PROTEOLYSIS-TARGETING VIRUS, LIVE VACCINE THEREOF, PREPARATION METHOD AND USE THEREOF

Abstract

Provided is a proteolysis-targeting virus, wherein one or more proteolysis-targeting molecules that can be recognized by the ubiquitin-proteasome system are comprised at one or more different sites of protein thereof, and the viral protein is linked to the proteolysis-targeting molecules by one or more linkers that can be selectively cleaved. Also provided are a nucleic acid molecule encoding the proteolysis-targeting virus, a nucleic acid vector expressing the proteolysis-targeting virus, a preparation method for the proteolysis-targeting virus, methods for the preparation of an attenuated live virus, replication-incompetentlive virus, replication-controllable live virus, and a relevant vaccine and medication for preventing and treating virus infections, a vaccine or pharmaceutical composition comprising the proteolysis-targeting virus, and a system for preparing the proteolysis-targeting virus.

Claims

1. A proteolysis-targeting virus, comprising one or more proteolysis-targeting molecules at one or more different sites of viral protein thereof that can be recognized by the ubiquitin-proteasome system, and the viral protein is linked to the proteolysis-targeting molecule by one or more linkers, wherein the linker can be selectively cleaved.

2. The proteolysis-targeting virus according to claim 1, wherein the site is the C-terminus and/or N-terminus of viral protein; preferably, the proteolysis-targeting molecule is any amino acid sequence selected from those as shown in SEQ ID NO: 1-110; preferably, the linker is a molecule that can be selectively cleaved; more preferably, the linker is an amino acid sequence that can be selectively cleaved; further preferably, the linker is selected from the group consisting of molecules that are selectively cleaved by tobacco etch virus protease, cleavable molecules that are sensitive to thrombin, molecules that are cleavable by coagulation factor Xa, molecules that are cleavable by enterokinase, molecules that are cleavable by 3C protease, molecules or sequences that are cleavable by SUMO protease, molecules that are cleavable by bacterial gelatinase, preferably such as GPLGV, and self-cleavable linker; still further preferably, the self-cleavable linker is a 2A short peptide, and preferably the 2A short peptide is selected from the group consisting of P2A of porcine teschovirus-1, E2A of equine rhinitis A virus, F2A of foot and mouth disease virus, and self-cleavable T2A; preferably, the linker selectively cleaved by tobacco etch virus protease is a sequences as shown in the following general formula I:
E-X.sub.aa-X.sub.aa-Y-X.sub.aa-Q-(G/S/M)   I; more preferably, the linker is any amino acid sequence selected from those as shown in SEQ ID NO: 111-137; preferably, a flexible connector is also comprised between the proteolysis-targeting molecule and the linker; more preferably, the proteolysis-targeting molecule, linker and flexible connector are linked in the following mode: flexible connector-linker-flexible connector-proteolysis-targeting molecule; further preferably, the flexible connector-linker-flexible connector-proteolysis-targeting molecule is any amino acid sequence selected from those as shown in SEQ ID NO: 138-149, 167 and 168.

3. The proteolysis-targeting virus according to claim 1 or 2, wherein the virus is selected from the group consisting of influenza virus, HIV, hand-foot-mouth virus, coxsackievirus, hepatitis C virus HCV, hepatitis B virus HBV, hepatitis A virus, hepatitis D virus, hepatitis E virus, EB virus, human papilloma virus HPV, herpes simplex virus HSV, cytomegalovirus, varicella-zoster virus, vesicular stomatitis virus, respiratory syncytial virus RSV, dengue virus, Ebola virus, Marburg virus, Zika virus, SARS, Middle East respiratory syndrome virus, rotavirus, rabies virus, measles virus, adenovirus, poliovirus, echovirus, encephalitis B virus, forest encephalitis virus, hantavirus, novel enterovirus, rubella virus, mumps virus, parainfluenza virus, blue ear virus, swine fever virus, foot-and-mouth disease virus, microvirus, prion virus, smallpox virus, tobacco mosaic virus, adeno-associated virus, phage, herpes virus, West Nile virus, Norovirus, human boca virus, coronavirus and novel coronavirus SARS-CoV-2; and more preferably, the virus is influenza virus or novel coronavirus SARS-CoV-2; preferably, the virus is a modified virus.

4. The proteolysis-targeting virus according to any of claims 1-3, which is a proteolysis-targeting influenza virus comprising one or more proteolysis-targeting molecules at one or more different sites of viral protein thereof that can be recognized by the ubiquitin-proteasome system, and the viral protein is linked to the proteolysis-targeting molecule by a linker, wherein the linker is E-X.sub.aa-X.sub.aa-Y-X.sub.aa-Q-(G/S/M), which can be specifically recognized and cleaved by tobacco etch virus protease; preferably, the virus is H1N1, H5N1, H7N9, H3N2 or influenza B virus; more preferably, one or more of PA, PB1, PB2, NP, HA, NA, M1, M2, NS1, and NEP proteins of influenza virus comprise a proteolysis-targeting molecule and linker; further preferably, both of PA and PB2 of the influenza virus comprise one or more proteolysis-targeting molecules and linkers; both of PA protein and PB1 protein of the influenza virus comprise one or more proteolysis-targeting molecules and linkers; both of PB2 protein and PB1 protein of the influenza virus comprise one or more proteolysis-targeting molecules and linkers; all of PA protein, PB2 protein, and PB1 protein of the influenza virus comprise one or more proteolysis-targeting molecules and linkers; all of PA protein, PB2 protein, PB1 protein, and M1 protein of the influenza virus comprise one or more proteolysis-targeting molecules and linkers; all of PA protein, PB2 protein, PB1 protein, M1 protein, and NP protein of the influenza virus comprise one or more proteolysis-targeting molecules and linkers; all of PB2 protein, PB1 protein, and M1 protein of the influenza virus comprise one or more proteolysis-targeting molecules and linkers; both of PB1 protein and M1 protein of the influenza virus comprise one or more proteolysis-targeting molecules and linkers; both of PB2 protein and M1 protein of the influenza virus comprise one or more proteolysis-targeting molecules and linkers; all of PB2 protein, PB1 protein, M1 protein, and NS1 protein of the influenza virus comprise one or more proteolysis-targeting molecules and linkers; all of PB2 protein, PB1 protein, M1 protein, and NEP protein of the influenza virus comprise one or more proteolysis-targeting molecules and linkers; or both of NS1 protein and NEP protein of the influenza virus comprises one or more proteolysis-targeting molecules and linkers; preferably, the proteolysis-targeting virus is a proteolysis-targeting coronavirus, comprising one or more proteolysis-targeting molecules at one or more different sites of viral protein thereof that can be recognized by the ubiquitin-proteasome system, and the viral protein is linked to the proteolysis-targeting molecule by a linker, wherein the linker is E-X.sub.aa-X.sub.aa-Y-X.sub.aa-Q-(G/S/M), which can be specifically recognized and cleaved by tobacco etch virus protease; preferably, the virus is novel coronavirus SARS-CoV-2; more preferably, one or more of spike protein, envelope glycoprotein, membrane glycoprotein, nucleocapsid protein, non-structural protein 1, non-structural protein 2, non-structural protein 3, non-structural protein 4, non-structural protein 5, non-structural protein 6, non-structural protein 7, non-structural protein 8, non-structural protein 9, non-structural protein 10, non-structural protein 11, non-structural protein 12, non-structural protein 13, non-structural protein 14, non-structural protein 15, non-structural protein 16, 3a protein, 3b protein, 6 protein, 7a protein, 7b protein, 8a protein, 8b protein, 9b protein, 3C-like proteinase, leader protein, 2′-O-ribose methyltransferase, endonuclease, 3′- to 5′-exonuclease, helicase, RNA-dependent RNA polymerase, orf1a polyprotein, ORF10 protein, ORF8 protein, ORF7a protein, ORF6 protein, and ORF3a protein of the coronavirus comprise one or more proteolysis-targeting molecules and linkers; preferably, the proteolysis-targeting virus is a proteolysis-targeting HIV virus, comprising one or more proteolysis-targeting molecules at one or more different sites of viral protein thereof that can be recognized by the ubiquitin-proteasome system, and the viral protein is linked to the proteolysis-targeting molecule by a linker, wherein the linker is E-X.sub.aa-X.sub.aa-Y-X.sub.aa-Q-(G/S/M), which can be specifically recognized and cleaved by tobacco etch virus protease; preferably, the virus is an HIV virus; still preferably, one or more of Gag polyprotein, pol polyprotein, gp160, HIV trans-activator of transcription, regulator of expression of virion protein, viral negative factor, lentiviral protein R, viral infectivity factor, viral protein U, matrix protein, capsid protein, spacer peptide 1, nucleocapsid protein, spacer peptide 2, P6, reverse transcriptase, ribonuclease H (Rnase H), integrase, HIV protease, gp120, and gp41 protein comprise one or more proteolysis-targeting molecules and linkers.

