleen (right panel) of aged mice; (c) is the titer of wild-type WSN influenza virus in lung tissue of immunized and unimmunized aged mice 3 days after infection with wild-type WSN influenza virus; (d) is the body weight and survival rate of immunized and unimmunized aged mice within 2 weeks after infection with wild-type WSN influenza virus; and (e) is the virus titer in nasal wash fluid of immunized and unimmunized ferrets 3 days after infection with wild-type WSN influenza virus. Data are displayed using M1-PTDβ-TrCP (M1-PTD94) as a representative. [Figure 8] This shows the tumor suppressive effect of proteolytically targeted influenza viruses, with data shown using M1-PTDβ-TrCP (M1-PTD94) as a representative. DETAILED DESCRIPTION OF THE INVENTION

[0052] The technical solution of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the above examples are only for understanding the present application and should not be considered as specifically limiting the present application.

[0053] Unless specific techniques or conditions are specified in the examples, they are performed according to techniques or conditions described in the literature in this field or according to the product instructions. Any reagents or equipment used that do not specify the manufacturer are ordinary products that can be purchased through official channels. [Example]

[0054] Example 1 This example provides a series of proteolytically targeted influenza viruses, which contain a hydrolysis targeted M1 protein, and a TEVp recognition site and a proteolytically targeted molecule recognized by the ubiquitin-proteasome system are sequentially inserted into the C-terminus of the hydrolysis targeted M1 protein. The amino acid sequences of the proteolytically targeted molecules recognized by the ubiquitin-proteasome system are as set forth in SEQ ID Nos. 1 to 353. The amino acid sequence of the TEVp recognition site is as set forth in SEQ ID No. 354, the C-terminus of the M1 protein and the TEVp recognition site are connected by flexible linker 1, the amino acid sequence of flexible linker 1 is as set forth in SEQ ID No. 355, the TEVp recognition site and the proteolytic target molecule are connected by flexible linker 2, the amino acid sequence of flexible linker 2 is GSG, and the amino acid sequence structure from the C-terminus to the stop codon of the M1 protein is M1 protein C-terminus-flexible linker 1-TEVp recognition site-flexible linker 2-proteolytic target molecule.

[0055] The amino acid sequence of SEQ ID No. 354 is ENLYFQG.

[0056] The amino acid sequence of SEQ ID No. 355 is GSGG.

[0057] The amino acid sequences of the proteolytic target molecules recognized by the ubiquitin-proteasome system are shown in Table 1.

[0058] [Table 1] TIFF2025506272000003.tif250159 TIFF2025506272000004.tif250156 TIFF2025506272000005.tif250156 TIFF2025506272000006.tif250161 TIFF2025506272000007.tif250157 TIFF2025506272000008.tif250157 TIFF2025506272000009.tif250157 TIFF2025506272000010.tif250157 TIFF2025506272000011.tif250157 TIFF2025506272000012.tif250157 TIFF2025506272000013.tif250157 TIFF2025506272000014.tif250157 TIFF2025506272000015.tif250157 TIFF2025506272000016.tif250157 TIFF2025506272000017.tif250157 TIFF2025506272000018.tif250157 TIFF2025506272000019.tif250157 TIFF2025506272000020.tif250157 TIFF2025506272000021.tif250157 TIFF2025506272000022.tif52169

[0059] The influenza viruses among the proteolytic target influenza viruses are type A, type B, and type C viruses, and the subtypes of the influenza viruses are H1N1, H1N2, H1N3, H1N8, H1N9, H2N2, H2N3, H2N8, H3N1, H3N2, H3N8, H4N2, H4N4, H4N6, H4N8, H5N1, H5N2, H5N3, H5N6, H5N8, H5N9, H6N1, H6N2, H These include 6N4, H6N5, H6N6, H6N8, H7N1, H7N2, H7N3, H7N7, H7N8, H7N9, H8N4, H9N1, H9N2, H9N5, H9N8, H10N3, H10N4, H10N7, H10N8, H10N9, H11N2, H11N6, H11N9, H12N1, H12N3, H12N5, H13N6, H13N8, H14N5, H15N2, H15N8, H16N3, H17N10, and H18N11.

