Proteolytic target virus, live vaccine therefor, and method for producing same and use thereof

Abstract

The present invention provides a proteolytically targeted virus in which one or more distinct protein sites contain one or more proteolytic target molecules recognizable by the ubiquitin-proteasome system, and the viral protein and the proteolytic target molecule are linked via one or more selectively cleavable linker chains. The present invention also provides a nucleic acid molecule encoding the proteolytically targeted virus, a nucleic acid vector expressing the proteolytically targeted virus, a method for producing the proteolytically targeted virus, methods for producing live attenuated vaccines, replication-incompetent live vaccines, and replication-controllable live vaccines, methods for producing related vaccines and drugs for preventing and treating viral infections, vaccines or pharmaceutical compositions containing the proteolytically targeted virus, and a system for producing the proteolytically targeted virus.

Description

[Technical field]

[0001] The present invention is in the field of biotechnology. In particular, the present invention relates to a proteolytic targeting virus and its application. The present invention further provides a method for producing said virus. [Background technology]

[0002] Influenza is an acute respiratory infection caused by influenza viruses, which can seasonally infect birds, mammals, and humans. Influenza viruses can also be divided into three types, A, B, and C, of ​​which influenza A (also called type A) viruses are the most frequent outbreaks. Type A influenza viruses belong to the Orthomyxoviridae family, and their genomes consist of a total of eight independent single-stranded RNA fragments, encoding ten proteins (hemagglutinin protein (HA), matrix protein (M) divided into M1 and M2, neuraminidase (NA), nucleocapsid protein (NP), nonstructural proteins (NS) including NS1 and NEP, and three polymerases, PB1, PB2, and PA). The proteins of each type have important biological functions for influenza viruses. Type A influenza viruses are classified into different subtypes according to the antigenic differences of their major surface antigens, HA and NA. At present, 18 subtypes of HA proteins and 11 subtypes of NA proteins have been discovered. Large-scale epidemics of influenza A virus can cause extremely high morbidity and mortality rates, and are a serious threat to human health (WHO 2003; Coleman 2007). In the 20th century, influenza A virus caused three major influenza epidemics, namely H1N1 in 1918, H2N2 in 1957, and H3N2 in 1968, which killed approximately 50 million people in total (Kilbourne 2006; Taubenberger, Hultin et al. 2007). The 2009 influenza A virus was also caused by the H1N1 influenza virus (Dawood, Jain et al. 2009; Zimmer and Burke 2009), and its rapid spread has attracted worldwide attention. According to statistics, the number of deaths from influenza in the world was an average of 300,000 to 500,000 per year (Fiore, Shay et al. 2007).

[0003] Since the discovery of influenza, scientists have been devoting themselves to the prevention and control of influenza viruses. Vaccination is currently the most advantageous means for preventing influenza and controlling its spread. In the late 1930s, influenza virus vaccines began to be used in humans. Current influenza virus vaccines can be divided into inactivated virus vaccines, attenuated virus vaccines, DNA vaccines, subunit vaccines, recombinant virus vector vaccines and virus-like particle vaccines.

[0004] The currently used inactivated influenza vaccines are trivalent vaccines, including H1N1, H3N2 and influenza B virus vaccines. In clinical use for many years, the influenza inactivated vaccine has very good immune effect and safety, and can stimulate the human body to produce appropriate antibodies after vaccination, but the disadvantage is that it cannot stimulate the production of secretory immunoglobulin A (sIgA). In addition, influenza viruses induce antigenic mutations when they are passaged in chicken embryos, and most chickens carry various viruses in their bodies, so the vaccine may be contaminated by these viruses. In order to be able to produce large quantities of vaccines, the newly epidemic antigenic mutant strains must be efficiently replicated in chicken embryos. As the wild-type virus used to make the vaccine grows in eggs, its immunogenicity will change or decrease to a certain extent. If the produced vaccine strain does not match the current epidemic strain, it will lose its immune protective effect. In recent years, one important development has been the use of mammalian cells instead of chicken embryo culture. Mammalian cells are mainly MDCK cells and Vero cells, which have the advantages of no foreign factor contamination, easy large-scale production, and antigen stability. Influenza vaccines cultured by mammalian cells have relatively good immunogenicity, mild side effects after vaccination, and are relatively safe. Although good experimental results have been obtained in the research stage, some problems have yet to be solved, and they have not yet been used in clinical practice.