5. A nucleic acid molecule encoding the proteolysis-targeting virus according to any one of claims 1-4.

6. A nucleic acid vector expressing the proteolysis-targeting virus according to any one of claims 1-4.

7. A method for preparing the proteolysis-targeting virus according to any one of claims 1-4, comprising the steps of: 1) construction of cell line: constructing a cell line that can stably express a protease capable of selective cleaving the linker of the proteolysis-targeting virus; preferably, the cell line is a mammalian cell line; more preferably, the cell line is selected from CHO cells, Vero cells, MDCK.2 cells, HEK293T cells, MDCK cells, A549 cells, BHK cells, BHK-21/BRS cells, Sp2/0 cells, HEK293 cells, 293F cells, HeLa cells, TZM-b1 cells, Sup-T1 cells, MRC-5 cells and VMK cells, LLC-MK2 cells, HCT-8 cells, Huh-7 cells, and Caco2 cells; further preferably, the cell line is selected from HEK293T cell line and MDCK cell line; preferably, the cell line is optionally a ubiquitin-proteasome system-deficient cell line; more preferably, the cell line is a cell line with E3 ligase knockout or knockdown; 2) site selection: determining a viral protein and site into which the proteolysis-targeting molecule and linker are introduced through statistical analysis on the expression distribution of the ubiquitin-proteasome system in a host, and bioinformatic and protein structural prediction of the virus; 3) gene mutation: introducing a nucleotide sequence encoding the proteolysis-targeting molecule and linker into the selected site of encoding gene of determined viral protein, using a genetic engineering method; 4) construction of expression vector: operably linking the encoding nucleotide sequence of genetically mutated viral protein obtained in step 3) to a vector to obtain an expression vector; preferably, the expression vector is a plasmid; 5) cotransfecting the expression vector in step 4) and other expression vector for rescue of influenza virus into the cell line constructed in step 1) capable of selectively cleaving the linker of the proteolysis-targeting virus using reverse genetic technology, to obtain the proteolysis-targeting virus; optionally, 6) replicating the proteolysis-targeting virus in the cell line obtained in step 1) to produce the proteolysis-targeting virus; preferably, a proteasome inhibitor is added during the preparation of the virus; more preferably, the proteasome inhibitor is MG132, MG-341 or lactacystin; or comprising the steps of: (i) site selection: determining a viral protein and site into which the proteolysis-targeting molecule and linker are introduced through statistical analysis on the expression distribution of the ubiquitin-proteasome system in a host, and bioinformatic and protein structural prediction of the virus; (ii) gene mutation: introducing a nucleotide sequence encoding the proteolysis-targeting molecule and linker into the selected site of encoding gene of determined viral protein, using a genetic engineering method; (iii) construction of expression vector for mutated sequence: operably linking the encoding nucleotide sequence of genetically mutated viral protein obtained in step ii) to a vector to obtain an expression vector; preferably, the expression vector is a plasmid; (iv) constructing an overexpression vector of a protease capable of selectively cleaving the linker of the proteolysis-targeting virus; constructing a cell line that can stably express a protease capable of selectively cleaving the linker of the proteolysis-targeting virus; (v) cotransfecting the expression vector obtained in step (iii), other expression vector required for rescue of the virus, and the expression vector obtained in step (iv) into host cells using reverse genetic technology, and culturing the host cells transfected successfully in a culture medium, to obtain the proteolysis-targeting virus.

8. The method according to claim 7, further comprising step 7): detection: determining whether the proteolysis-targeting virus has been successfully modified by measuring the replication capacity of the proteolysis-targeting virus obtained in step 5) in the cell line obtained in step 1) and in normal host cells without being modified, wherein the proteolysis-targeting virus that replicates in the cell line obtained in step 1) and has or no reduced replication capacity in normal host cells without being modified is a successfully modified proteolysis-targeting virus; optionally, the method further comprises step 8): using the successfully modified proteolysis-targeting virus, repeating steps 2)-5), so that the proteolysis-targeting molecule and linker are introduced into multiple viral proteins of the proteolysis-targeting virus, or multiple proteolysis-targeting molecules and linkers are introduced into any viral protein of the proteolysis-targeting virus; optionally, determining whether the proteolysis-targeting virus has been successfully modified by measuring the replication capacity of obtained proteolysis-targeting virus in the cell line obtained in step 1) and in normal host cells without being modified, wherein the proteolysis-targeting virus that replicates in the cell line obtained in step 1) and has reduced or no replication capacity in normal host cells without being modified is a successfully modified proteolysis-targeting virus.