[0060] The preparation process of the proteolytic target influenza virus is shown in FIG. 1. The preparation method of the proteolytic target influenza virus includes the following steps:

[0061] (1) An expression vector was constructed to prepare proteolytically targeted influenza viruses.

[0062] Using a plasmid expressing the influenza virus M gene in the influenza virus rescue system as a template, a gene sequence capable of expressing the amino acid sequence "flexible linker 1 (SEQ ID No. 355: GSGG)-TEVp recognition site (SEQ ID No. 354: ENLYFQG)-flexible linker 2 (GSG)-proteolytic target molecule" was inserted by site-directed mutagenesis into the C-terminus of the gene sequence expressing the M1 protein, before the stop codon. If the amino acid sequence of the proteolytic target molecule is PTD3, the constructed vector is named M1-PTD3; if the amino acid sequence of the proteolytic target molecule is PTD4, the constructed vector is named M1-PTD4. The vector naming convention is as follows. The obtained target vector was subjected to sequencing verification, confirming the successful construction of an expression vector.

[0063] (2) The plasmid expressing the influenza virus M gene in the influenza virus rescue system was replaced with the expression vector obtained in step (1), and the replaced influenza virus rescue system was co-transfected with a cell line to obtain the proteolytic target influenza virus.

[0064] Rescue of WSN influenza virus was performed using a 12-plasmid rescue system for WSN virus. When rescuing the proteolytic target influenza virus, the plasmid for expressing the M gene in the 12-plasmid rescue system was replaced with the expression vector constructed in step (1). The virus strain obtained by rescue was named after the corresponding vector in step (1).

[0065] TEVp-expressing cells were seeded into 6-well plates. The next day, the M1-PTD3 vector from step (1) and the other 11 plasmids in the WSN influenza virus rescue system were co-transfected into a cell line (HEK293T cell line) expressing TEVp protein, with 0.2 μg of each plasmid added to each well of the 6-well plate. Six hours after transfection, the medium was replaced with fresh DMEM containing 0.5% FBS, 1 μg / mL TPCK-trypsin, and bispecific antibodies. Cell lesions were then monitored daily. When 80% lesions were reached, the viral supernatant was collected and the proteolytic target virus, designated M1-PTD3, was isolated.

[0066] Other proteolytic target influenza viruses can be obtained by following the same method and named according to the same rules. The obtained proteolytic target influenza viruses are as shown in Table 2.

[0067] [Table 2] TIFF2025506272000024.tif250157 TIFF2025506272000025.tif250158 TIFF2025506272000026.tif215170

[0068] The resulting viral supernatant was used to infect cell lines expressing the new TEVp protein, and the proteolytically targeted viruses constructed according to the standard were examined for their ability to cause cytopathology.

[0069] If the virus can induce lesions in cell lines expressing TEVp protein (e.g., HEK293T cells expressing TEVp and / or MDCK cells expressing TEVp), this indicates successful rescue of the proteolytic target virus.

[0070] Failure to induce lesions in TEVp-expressing cells (e.g., TEVp-expressing HEK293T cells and / or TEVp-expressing MDCK cells) indicates failure to rescue the proteolytic target virus.

[0071] Results showed that all proteolytically targeted influenza viruses were successfully rescued.

[0072] Test Example 1 The proteolytic target influenza virus was evaluated for preparation efficiency and safety.

[0073] (1) Evaluation of the efficiency and safety of preparation of proteolytically targeted influenza viruses at the cellular level

[0074] We investigated the cytopathic effects of influenza virus strains M1-PTD3 to M1-PTD413 on TEVp-expressing MDCK cell lines (MDCK-TEVp cell lines) and normal MDCK cell lines, and examined the efficiency and safety of the preparation of virulent strains. Wild-type influenza virus was used as a control.