[0005] There are five main types of attenuated live vaccines: temperature-sensitive vaccines, reassortant vaccines, cold-adapted attenuated live influenza vaccines, reverse genetics vaccines, and replication-deficient influenza vaccines. Currently, the cold-adapted attenuated live influenza vaccine has been successfully developed. This vaccine is an influenza virulence strain that can grow at the optimal adaptation temperature with reduced toxicity. Since this virus can only replicate at about 25°C and cannot be passaged at 37°C, its infection is limited to the upper respiratory tract and there are no obvious influenza symptoms clinically. By performing genetic recombination using an attenuated strain and a current epidemic virulence strain, a recombinant virus with attenuated strain fragments and the HA and NA genes of the epidemic strain is obtained. Many studies have proven that the biological properties of the cold-adapted virulence strain have very good stability. The antibody seroconversion rates after immunization with the attenuated influenza vaccine and the inactivated vaccine are both 50%-70%, which can effectively suppress influenza epidemics. The cold-adapted live attenuated vaccine is used in Russia and is expected to be approved for use in the United States. It has certain advantages over inactivated vaccines in terms of the route of administration and immune effect, for example, it can be immunized through nasal spray or infusion. Cold-adapted live attenuated vaccines can replicate in the upper respiratory tract, induce mucosal sIgA and systemic humoral and cellular immune responses, and produce broader and longer-lasting protection than inactivated vaccines. However, cold-adapted live attenuated vaccines can obtain virulent reassortant strain viruses after genetic reassortment with other influenza viruses, and interference phenomena may occur in bivalent or trivalent cold-adapted live attenuated vaccines.

[0006] An ideal vaccine should have the following characteristics: (1) strong immunogenicity, (2) minimal toxic reactions, (3) genetic stability, and (4) the ability to rapidly acquire antigenicity consistent with epidemic strains during epidemic periods. In view of the advantages of live vaccines over inactivated vaccines and the safety considerations of live vaccines, the production of novel, replication-controllable, genetically stable, safe and effective live influenza virus vaccines using mammalian cells and the improvement of vaccine safety and efficacy will promote the rapid development of influenza vaccines.

[0007] Ubiquitin-proteasome system Protein degradation is crucial to maintain normal cell functions (e.g., proliferation, differentiation, death). The main pathways for protein degradation in eukaryotic cells are the lysosomal pathway, the hydrolytic system of specialized cell apparatus, the cell membrane surface hydrolytic system, the caspase protease system, and the highly conserved ubiquitin-proteasome pathway (UPP). The ubiquitin-proteasome pathway is a proteolytic pathway that depends on ATP in the cytoplasm and cell nucleus, and is different from the lysosomal pathway. It can degrade proteins produced by cells under stress and non-stress conditions, especially functional proteins with short half-lives, oncogene products, denatured proteins, and allosteric proteins, with high efficiency and selectivity, and can also degrade intracellular structural proteins under stress conditions. The discovery of this proteolytic pathway is considered a turning point in the research of cell cycle and cell transformation, and is a very important regulator of cell function.

[0008] The ubiquitin-proteasome system consists of ubiquitin, ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), ubiquitin-protein ligases (E3), and the proteasome. Ubiquitin is a highly conserved small protein consisting of 76 amino acids with a molecular weight of 8.45 kDa. It is widely distributed from the cell nucleus to the cytoplasm, from germ cells to various somatic cells, and is highly expressed in actively proliferating tumor cells and germ cells. To facilitate recognition and hydrolysis of target proteins by the proteolytic enzyme complex, the glycine (Gly) carboxyl group at the C-terminus of ubiquitin is linked to the lysine (Lys) α- or ε-amino groups of the target protein to be degraded, with the cooperation of ubiquitin ligase, to form polyubiquitin chains. Ubiquitin-activating enzyme (E1) catalyzes the conjugation of ubiquitin to protein substrates. When the ubiquitin pathway is initiated, with the assistance of ATP, the C-terminal glycine residue of ubiquitin is conjugated to the active site cysteine ​​(Cys) of ubiquitin-conjugating enzyme (E2), and then ubiquitin is activated. After ubiquitin is activated, ubiquitin-conjugating enzyme (E2) transfers the activated ubiquitin to the active site cysteine ​​residue of ubiquitin-conjugating enzyme. Ubiquitin protein ligase (E3) plays an important role in determining the selectivity of substrate proteolysis mediated by ubiquitin. E3 must have affinity for the enzyme substrate and bind to E2 to transfer ubiquitin from E2 to the target protein. After ubiquitin is conjugated to the enzyme substrate, a single polyubiquitin chain is generally formed. In this chain, the C-terminus of each ubiquitin monomer is bound to a specific lysine residue of the previous ubiquitin. The proteasome is the key enzyme that catalyzes the degradation of ubiquitin-substrate protein conjugates, and it includes the 20S proteasome and the 26S proteasome. The 20S α subunit is mainly used for substrate recognition, and the β subunit is mainly used for substrate degradation.The action of the 26S proteasome may involve the production of free peptides, free ubiquitin, or reusable ubiquitin. Therefore, the action of the ubiquitin-proteasome system consists of two consecutive steps: (1) covalent binding of ubiquitin molecules to target proteins, and (2) degradation of polyubiquitinated proteins by the 26S proteasome with simultaneous reactivation of ubiquitin. This process requires the cooperation of three enzymes, E1, E2, and E3, and penetrates the entire plasma membrane system of the cell, including the plasma membrane, endoplasmic reticulum, and nuclear membrane. The ubiquitin-proteasome pathway is an important protein regulation system in eukaryotic cells, and participates in various cellular physiological processes, such as the regulation of cell cycle processes, cell proliferation and differentiation, and signal transduction, so that protein ubiquitination and degradation in cells is an important post-transcriptional modification method for proteins.