9. The method according to claim 7 or 8, wherein the proteolysis-targeting virus is a proteolysis-targeting influenza virus, comprising the steps of: 1) construction of cell line: stably transducing the tobacco etch virus protease TEVp into a mammalian cell line, and constructing a cell line that can stably express the tobacco etch virus protease TEVp; preferably, the mammalian cell line is HEK293T cell line or MDCK cell line; 2) site selection: determining the gene fragment and site into which the proteolysis-targeting molecule and linker are introduced, through statistical analysis on the expression distribution of the ubiquitin-proteasome system in a host, and bioinformatic and protein structural prediction of influenza virus, predicting and analyzing the protein structure of influenza virus into which a sequence of the proteolysis-targeting molecule and linker that is cleaved by tobacco etch virus protease are introduced; preferably, one or more insertion sites are selected from the gene fragments encoding different proteins of influenza virus; 3) gene mutation: introducing a nucleotide sequence encoding the proteolysis-targeting molecule and linker into the selected site of encoding gene of determined influenza viral protein, using a genetic engineering method; 4) construction of plasmid: operably linking the encoding nucleotide sequence of genetically mutated viral protein obtained in step 3) to a plasmid to obtain an encoding plasmid; 5) cotransfecting the plasmid in step 4) and other plasmid for rescue of influenza virus in the cell line that stably expresses TEVp obtained in step 1) using reverse genetic technology, to obtain the proteolysis-targeting influenza virus; optionally, 6) producing the proteolysis-targeting influenza virus in a stable cell line that stably expresses TEVp; preferably, the method further comprises step 7): detection: determining whether the proteolysis-targeting influenza virus has been successfully modified by determining the dependence of proteolysis-targeting influenza virus obtained in step 5) on TEVp and the dependence of inactivation of its packaged product on the proteasome pathway; optionally, the method further comprises step 8): using the successfully modified proteolysis-targeting influenza virus vector, repeating steps 2)-5), so that the proteolysis-targeting molecule and linker are introduced into each of multiple viral proteins of the proteolysis-targeting influenza virus, or multiple proteolysis-targeting molecules and linkers are introduced into any viral protein of the proteolysis-targeting influenza virus; preferably, determining whether the proteolysis-targeting virus has been successfully constructed by determining the dependence of obtained proteolysis-targeting influenza virus on TEVp and the dependence of inactivation of its packaged product on the proteasome pathway, and retaining the proteolysis-targeting virus that still maintains the dependence on TEVp after a long-term passage as a successfully modified candidate; optionally, the method further comprises: step 9): selecting the successfully modified candidate and purifying the product; step 10): performing a safety or immunogenicity detection on the proteolysis-targeting influenza virus in step 9), and compared with the wild type virus, the safer influenza virus is a successfully modified influenza virus.

10. A method for preparing an attenuated live virus, replication-incompetent live virus, replication-controllable live virus, and for preparing a relevant vaccine and medicament for preventing and treating viral infections, comprising a step of using the proteolysis-targeting virus of any of claims 1-4, or a step of preparing a proteolysis-targeting virus using the method of any of claims 7-9.

11. A vaccine or pharmaceutical composition, comprising the proteolysis-targeting virus according to any one of claims 1-4; preferably, the vaccine is an attenuated live vaccine, replication-incompetent live vaccine, or replication-controllable live vaccine.

12. A system for preparing the proteolysis-targeting virus according to any one of claims 1-4, comprising: a cell line that stably expresses a protease capable of selectively cleaving the linker of the proteolysis-targeting virus; preferably, the cell line is a cell line stably expressing the tobacco etch virus protease TEVp; more preferably, the cell line is HEK293T cell line or MDCK cell line stably expressing the tobacco etch virus protease TEVp; further preferably, the cell line is optionally a ubiquitin-proteasome system-deficient cell line; preferably, the cell line is a cell line with E3 ligase knockout or knockdown; still further preferably, the system further comprises a nucleic acid vector expressing the proteolysis-targeting virus according to any one of claims 1-4.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Hereinafter, the embodiments of the present invention are illustrated in detail in conjunction with the accompanying drawings, wherein:

[0133] FIG. 1 is a map of TEVp overexpression lentiviral vector.

[0134] FIG. 2A is an electrophoretogram of RT-PCR identification of potential HEK293T-TEVp cells (the cells indicated by the arrows are those with a relatively high mRNA expression level of the target gene TEVp); FIG. 2B is an electrophoretogram of RT-PCR identification of potential MDCK-TEVp cells (O (old) represents the first batch of MDCK cells, and N (new) represents the second batch of MDCK cells; and the cells indicated by the arrows are those with a relatively high mRNA expression level of the target gene TEVp).

[0135] FIG. 3 are graphs of protein expression assay of the target protein TEVp and virus packaging efficiency for HEK293T-TEVp and MDCK-TEVp cell lines prepared according to the method of the present invention.

[0136] FIG. 4 is a histogram of replication titers of proteolysis-targeting influenza viruses (PROTAC influenza viruses) prepared according to the method of the present invention 2 days after the infection in an MDCK-TEVp cell line and normal cell line.

[0137] FIG. 5 is a histogram of replication titers of proteolysis-targeting influenza viruses (PROTAC influenza viruses) comprising multiple proteolysis molecules prepared according to the method of the present invention after the infection in an MDCK-TEVp cell line and normal cell line.

[0138] FIG. 6 are histograms comparing the replication capacities of proteolysis-targeting influenza viruses (PROTAC influenza viruses) prepared according to the method of the present invention in an MDCK-TEVp cell and normal cell line.

[0139] FIG. 7 shows an investigation of genetic stability of proteolysis-targeting influenza viruses (PROTAC influenza viruses) prepared according to the method of the present invention, demonstrating that the prepared PROTAC influenza viruses remain stable after multiple passages.

[0140] FIG. 8 is a graph evaluating that the PROTAC influenza viruses according to the present invention can be degraded by the proteasome in normal cells using western blot assay.

[0141] FIG. 9 is a graph of immunofluorescence detection for evaluating the replication of PROTAC influenza viruses according to the present invention in an MDCK-TEVp cell line and normal cells using immunofluorescence assay.

[0142] FIG. 10 shows the immunogenicity and protection results for the PROTAC viruses according to the present invention at an animal level.

[0143] FIG. 11 provides a flow chart of the preparation method for preparing a proteolysis-targeting virus according to the method of the present invention.

BEST MODES FOR CARRYING OUT THE INVENTION

[0144] In order to better understand the invention, the inventors illustrate and set forth specific experiments with examples, wherein the examples are used for illustrative purposes only and do not limit the protection scope of the present invention. Any variants or embodiments equivalent to the invention are included in the present invention.

Example 1 Establishment of Mammalian Stable Cell Lines HEK293-TEVp and MDCK-TEVp that can Stably Express TEVp

[0145] The mammalian cell lines stably expressing TEVp were completed with the assistance of Beijing CorreGene Biotechnology Co. Ltd. through a delegate.

[0146] (1) Construction of TEVp overexpression lentiviral vector:

[0147] a map of TEVp overexpression lentiviral vector is shown in FIG. 1, and the lentiviral vector has puromycin resistance.

[0148] (2) Lentiviral packaging, purification and quantification

[0149] (i) cell inoculation on day 0: 293T was inoculated into 10 cm/15 cm culture dishes (the inoculation number was determined by the desired amount of virus, 1×10{circumflex over ( )}8/15 cm dish), the inoculation density was controlled, and the cells grew to a confluence of 80% the next day;

[0150] (ii) plasmid transfection on day 1: 293T cells were cotransfected using the TEVp overexpression lentiviral vector described in (1), psPAX2 plasmid (which is available from Addgene) and pVSVG plasmid (which is available from Addgene);

[0151] (iii) medium replacement on day 2: the medium was replaced after 18 hours of transfection (the transfection system is toxic), the medium was completely suck off, and 20 mL of fresh complete medium was carefully added (15 cm culture dish);

[0152] (iv) viral supernatant pretreatment: after collecting the viral supernatant on days 3, 4 and 5, the culture supernatant was transferred into a 50 mL centrifuge tube and subjected to centrifugation at the highest speed for 10 min; the viral supernatant was filtered using a 0.45 μm filter and collected directly using a sterilized 250 mL centrifuge bottle;

[0153] (v) virus purification by high-speed centrifugation: by using a 20 mL syringe, 10% sucrose solution was slowly injected into the bottom of centrifuge bottle, a volume ratio of 4:1 (4 parts of virus supernatant, 1 part of sucrose solution) was maintained, and centrifugation was performed at 4° C., 14000 rpm for 2 hours;

[0154] (vi) virus resuspension: the supernatant was discarded and sucked off, and 100 ul-1000 ul PBS was added and mixed uniformly by sucking and blowing;

[0155] (vii) virus supernatant was subpackaged and cryopreserved at −80° C.; about 30 ul of the remaining was cryopreserved separately at the time of subpackage for titer determination;

[0156] (viii) chemiluminescence assay (CMIA) was used to determine the lentivirus titer, and the titer of pLenti-CMV-puro-2A-TEVp-8534 bp was determined as 5.12E+7TU/mL, in a volume of 1000 ul with a total amount of 5.12 E+7TU, which meeted the transduction requirements.