[0075] The standards for determining the safety of toxic strains are as follows:

[0076] Observation and confirmation based on cytopathology: Wild-type influenza virus can cause complete cytopathology (100%) in both MDCK-TEVp cells and MDCK cells. If a virulent strain can cause obvious cytopathology in the MDCK-TEVp cell system (cytopathology reaches 50-100%), it indicates that the preparation efficiency of the virulent strain is high. If a virulent strain cannot cause cytopathology or causes less cytopathology (cytopathology lower than 100% cytopathology caused by wild-type virus) in the normal MDCK cell system compared to the wild-type virus, it indicates that the virulent strain is safe.

[0077] Considerations based on the growth curve: Compared with wild-type viruses, if the toxic strain can replicate highly in the MDCK-TEVp cell line (the virus titer is higher than, equivalent to, or more than one thousandth of the wild-type virus), it means that the preparation efficiency of the toxic strain is high. Compared with wild-type viruses, if the toxic strain has a reduced replication ability or even cannot replicate in the normal MDCK cell line (the virus titer is lower than that of the wild-type virus), it means that the toxic strain is safe.

[0078] (a) The steps for detection by cellular lesions are as follows:

[0079] The prepared proteolytic target influenza virus and wild-type influenza virus were infected into MDCK-TEVp cell lines and normal MDCK cell lines at an MOI of 0.01, respectively, and the lesion status of the cells was observed and recorded daily for 4 days.

[0080] The test results show that in MDCK-TEVp cell line, all proteolytic target influenza viruses and wild-type influenza viruses can cause significant cytopathology, while in normal MDCK cell line, only wild-type influenza viruses can cause significant cytopathology, and the lesions caused by proteolytic target influenza viruses are reduced and even eliminated.These results indicate that the prepared proteolytic target influenza viruses are safe at the cellular level.

[0081] (b) The steps of growth curve detection are as follows:

[0082] The prepared proteolytic target influenza virus and wild-type influenza virus were infected into MDCK-TEVp cell line and normal MDCK cell line, respectively, at an MOI of 0.001. 24, 48, 72, and 96 hours after infection, the cell culture supernatants were collected and analyzed by TCID 50By detecting the virus titer in the supernatant in experiments and plaque experiments, we were able to determine the replication ability of the virus in the two types of cells.

[0083] According to the results in Figure 2, all proteolytically targeted influenza viruses and wild-type influenza viruses had good replication ability in the MDCK-TEVp cell line, while only wild-type influenza viruses showed good replication ability in the normal MDCK cell line, indicating that the replication ability of the proteolytically targeted influenza viruses was reduced and thus defective in replication.These results indicated that the prepared proteolytically targeted influenza viruses were safe at the cellular level.

[0084] (2) Safety evaluation of proteolytic target influenza virus in animals

[0085] The safety of the proteolytically targeted influenza virus was evaluated at the animal level using BALB / c and C57BL / 6J mice. Six proteolytically targeted influenza virus virulence strains (M1-PTD) were used as representatives of the proteolytically targeted influenza virus. KLHDC2 , M1-PTD KLHDC3 , M1-PTD APPBP2 , M1-PTD KLHL20 , M1-PTD FBXO31 , M1-PTD β-TrCP ) were selected and evaluated for viral safety.

[0086] The specific steps for safety evaluation are as follows:

[0087] (a) Eighty 7-week-old female BALB / c or C57BL / 6J mice were divided into eight groups, with 10 mice per group.

[0088] (b) Each mouse in the first group was inoculated with PBS (vehicle) via nasal instillation, and each mouse in the second group was inoculated with 1 × 10 5 TCID 50Wild-type WSN influenza virus (WT) was inoculated via nasal drop in groups 3 to 8, with 1 × 10 5 TCID 50 M1-PTD KLHDC2 , M1-PTD KLHDC3 , M1-PTD APPBP2 , M1-PTD KLHL20 , M1-PTD FBXO31 , or M1-PTD β-TrCP was administered by nasal drop.

[0089] (c) Three days after inoculation, five mice were selected from each group, and their lung tissues were collected to detect the virus titer therein.

[0090] (d) The weight and mortality of the remaining 5 mice in each group were monitored for 14 days.