[0157] (3) Construction of stable monoclonal cell line

[0158] (i) cell inoculation at Day 0: HEK293T or MDCK was inoculated into a 12/6-well plate culture dish (the inoculation number was determined by the desired amount of virus, 1×10{circumflex over ( )}6/6-well plate culture dish), the inoculation density was controlled, and the cells grew to a confluence of 20-30% the next day;

[0159] (ii) virus thawing on day 1: the virus was taken from −80° C. refrigerator and thawed on ice; the cells were infected using an appropriate moi (MOI=10); (iii) virus transduction on day 2: the cells were infected using an appropriate moi for 24 hours and then 2 μg/mL puro resistance medium was replaced for resistance screening;

[0160] (iv) continuous resistance screening was performed for about 7 days (blank controls were all killed);

[0161] (v) the surviving cells were digested and counted, and 100/200 cells were distributed equally into a 96-well plate (on average, 1-2 cells per well);

[0162] (vi) clonal proliferation was visible in 3-5 days, the monoclonal cells contained the wells were selected and subjected to proliferation, and step-by-step expanded to 24-well plate and 12-well plate;

[0163] (vii) monoclone identification:

TABLE-US-00005   identification primers TEVp-F SEQ ID NO: 151 TCATTACAAACAAGCACTTG TEVp-R: SEQ ID NO: 152 TAGGCATGCGAATAATTATC Fragment size: 144 bp 293t-GAPDH-eF: SEQ ID NO: 153 CCACATCGCTCAGACACCAT 293t-GAPDH--eR: SEQ ID NO: 154 GGCAACAATATCCACTTTACCAGAGT

[0164] Fragment size: 114 bp

[0165] The preferred monoclonal cells were subjected to digestion, RNA extraction and RT-PCR identification to verify whether the cells integrated and expressed TEVp. The amplification conditions are shown in Table 1 below:

TABLE-US-00006 TABLE 1 Amplification conditions 1 cycle 40 cycles 1 cycle 1 cycle 94° C. 94° C. 55° C. 72° C. 72° C. 25° C. 5 min 15 s 15 s 15 s 5 min ∞

[0166] The results are shown in FIG. 2. FIG. 2A is an electrophoretogram of RT-PCR identification for potential HEK293T-TEVp cells (the cells indicated by the arrows are those with a relatively high mRNA expression level of the target gene TEVp). The verification work was completed with the assistance of Beijing CorreGene Biotechnology Co. Ltd. through a delegation. FIG. 2B is an electrophoretogram of RT-PCR identification of potential MDCK-TEVp cells (O (old) represents the first batch of MDCK cells, and N (new) represents the second batch of MDCK cells; and the cells indicated by the arrows are those with a relatively high mRNA expression level of the target gene TEVp). The verification work was completed with the assistance of Beijing CorreGene Biotechnology Co. Ltd. through a delegation.

[0167] (viii) amplification culture of preferred monoclonal cell lines

[0168] preferably, the cell lines with a high TEVp expression efficiency were expanded and cryopreserved for use, which were designated as HEK293T-TEVp and MDCK-TEVp, respectively.

[0169] The results are shown in FIG. 3, western blot assays were performed on HEK293T-TEVp and MDCK-TEVp cell lines using an anti-TEVp antibody. The results showed that the tobacco etch virus protease TEVp could be efficiently and stably expressed in the HEK293T-TEVp and MDCK-TEVp stable cell lines. Furthermore, the modification of cells did not cause changes of virus production efficiency, indicating the compatibility of TEVp in the cells.

Example 2 Construction of Gene Vector for Influenza Virus WSN Comprising Cleavable Proteolysis-Targeting Molecule

[0170] (1) Acquisition of plasmids for rescuing wild type influenza virus WSN:

[0171] According to the gene sequences of influenza virus A/WSN/1933 as published by pubmed

https://www.ncbi.nlm.nih.gov/nuccore/?term=WSN+PB2;
https://www.ncbi.nlm.nih.gov/nuccore/?term=WSN+PB1;
https://www.ncbi.nlm.nih.gov/nuccore/?term=WSN+PA;
https://www.ncbi.nlm.nih.gov/nuccore/?term=WSN+HA;
https://www.ncbi.nlm.nih.gov/nuccore/?term=WSN+NA;
https://www.ncbi.nlm.nih.gov/nuccore/?term=WSN+NS,
various gene fragments of this influenza virus gene were obtained by total gene synthesis. Then, they were linked to pHH21, pCDNA 3 (neo), and pcAAGGS/MCS vectors (obtained from Beijing Zhongke Yubo Biotechnology Co., Ltd.) respectively, to obtain the plasmids for rescuing wild type influenza virus WSN. The nomenclature and composition of obtained plasmids are shown in Table 2 below.

TABLE-US-00007 TABLE 2 Name of Key Restriction Structure of Abbreviation plasmid gene enzyme site constructed plasmid Ben1 PHH21 PB2 BsmBI pPolI-WSN-PB2 Ben2 PHH21 PB1 BsmBI pPolI-WSN-PB1 Ben3 PHH21 PA BsmBI pPolI-WSN-PA Ben4 PHH21 HA BsmBI pPolI-WSN-HA Ben5 PHH21 NP BsmBI pPolI-WSN-NP Ben6 PHH21 NA BsmBI pPolI-WSN-NA Ben7 PHH21 M BsmBI pPolI-WSN-M Ben8 PHH21 NS BsmBI pPolI-WSN-NS Ben9 pcDNAS(neo) PB2 EcoRI pcDNA3(neo)-PB2 Ben10 pcDNA3(neo) PB1 EcoRI pcDNA3(neo)-PB1 Ben11 pcDNA3(neo) PA EcoRI pcDNA3(neo)-PA Ben13 pcAGGS/MCS NP EcoRI pcAGGS/MCS-NP

[0172] (2) Construction of viral vectors having introduced cleavable proteolysis-targeting molecule

[0173] The inventors introduced a gene sequence of proteolysis-targeting molecule that can be cleaved by TEVp into the C-terminus of gene coding region corresponding to each viral protein (PA, PB2, PB1, NP, HA, NA, M1, M2, NS1, and NEP) of the influenza virus WSN before the termination codon respectively, and constructed the following viral vectors.