[0091] The results in Figure 3 show that the wild-type WSN influenza virus replicates highly in the lungs of mice, causing significant weight loss and death in the mice. On the other hand, the proteolytically targeted influenza virus has a weak replication ability in the lungs of mice (below the detection limit), and does not cause weight loss or death in the mice. Therefore, the proteolytically targeted virus vaccine has good safety.

[0092] Test Example 2 The replication ability of proteolytically targeted influenza viruses was examined.

[0093] (1) The expression level of M1 protein of the proteolytic target influenza virus was detected using Western blotting, and five strains of the proteolytic target virus were selected as representative virulent strains to examine the replication ability of the proteolytic target influenza virus in normal cells.

[0094] Normal MDCK cell lines were infected with proteolytically targeted influenza virus and wild-type influenza virus (MOI = 0.1), respectively, and the medium was supplemented with 25 nM, 50 nM, and 100 nM of the proteasome inhibitor MG-132, respectively. DMSO (at the same dilution as the virus) and a mixture of normal MDCK cell lines served as a control. Cell samples were collected 24 h, 48 h, and 72 h after infection, respectively, and the expression levels of the viral M1 protein were detected by Western blotting.

[0095] (2) The expression level of the proteolytic target influenza virus M1 protein was detected by immunofluorescence experiments, and the replication ability of the proteolytic target influenza virus was examined.

[0096] The proteolytically targeted influenza virus and wild-type influenza virus were infected into MDCK-TEVp cell line and normal MDCK cell line (MOI = 0.01), respectively. The medium was supplemented with 0 nM, 25 nM, 50 nM, and 100 nM of the proteasome inhibitor MG-132, respectively. DMSO (at the same dilution as the virus) and a mixture of normal MDCK cell line were used as a control. 48 h after infection, the cells were fixed with 4% PFA, and the expression level of the proteolytically targeted influenza virus M1 protein was detected by immunofluorescence.

[0097] The experimental results shown in Figure 4 indicate that the proteolytically targeted influenza virus can replicate in large quantities and synthesize large amounts of viral protein after infection of MDCK-TEVp cells. Meanwhile, the proteolytically targeted influenza virus cannot replicate in large quantities after infection of normal MDCK cells, and only a small amount of viral protein M1 signal is detected. After the cellular proteasome system is inhibited, the viral protein M1 signal increases, indicating that the viral replication ability is enhanced after the proteasome system is inhibited. The detection results are consistent with the Western blot detection results in Test Example 2, further demonstrating that the introduction of the proteolytically targeted molecule can mediate the degradation of viral proteins by the cellular proteasome, thereby inhibiting the viral replication ability. After the cellular proteasome system is inhibited, the viral replication ability is restored, which is consistent with the design principle of the proteolytically targeted influenza virus.

[0098] Test Example 3 Immunogenicity and protection of proteolytically targeted influenza viruses at the animal level

[0099] The immunogenicity and protective properties of the proteolytically targeted influenza virus were evaluated in animals. Inactivated influenza vaccine (IIV) was used as a control (the inactivated influenza virus vaccine was prepared by the inventors using the same type of influenza virus particles according to the method of the Chinese Pharmacopoeia), and a clinically used cold-adapted attenuated vaccine was used as a control. Six proteolytically targeted influenza virus virulence strains (M1-PTD) were used as representatives of the proteolytically targeted influenza virus. KLHDC2 , M1-PTD KLHDC3 , M1-PTD APPBP2 , M1-PTD KLHL20 , M1-PTD FBXO31 , M1-PTD β-TrCP ) were selected for evaluation of their immunogenicity and protection against proteolytically targeted influenza viruses.

[0100] Specific steps for considering immunogenicity and protection are as follows:

[0101] (1) One hundred and eighty 7-week-old female BALB / c or C57BL / 6J mice were divided into nine groups, with 20 mice per group.