[0174] Specifically, the gene sequences of proteolysis-targeting molecules that were introduced to any protein of the virus and can be cut off by TEVp, and the amino acid sequences expressed by them were as follows, but not limited to the following sequences. The gene sequences corresponding to the amino acid sequences used were optimized for humanization and inserted into the C-terminus of the encoding region of the target protein gene before the termination codon. This work was completed with the assistance of Beijing Tsingke Biotechnology Co., Ltd. through a delegation, and the successful construction of mutant was verificated by sequencing, wherein:

[0175] (1) the sequence as shown in SEQ ID NO: 138 was introduced into the C-terminus of PA protein of the influenza virus, i.e., the nucleotide sequence as shown in SEQ ID NO: 155 was introduced into the C-terminus of its PA protein gene encoding region before the termination codon, and the resultant sequence was named as PA-TEVcs+PROTAC-1 (or named as PA-PTD1);

TABLE-US-00008 SEQ ID NO: 138 GSGGENLYFQGGSGALAPYIP; SEQ ID NO: 155 GGTTCTGGTGGTGAGAAT CTGTAC TTC CAA GGTGGATCTGGAGCA TTG GCC CCC TAC ATTCCA;

[0176] 2) similarly, the sequence as shown in SEQ ID NO: 138 was introduced into the C-terminus of M1 protein of the influenza virus, i.e., the nucleotide sequence as shown in SEQ ID NO: 155 was introduced into the C-terminus of its PA protein gene encoding region before the termination codon, and the resultant sequence was named as M1-TEVcs+PROTAC-1 (or named as M1-PTD1);

[0177] (3) the sequence as shown in SEQ ID NO: 139 was introduced into the C-terminus of PB1 protein of the influenza virus, i.e., the nucleotide sequence as shown in SEQ ID NO: 156 was introduced into the C-terminus of its PB1 protein gene encoding region before the termination codon, and the resultant sequence was labeled as PB1-TEVcs+PROTAC-2 (or named as PB1-PTD2):

TABLE-US-00009 SEQ ID NO: 139 GSGGENLYFQGGSGDRHDSGILDSM SEQ ID NO: 156 GGTTGTGGTGGTGAG AAT CTGTAC TTC CAA GGTGGATCTGGAGAT CGC CAC GAT TCA GGG CTC GAT TCC ATG

[0178] 4) the sequence as shown in SEQ ID NO: 139 was introduced into the C-terminus of PB2 protein of the influenza virus, i.e., the nucleotide sequence as shown in SEQ ID NO: 156 was introduced into the C-terminus of its PB2 protein gene encoding region before the termination codon, and the resultant sequence was labeled as PB2-TEVcs+PROTAC-2 (or named as PB2-PTD2);

[0179] 5) the sequence as shown in SEQ ID NO: 139 was introduced into the C-terminus of M1 protein of the influenza virus, i.e., the nucleotide sequence as shown in SEQ ID NO: 156 was introduced into the C-terminus of its M1 protein gene encoding region before the termination codon, and the resultant sequence was labeled as M1-TEVcs+PROTAC-2 (or named as M1-PTD2);

[0180] 6) the sequence as shown in SEQ ID NO: 140 was introduced into the C-terminus of PA protein of the influenza virus, i.e., the nucleotide sequence as shown in SEQ ID NO: 157 was introduced into the C-terminus of its PA protein gene encoding region before the termination codon, and the resultant sequence was named as PA-TEVcs+PROTAC-3 (or named as PA-PTD3);

TABLE-US-00010 SEQ ID NO: 140: GSGGENLYFQGGGGSSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACAS ARINV; SEQ ID NO: 157 GGTTCTGGTGGTGAGAATCTGTACTTCCAAGGTGGAGGAGGATCCAGCCA TGGCTTCCCGCCGGAGGTGGAGGAGCAGGATGATGGCACGCTGCCCATGT CTTGTGCCCAGGAGAGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCT GCTAGGATCAATGTG

[0181] (7) PA-TEVcs+PROTAC-1 (PA-PTD1) and PB1-TEVcs+PROTAC-2 (PB11-PTD2) were combined to construct a virus carrying a cleavable proteolysis molecule in PA and PB1, respectively.

Further, the inventors also constructed the following viral vectors:

TABLE-US-00011 TABLE 3 Viral vectors Viral vector Introduced naming amino acids Introduced nucleotides PA-TEVcs + TEVcs + SEQ ID NO: 141: SEQ ID NO: 158 PROTAC-1 GGTTCTGGTGGTGAGAATCTGTACTFCCAAGGTG GATCTGGAGAAAACCTCTATTTTCAGTCAGGTAG TGGTGCATTGGCCCCCTACATTCCA PB2/PB1-TEVcs + SEQ ID NO: 142 SEQ ID NO: 159 TEVcs + PROTAC-2 GGTTCTGGTGGTGAGAATCTGTACTTCCAAGGTG GATCTGGAGAAAACCTCTATTTTCAGTCAGGTAG TGGTGATCGCCACGATTCAGGGCTCGATTCCATG PA-TEVcs + SEQ ID NO: 143 SEQ ID NO: 160 TEVcs + PROTAC-3 GGTTCTGGTGGTGAGAATCTGTACTTCCAAGGTG GAGGAGGATCCGAAAACCTCTATTTTCAGTCAGG TAGTGGTAGCCATGGCTTCCCGCCGGAGGTGGAG GAGCAGGATGATGGCACGCTGCCCATGTCTTGTG CCCAGGAGAGCGGGATGGACCGTCACCCTGCAGC CTGTGCTTCTGCTAGGATCAATGTG PA-TEVcs + SEQ ID NO: 144 SEQ ID NO: 161 PROTAC-1 + GGTTCTGGTGGTGAGAATCTGTACTTCCAAGGTG PROTAC-1 (or GATCTGGAGCATTGGCCCCCTACATTCCAGGTAG named as TGGTGCCCTTGCACCATATATCCCC PA-PTD4) PB2/PB1-TEVcs + SEQ ID NO: 145 SEQ ID NO: 162 PROTAC-2 + GGTTCTGGTGGTGAGAATCTGTACTTCCAAGGTG PROTAC-1 (or GATCTGGAGATCGCCACGATTCAGGGCTCGATTC named as CATGGGATCTGGAGCATTGGCCCCCTACATTCCA PB2/PB1-P1D5) PA-TEVcs + SEQ ID NO: 146 SEQ ID NO: 163 PROTAC-3 + GGTTCTGGTGGTGAGAATCTGTACTTCCAAGGTG PROTAC-1 GAGGAGGATCCAGCCATGGCTTCCCGCCGGAGGT GGAGGAGCAGGATGATGGCACGCTGCCCATGTCT TGTGCCCAGGAGAGCGGGATGGACCGTCACCCTG CAGCCTGTGCTTCTGCTAGGATCAATGTGGGATC TGGAGCATTGGCCCCCTACATTCCA PA-TEVcs + SEQ ID NO: 147 SEQ ID NO: 164 TEVes + PROTAC-1 + GGTTCTGGTGGTGAGAATCTGTACTTCCAAGGTG PROTAC-1 GATCTGGAGAAAACCTCTATTTTCAGTCAGGTAG (or named as TGGTGCATTGGCCCCCTACATTCCAGGTAGTGGT PA-PTD8) GCCCTTGCACCATATATCCCC PB2/PB1-TEVcs + SEQ ID NO: 148 SEQ ID NO: 165 TEVcs + PROTAC-2 + GGTTCTGGTGGTGAGAATCTGTACTTCCAAGGTG PROTAC-1 GATCTGGAGAAAACCTCTATTTTCAGTCAGGTAG (or named as TGGTGATCGCCACGATTCAGGGCTCGATTCCATG PB2/PB1-PTD9) GGTAGTGGTGCCCTTGCACCATATATCCCC PA-TEVcs + SEQ ID NO: 149 SEQ ID NO: 166 TEVcs + PROTAC-3 + GGTTCTGGTGGTGAGAATCTGTACTTCCAAGGTG PROTAC-1 GAGGAGGATCCGAAAACCTCTATTTTCAGTCAGG (or named as TAGTGGTAGCCATGGCTTCCCGCCGGAGGTGGAG PA-PTD10) GAGCAGGATGATGGCACGCTGCCCATGTCTTGTG CCCAGGAGAGCGGGATGGACCGTCACCCTGCAGC CTGTGCTTCTGCTAGGATCAATGTGGGTAGTGGT GCCCTTGCACCATATATCCCC M1-TEVcs + SEQ ID NO: 167 SEQ ID NO: 169 PROTAC-1 + GSGGENLYFQG GGTTCTGGTGGTGAGAATCTGTACTTCCAAGGTG PROTAC-2 GSGALAPYIPD GATCTGGAGCATTGGCCCCCTACATTCCAGATCG (M1-PTD6) RHDSGLDSM CCACGATTCAGGGCTCGATTCCATG PA-TEVcs + SEQ ID NO: 168 SEQ ID NO: 170 PROTAC-3 + GSGGENLYFQG GGTTCTGGTGGTGAGAATCTGTACTTCCAAGGTG PROTAC-1 GGGSSHGFPPE GAGGAGGATCCAGCCATGGCTTCCCGCCGGAGGT (PA-PTD7) VEEQDDGTLPM GGAGGAGCAGGATGATGGCACGCTGCCCATGTCT SCAQESGMDRH TGTGCCCAGGAGAGCGGGATGGACCGTCACCCTG PAACASARINV CAGCCTGTGCTTCTGCTAGGATCAATGTGGGATC GSGALAPYIP TGGAGCATTGGCCCCCTACATTCCA PB2-TEVcs + PROTAC-1 SEQ ID NO: 138 SEQ ID NO: 155 (named as PB2-PTD1) PBI-TEVcs + PROTAC-1 SEQ ID NO: 138 SEQ ID NO: 155 (named as PB1-PTD1) HA-TEVcs + PROTAC-1 SEQ ID NO: 138 SEQ ID NO: 155 (named as HA-PTD1) NP-TEVcs + PROTAC-1 SEQ ID NO: 138 SEQ ID NO: 155 (named as NP-PTD1) NA-TEVcs + PROTAC-1 SEQ ID NO: 138 SEQ ID NO: 155 (named as NA-PTD1) M2-TEVcs + PROTAC-1 SEQ ID NO: 138 SEQ ID NO: 155 (named as M2-PTD1) NS-TEVcs + PROTAC-1 SEQ ID NO: 138 SEQ ID NO: 155 (named as NS-PTD1) NEP-TEVcs + PROTAC-1 SEQ ID NO: 138 SEQ ID NO: 155 (named as NEP-PTD1) PA-TEVcs + PROTAC-2 SEQ ID NO: 139 SEQ ID NO: 156 (or named as PA-PTD2) HA-TEVcs + PROTAC-2 SEQ ID NO: 139 SEQ ID NO: 156 (or named as HA-PTD2) NP-TEVcs + PROTAC-2 SEQ ID NO: 139 SEQ ID NO: 156 (or named as NP-PTD2) NA-TEVcs + PROTAC-2 SEQ ID NO: 139 SEQ ID NO: 156 (or named as NA-PTD2) M2-TEVcs + PROTAC-2 SEQ ID NO: 139 SEQ ID NO: 156 (or named as M2-PTD2) NS-TEVcs + PROTAC-2 SEQ ID NO: 139 SEQ ID NO: 156 (or named as NS-PTD2) NEP-TEVcs + PROTAC-2 SEQ ID NO: 139 SEQ ID NO: 156 (or named as NEP-PTD2) M1-TEVcs + PROTAC-3 SEQ ID NO: 140 SEQ ID NO: 157 (or named as M1-PTD3) PB2-TEVcs + PROTAC-3 SEQ ID NO: 140 SEQ ID NO: 157 (or named as PB2-PTD3) PB1-TEVcs + PROTAC-3 SEQ ID NO: 140 SEQ ID NO: 157 (or named as PB1-PTD3) HA-TEVcs + PROTAC-3 SEQ ID NO: 140 SEQ ID NO: 157 (or named as HA-PTD3) NP-TEVcs + PROTAC-3 SEQ ID NO: 140 SEQ ID NO: 157 (or named as NP-PTD3) NA-TEVcs + PROTAC-3 SEQ ID NO: 140 SEQ ID NO: 157 (or named as NA-PTD3) M2-TEVcs + PROTAC-3 SEQ ID NO: 140 SEQ ID NO: 157 (or named as M2-PTD3) NS-TEVcs + PROTAC-3  SEQ ID NO: 140 SEQ ID NO: 157 (or named as NS-PTD3) NEP-TEVcs + PROTAC-3 SEQ ID NO: 140 SEQ ID NO: 157 (or named NEP-PTD3) M1-TEVcs + PROTAC-2 + SEQ ID NO: 145 SEQ ID NO: 162 PROTAC-1 (or named as M1-PTD5)

Example 3: Rescue of PROTAC Influenza Viruses Modified by Site-Directed Mutation

[0182] 12 plasmids used for rescue of influenza virus were cotransfected into a stable cell line according to a normal method for rescue of influenza virus, and the corresponding plasmids of these 12 plasmids were replaced with the plasmids modified by site-directed mutation in Example 2. For each well of six-well plate, 0.2 μg of each plasmid was added. After transfection, the cells were observed for cytopathic alteration and screened for insertion sites, proteolysis-targeting molecules, linkers cleavable by TEVp, and combinations thereof that could rescue the virus and were TEVp-dependent. The screened strains were named according to the protein and the introduced cleavable proteolysis-targeting molecule.