[0102] (2) Each mouse in the first group was inoculated with PBS (vehicle) via nasal instillation, and each mouse in the second group was inoculated with 1 × 10 5 TCID 50 Each mouse in the third group was inoculated with 1 × 10 cold-adapted attenuated influenza vaccine (CAIV) via nasal instillation. 5 TCID 50 Inactivated influenza vaccine (IIV) was administered intramuscularly to groups 4 to 9, with 10 doses each. 5 TCID 50 M1-PTD KLHDC2 , M1-PTD KLHDC3 , M1-PTD APPBP2 , M1-PTD KLHL20 , M1-PTD FBXO31 , or M1-PTD β-TrCP was inoculated.

[0103] (3) One week after inoculation, five mice from each group were selected, and their lung tissues and spleens were collected to detect the immune response of T cells therein.

[0104] (4) Three weeks after inoculation, five mice were selected from each group, and blood samples were collected for hemagglutination inhibition (HI) tests, neutralizing (NT) antibody detection, and ELISA detection, respectively, to detect the antibody immune responses therein.

[0105] (5) Three weeks after inoculation, mice in each group were treated with 2 × 10 5 PFU of the homologous wild-type WSN influenza virus or the heterologous wild-type influenza virus A / X-31 (H3N2) was administered via nasal inoculation.

[0106] (6) Three days after inoculation with wild-type influenza virus, five mice were selected from each group, and their lung tissues were collected to detect the virus titer.

[0107] (7) The weight and mortality of the remaining five mice in each group were monitored for 14 days.

[0108] The results in Figure 5 show that all proteolytically targeted influenza viruses induced high levels of hemagglutination-inhibition (HI) antibody titers, neutralizing (NT) antibody titers, anti-HA IgG, anti-NP IgG, anti-NP IgA, and T cell immune responses in animals. The levels of hemagglutination-inhibition antibodies, neutralizing antibodies, anti-NP IgG, anti-NP IgA, and T cell immune responses were significantly higher than those induced by inactivated influenza vaccines. The immune response levels induced by some virulent strains were also higher than those induced by cold-adapted attenuated vaccines. The results in Figure 6 also show that vaccination with the proteolytically targeted influenza virus vaccine significantly reduced wild-type WSN influenza virus and X-31 (H3N2) virus titers in animal lung tissue and protected the survival of all animals. Therefore, the proteolytically targeted influenza virus vaccine can provide cross-immunoprotection, and the protection provided by the proteolytically targeted influenza virus vaccine is significantly superior to that provided by inactivated influenza vaccines. Therefore, the proteolysate-targeted influenza virus vaccine has superior immunogenicity and protective effect. Furthermore, the results in Figure 7 show that the proteolysate-targeted influenza virus vaccine can exert a high level of immune protection even in aged (15-month-old) mouse and ferret models.

[0109] Test Example 4 The potential of proteolytically targeted influenza virus as an oncolytic virus was discussed.

[0110] The oncolytic effect of the proteolytically targeted influenza virus was evaluated using a C57BL / c melanoma tumor-bearing model.

[0111] The specific test steps are as follows:

[0112] (1) Mice were injected subcutaneously on the back with melanoma tumors and raised for 9 days until the tumor volume reached approximately 100 mm 3 When this occurs, continue the experiment.

[0113] (2) 50 μL of proteolytically targeted influenza virus M1-PTD delivered intratumorally β-TrCP (M1-PTD94) and PBS were injected, one time every other day, for a total of four injections.

[0114] (3) Tumor volume was detected daily.

[0115] As shown in the results in Figure 8, it was found that the proteolytically targeted influenza virus could effectively inhibit the increase in tumor volume, demonstrating that the proteolytically targeted influenza virus has the potential to become an oncolytic virus.

[0116] In summary, the proteolytically targeted influenza virus of the present application can be recognized and hydrolyzed by the ubiquitin-proteasome system, has a weak replication ability, is highly safe, and can be used to prevent influenza as a live vaccine or attenuated vaccine. The proteolytically targeted influenza virus of the present application can be used as an oncolytic virus in tumor therapy. The proteolytically targeted influenza virus of the present application has a wide variety of subtypes, which plays an important role in the research and development of influenza virus vaccines and tumor therapy.

[0117] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto, and the applicant declares that it should be understood that any modifications or replacements that a person skilled in the art can easily conceive within the technical scope disclosed in the present application are all included in the scope of protection and disclosure of the present application.

Claims

1. 1. A proteolytically targeted influenza virus comprising a hydrolysis targeted M1 protein, The hydrolysis target M1 protein is sequentially inserted at its C-terminus with a TEVp recognition site and a proteolytic target molecule recognized by the ubiquitin-proteasome system, and the proteolytic target molecule recognized by the ubiquitin-proteasome system comprises an amino acid sequence represented by SEQ ID No. 1-353; Proteolytic targeting of influenza virus.

2. The TEVp recognition site comprises the amino acid sequence represented by SEQ ID No. 354; The proteolytic targeted influenza virus of claim 1.

3. The C-terminus of the M1 protein and the TEVp recognition site are linked by a flexible linker 1; Preferably, the flexible linker 1 comprises the amino acid sequence shown in SEQ ID No. 355; Preferably, the TEVp recognition site and the proteolytic target molecule are linked by a flexible linker 2; Preferably, the flexible linker 2 comprises the amino acid sequence shown in SEQ ID No. 356; 3. The proteolytic target influenza virus of claim 1 or 2.

4. The proteolytic target influenza virus is a type A, type B, or type C virus; Preferably, the subtypes of the proteolytic target influenza virus are H1N1, H1N2, H1N3, H1N8, H1N9, H2N2, H2N3, H2N8, H3N1, H3N2, H3N8, H4N2, H4N4, H4N6, H4N8, H5N1, H5N2, H5N3, H5N6, H5N8, H5N9, H6N1, H6N2, H6N4, H6N5, H6N6, H6N8, H7N1, H7N2, H7N 3. H7N7, H7N8, H7N9, H8N4, H9N1, H9N2, H9N5, H9N8, H10N3, H10N4, H10N7, H10N8, H10N9, H11N2, H11N6, H11N9, H12N1, H12N3, H12N5, H13N6, H13N8, H14N5, H15N2, H15N8, H16N3, H17N10 or H18N11, or a combination of at least two of these. The proteolytic target influenza virus according to any one of claims 1 to 3.

5. A gene encoding the hydrolysis target M1 protein according to any one of claims 1 to 4, Nucleic acid molecule.

6. comprising at least one copy of the nucleic acid molecule of claim 5, Recombinant vectors.

7. A method for preparing a proteolytic target influenza virus according to any one of claims 1 to 4, comprising the steps of: (1) constructing an expression vector for preparing a proteolytic target influenza virus; and (2) replacing a plasmid expressing the M gene of influenza virus in the influenza virus rescue system with the expression vector obtained in step (1), and co-transfecting the expression vector into a cell line to obtain the proteolysis target influenza virus. Methods for preparing proteolytically targeted influenza viruses.

8. In step (1), the expression vector comprises a coding sequence for the hydrolysis target M1 protein; Preferably, in step (2), the cell line is an artificial cell line overexpressing TEVp; Preferably, in step (2), the influenza virus rescue system comprises a 12-plasmid rescue system of WSN influenza virus; A method for preparing the proteolytic target influenza virus of claim 7.

9. The method further comprises replicating the proteolytic target influenza virus in an artificially modified cell line that overexpresses TEVp and producing it on a large scale. A method for preparing a proteolytic target influenza virus according to claim 7 or 8.

10. An influenza virus vaccine comprising a proteolytic target influenza virus according to any one of claims 1 to 4, Preferably, the influenza virus vaccine is one of an attenuated vaccine, a replication-defective live virus vaccine, or a replication-regulatable live virus vaccine. Influenza virus vaccine.

11. Use of any one or a combination of at least two of the proteolytically targeted influenza virus according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the recombinant vector according to claim 6, the method for preparing the proteolytically targeted influenza virus according to any one of claims 7 to 9 or the influenza virus vaccine according to claim 10 in the preparation of a drug for treating influenza and / or an oncolytic drug.

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