[0183] For illustration, after introducing the cleavable proteolysis-targeting molecule TEVcs+PROTAC-1 into Ben3 pPolI-WSN-PA plasmid, this plasmid and the other plasmid Ben1 pPolI-WSN-PB2, Ben2 pPolI-WSN-PB1, Ben4 pPolI-WSN-HA, Ben5 pPolI-WSN-NP, Ben6 pPolI-WSN-NA, Ben7 pPolI-WSN-M, Ben8 pPolI-WSN-NS, Ben9 pcDNA 3 (neo)-PB2, Ben10 pcDNA 3 (neo)-PB1, Ben11 pcDNA 3 (neo)-PA, or Ben13 pcAGGS/MCS-NP that rescues influenza virus were cotransfected into the stable cell line established in Example 1, thereby rescuing a mutant influenza virus with TEVCs+PROTAC-1 introduced into the PA gene fragment of influenza virus, which was named as PA-TEVcs+PROTAC-1 (or named as PA-PTD1). After the cleavable proteolysis-targeting molecule TEVcs+PROTAC-1 was introduced into the C-terminus of M1 protein encoding region in the Ben7 pPolI-WSN-M plasmid, the plasmid and the other plasmid Ben1 pPolI-WSN-PB2, Ben2 pPolI-WSN-PB1, Ben3 pPolI-WSN-PA, Ben4 pPolI-WSN-HA, Ben5 pPolI-WSN-NP, Ben6 pPolI-WSN-NA, Ben8 pPolI-WSN-NS, Ben9 pcDNA 3 (neo)-PB2, Ben10 pcDNA 3 (neo)-PB1, Ben11 pcDNA 3 (neo)-PA, or Ben13 pcAGGS/MCS-NP that rescues influenza virus were cotransfected into the stable cell line established in Example 1, thereby rescuing a mutant influenza virus with TEVCs+PROTAC-1 introduced into the M1 gene fragment of influenza virus, which was named as M1-TEVcs+PROTAC-1 (or named as M1-PTD1).

[0184] Following the same approach, mutant influenza viruses with cleavable proteolysis-targeting molecule introduced into other sites can be obtained and named according to the same rules.

[0185] All the constructed PROTAC virus vectors were investigated according to the criterion whether they could cause cytopathic alteration: if they could cause cytopathic alteration in HEK293T-TEVp and/or MDCK-TEVp, it demonstrated that the rescue of PROTAC virus was successful; if they fail to cause cytopathic alteration in HEK293T-TEVp and/or MDCK-TEVp, it demonstrated that the rescue of PROTAC virus failed. The examples of rescue of partial mutant influenza viruses can be seen in Table 4 and FIG. 4. A portion of PROTAC influenza strains that can cause cytopathic alteration in MDCK-TEVp cells are summarized in Table 4. FIG. 4 shows the detected virus titers of cell supernatants the next day after infection after a portion of strains were inoculated into MDCK-TEVp cells and normal MDCK cells (MOI=0.01). The results indicate that selectively cleavable proteolysis-targeting molecule can be introduced into each of eight gene fragments of influenza virus, wherein the PROTAC influenza virus can be rescued in terms of PA, PB2, PB1, M1, and etc.

TABLE-US-00012 TABLE 4 Partial strains of PROTAC influenza virus causing cytopathic alteration in MDCK-TEVp cells Whether it causes Second name cytopathic Name of strain of strain alteration? PA-TEVcs + PROTAC-1 PA-PTD1 yes M1-TEVcs + PROTAC-1 M1-PTD1 yes PB2-TEVcs + PROTAC-2 PB2-PTD2 yes PB1-TEVcs + PROTAC-2 PB1-PTD2 yes M1-TEVcs + PROTAC-2 M1-PTD2 yes PA-TEVcs + PROTAC-3 PA-PTD3 yes PA-TEVcs + PROTAC-1 + PA-PTD4 yes PROTAC-1 M1-TEVcs + PROTAC-2 + M1-PTD5 yes PROTAC-1 M1-TEVcs + PROTAC-1 + M1-PTD6 yes PROTAC-2 PA-TEVcs + TEVcs + PA-PTD8 yes PROTAC-1 + PROTAC-1 PA-TEVcs + PROTAC-1 + PTOTAC-V1 yes PB1-TEVcs + PROTAC-2

[0186] From the above-mentioned mutation sites and cleavable proteolysis-targeting molecules, the inventors selected the mutational modifications that were highly efficient in rescuing influenza virus, genetically stable, and resulted in a significantly reduction or even complete loss of eventual replicate capacity in normal host cells, and combined them to prepare influenza viruses comprising multiple proteolysis-targeting molecules, further selected preferred strains therefrom, and named as M1-TEVcs+PROTAC-1+PROTAC-2 (or M1-PTD6), M1-TEVcs+PROTAC-2+PROTAC-1 (or M1-PTD5), and PROTAC-V1 (Table 4 and FIG. 5). M1-TEVcs+PROTAC-1+PROTAC-2 is a PROTAC strain comprising two proteolysis-targeting molecules, which was prepared by introducing PROTAC-1 and PROTAC-2 in tandem into the C-terminus of M1 protein of the virus. M1-TEVcs+PROTAC-2+PROTAC-1 is a PROTAC strain comprising two proteolysis-targeting molecules, which was prepared by introducing PROTAC-2 and PROTAC-1 in tandem into the C-terminus of M1 protein of the virus. PROTAC-V1 is a PROTAC strain comprising two proteolysis-targeting molecules, which was prepared by introducing TEVcs-PROTAC-1 into the C-terminus of PA protein of the virus and TEVcs-PROTAC-2 into the C-terminus of PB1 protein of the virus (Table 4). Further, FIG. 11 provides a flow chart of the preparation of proteolysis-targeting virus according to the method of the present invention.

Example 4 Expression and Purification of Influenza Viruses with Site-Directed Mutation

[0187] 1) Rescue of PROTAC viruses comprising a cleavable proteolysis-targeting molecule

[0188] The packaging plasmids of mutant influenza virus obtained in the rescue of influenza virus modified by site-directed mutation in the step of Example 3 were cotransfected into the stable cell line in Example 1, the medium was replaced with a new medium containing 1% FBS and 2 μg/mL of TPCK-trypsin after 6 hours, and normal cells were used as a control. The positive control used for this rescue experiment was wild type influenza virus WSN, and the conditions were the same as those for the rescue of mutant influenza virus, except that the plasmids for rescue of the virus were different. After the transfection was completed, the status of the cells was observed daily, and cytopathic alteration appeared in the TEVp stable cell line, while in normal cells the mutants showing no or fewer cytopathic alteration were positive mutants. However, wild type influenza virus showed cytopathic alteration in both the TEVp stable cell line and normal cell line.

[0189] 2) Purification of PROTAC influenza virus

[0190] a. When the stable cell line for rescue of mutant PROTAC influenza virus in step 1) became completely cytopathic, or about 4 days after the transfection, the cell supernatant was collected and centrifuged at 5000 g for 10 min. A massive amplification was performed with a new stable cell line, and when the cells became completely cytopathic or about 4 days after the amplification, the cell supernatant was collected and passed through a 0.45 μm filter membrane.

[0191] b. The influenza virus was purified using a method of sucrose gradient density centrifugation. The specific steps were as follows: centrifuging the viral solution in 1) in a 50 mL centrifuge tube (specific for a high speed) at 105 g for 2 h, and resuspending the precipitate with 1 mL PBS.

[0192] c. Sucrose was dissolved with NTE Buffer (100 mM NaCl, 10 mM Tris-Cl, pH 7.4, 1 mM EDTA) to formulate a 20% sucrose solution, which was passed through a 0.45 μm filter membrane.

[0193] d. Sucrose from step 3) was added into a 50 mL or 15 mL centrifuge tube, and the PBS resuspension from 2) was dropped into the sucrose solution, and centrifuged at 11×10.sup.4 g for 2 h.

[0194] e. The precipitate is added with about 15 mL NTE buffer, and centrifugation is continued at 11×10.sup.4 g for 2 h.

[0195] f. The precipitate from step 5) was resuspended with PBS.

Example 5 Investigation on Safety of Proteolysis-Targeting Influenza Viruses at a Cell Level

[0196] The inventors confirmed the safety of PROTAC viruses by investigating the TEVp protein dependence of the prepared mutant PROTAC influenza viruses M1-TEVcs+PROTAC-1, M1-TEVcs+PROTAC-2, M1-TEVcs+PROTAC-1+PROTAC-2, and M1-TEVcs+PROTAC-2+PROTAC-1, and conducted a long-term passaging culture to investigate the stability of the cleavable proteolysis-targeting molecules in the mutant viruses.

[0197] Specific experiment 1: the prepared mutant PROTAC influenza virus was used to infect MDCK-TEVp cells and normal MDCK cells at a ratio of MOI=0.01, and the supernatant was taken after 3-4 days to measure the titers of the virus. The virus titer of PROTAC virus in MDCK-TEVp cells was set as 100%, and a relative virus titer of PROTAC virus of MDCK-TEVp cells versus normal MDCK cells was determined, so that the difference of replication capacity of the virus in the two types of cells can be known.

[0198] Specific experiment 2: freshly prepared mutant PROTAC influenza virus was inoculated in a new medium at MOI=0.01 and infected a stable cell line, the new medium contained 1% FBS and 2 μg/mL of TPCK-trypsin, and a normal cell line was used as a control. When the TEVp stable cell line became cytopathic completely, the supernatant was taken and passed through a 0.45 μm filter membrane, and then inoculated into a new medium at a ratio of MOI=0.01 and infected a stable cell line. Similarly, a normal cell line was used as a control. This procedure was repeated for a long-term virus passage. The introduced proteolysis-targeting molecule was tested for mutagenesis by gene sequencing.

[0199] As can be seen from FIG. 6, PROTAC virus can replicate and proliferate in MDCK-TEVp cells, while its replication capacity was significantly reduced or even disappeared in normal MDCK cells, i.e., it has a dependence on TEVp, indicating that the virus is safe. As can be seen from FIG. 7, the proteolysis-targeting molecule introduced into the mutant PROTAC virus strain was not mutated after a long-period passage. This indicates that the cleavable proteolysis-targeting molecule introduced into an influenza virus gene is stably present, and in turn that it is genetically stable.

Example 6 Investigation of Whether Diminishment of Replication Capacity of Proteolysis-Targeting Influenza Viruses is Regulated by Proteasome in Cells

[0200] Using M1-TEVcs+PROTAC-1 and M1-TEVcs+PROTAC-2 as representative strains, the inventors investigated whether the diminishment of replication capacity of the designed PROTAC influenza virus in normal cells was mediated by the intracellular ubiquitin-proteasome system.

[0201] Specific experiment 1: the prepared mutant PROTAC influenza virus or wild type virus was used to infect normal MDCK cells (MOI=0.1), with a medium supplemented with 100 nM proteasome inhibitor MG-132 or DMSO diluted at the same ratio as a control. Cell samples were collected at 24 h and 48 h after the infection, respectively, and viral M1 protein levels were detected by western blot.

[0202] Specific experiment 2: the prepared mutant PROTAC influenza virus or wild type virus was used to infect MDCK-TEVp cells and normal MDCK cells (MOI=0.01), with a medium supplemented with different concentrations (0, 50, or 100 nM) of the proteasome inhibitor MG-132 or DMSO diluted at the same ratio as a control. At 48 h after the infection, the cells were fixed with 4% PFA, and the viral NP protein levels were detected by immunofluorescence assay.

[0203] As can be seen from FIG. 8, wild type virus can replicate in a large quantity and produce a large amount of viral proteins after infecting MDCK cells. In contrast, after the viral protein M1 of PROTAC virus was modified with the proteolysis-targeting molecule TEVcs+PROTAC-1 or TEVcs+PROTAC-2, the viral M1 protein was degraded after 48 hours post-infection. The proteasome-mediated degradation of viral protein M1 was inhibited after the addition of the proteasome inhibitor MG-132 into the medium. The results of this experiment indicate that the proteolysis-targeting molecule introduced by the inventors can mediate the degradation of viral protein, and the reduction of replication capacity of PROTAC virus in normal cells is caused by proteasome-mediated degradation of viral protein, which are in accordance with the principles of the present invention.

[0204] As can be seen from the results of immunofluorescence experiments in FIG. 9, PROTAC viruses can replicate in a large quantity and a large amount of viral proteins were synthesized after infecting MDCK-TEVp cells. However, PROTAC viruses could not replicate in a large quantity after infecting normal MDCK cells, so less signals of viral protein NP were detected, while after the proteasome system in the cells was inhibited, the signals of viral protein NP increased, indicating that after the proteasome system was inhibited, the replication capacity of the viruses was enhanced. These results are consistent with those in FIG. 8, further demonstrating that the introduction of proteolysis-targeting molecule will mediate the degradation of viral proteins by the proteasome in cells, which in turn inhibit the replication capacity of the virus, and after the proteasome system in cell is inhibited, the replication capacity of the virus will be restored.

[0205] The above results demonstrate that the reduction or defect in the replication capacity of PROTAC virus is mediated by the ubiquitin-proteasome system in cells, and this is in accordance with the inventors' design expectations for PROTAC virus.

Example 7 Investigation of Immunogenicity and Protection of Proteolysis-Targeting Influenza Virus (PROTAC Influenza Virus) at an Animal Level

[0206] The immunogenicity and efficacy of PROTAC virus at an animal level was evaluated in this study using ferret (provided by Cay Ferret Farm, Wuxi, Jiangsu, China). With an inactivated influenza vaccine (IIV) as a control (the inactivated influenza virus vaccine was prepared by the inventors using homologous influenza virus particles according to the method provided by the Chinese Pharmacopoeia), M1-TEVcs+PROTAC-1 was selected as a representative of PROTAC virus for the evaluation of immunogenicity and protection of PROTAC virus.

[0207] Specific Experiment:

[0208] 1) Nine female ferrets aged 4-6 months were divided into three groups with three animals in each group.

[0209] 2) Virus vaccination: the first group was vaccinated intranasally with PBS, the second group was vaccinated intranasally with 10.sup.5 PFU M1-TEVcs-PROTAC-1, and the third group was vaccinated with the same dose of inactivated virus.

[0210] 3) Three weeks after the vaccination, serum was collected from each group for hemagglutination inhibition (HI) test and neutralizing (NT) antibody detection.

[0211] 4) Three weeks after the vaccination, each group of animals were vaccinated intranasally with 107 PFU of wild type virus WSN.

[0212] 5) Three days after the vaccination with wild type virus, lung tissue was taken and the virus content therein was measured by plaque assay.

[0213] The results are shown in FIG. 10. PROTAC virus could induce high levels of hemagglutination-inhibiting antibody titer (A) and neutralizing antibody titer (B) in the animals. The levels of hemagglutination-inhibiting and neutralizing antibodies induced by PROTAC virus were significantly higher than those induced by the inactivated vaccine. PROTAC virus vaccination significantly reduced the titer of wild type virus (C) in the lung tissues of the animals, and PROTAC virus vaccine provided a significantly better protection than that provided by the inactivated vaccine.

[0214] The above description is only some embodiments of the present invention. For a person of ordinary skill in the art, several variations and modifications can be made without departing from the inventive concept of the present invention, and all these fall within the protection scope of the present invention.

REFERENCES

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