[0009] In summary, cells in an organism strive to maintain adequate protein levels, producing and degrading thousands of proteins at any given time. A key factor in maintaining protein balance is a small protein called ubiquitin. When ubiquitin is linked to proteins, these proteins are delivered to the proteasome for degradation. Using this principle, researchers have developed protein degraders based on the Proteolysis-Targeting Chimeras (PROTACs) technology. That is, researchers have designed small molecules with two active ends, one of which can bind to a target protein and the other to a protein called E3 ubiquitin ligase. This double-function small molecule can force the binding of ubiquitin to the target protein and deliver it to the cell's garbage disposal station. Even after a protein has been degraded, it can continue to target other proteins, thereby quickly reducing the levels of unwanted proteins.

[0010] However, there are currently no reports of using this technology to modify viruses. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Kathleen M. Sakamoto et al. Protacs:Chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. PNAS(2001). [Non-Patent Document 2] Ashley R. Schneekloth et al. Targeted intracellular protein degradation induced by a small molecule: En route to chemical proteomics.Bioorganic & Medicinal Chemistry Letters(2008). [Non-Patent Document 3] Dennis L. Buckley et al. Small‐Molecule Inhibitors of the Interaction between the E3 Ligase VHL and HIF1α. Angewandte Chemie International Edition(2012). [Non-Patent Document 4] Daniel P Bondeson et al. Catalytic in vivo protein knockdown by small-molecule PROTACs. Nature Chemical Biology (2015). [Non-Patent Document 5] George E. Winter et al. Phthalimide conjugation as a strategy for in vivo target protein degradation.Science(2015).

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[0012] In view of the shortcomings of the prior art, the present invention provides a proteolytic targeting virus, which comprises a molecule that can be recognized by the ubiquitin-proteasome system and a linker. The present invention further provides a method for producing the virus and uses of the virus.

[0013] In this application, the term "proteolysis-targeting molecules" refers to various molecules, such as polypeptides, amino acid sequences, proteins, or other molecules, that can be recognized by the ubiquitin-proteasome system. The applicant has found that when the coding nucleotide sequence of the proteolysis-targeting molecule is introduced into the genome of a virus, the proteolysis-targeting molecule can be replicated with the replication of the viral genome and fused and expressed in the viral protein with the translation of the viral protein, thereby obtaining a virus that is site-specifically modified by the proteolysis-targeting molecule, i.e., a proteolysis-targeting chimeric virus (PROTAC virus).

[0014] Since the ubiquitin-proteasome system is widely present in host cells, in order to avoid the PROTAC virus being degraded by the ubiquitin-proteasome system in cells during the manufacturing process, resulting in a decrease in production efficiency, the inventors introduce a selectively cleavable linker between the viral protein and the proteolytic target molecule, which can be cleaved in a specific artificially modified cell system, thereby separating the viral protein from the proteolytic target molecule, and the viral protein is retained without being degraded by the ubiquitination-proteasome system, so that the PROTAC virus can be efficiently replicated and mass-produced in this specific artificially modified cell system. In normal cells, the ubiquitin-proteasome system can recognize the proteolytic target molecule fused with the viral protein, thereby degrading the viral protein and reducing the viral replication ability, or even completely losing the replication ability. Therefore, the PROTAC virus has a very high safety. Moreover, the PROTAC viruses may be further modified to obtain viruses with improved performance, for example, by introducing certain immune enhancing agents into specific regions or specific amino acids of the viral proteins, thereby obtaining PROTAC viruses with enhanced immunogenicity.

[0015] Another object of the present invention is to introduce a coding nucleotide sequence of a conditionally cleavable proteolytic target molecule into the influenza virus genome so that the influenza virus can only replicate in a specific virus production system that can cleave or inactivate the proteolytic target molecule. The dependence of influenza virus on this specific virus production system can be used to mass-produce influenza virus in this system, and the ubiquitin-proteasome pathway exists in normal cells such as human and animal bodies, and can recognize the proteolytic target molecule fused with the viral protein, thereby degrading the viral protein, so that the influenza virus produced cannot replicate and grow in the animal or human body, increasing the safety of the virus and making the influenza virus a live influenza virus vaccine in name and reality. Note that the ubiquitin-proteasome system in animals and human bodies can recognize various proteolytic target molecules, so different types and different quantities of proteolytic target molecules can be introduced into the viral protein. There are also various selectively cleavable linkers, so selective cleavage of the proteolytic target molecule can be achieved by introducing different types and different quantities of linkers. These different types and quantities of proteolytic target molecules and linking chains may be arbitrarily combined, providing promise for the preparation of viral vaccines with different replication efficiencies and different degrees of attenuation, which are of great importance to the production efficiency and immunogenicity of influenza virus vaccines.

[0016] The principle of this proteolytic target virus is that (1) the proteolytic target molecule introduced into the viral protein specific site can be recognized by the ubiquitin-proteasome system in normal host cells, thereby degrading and inactivating the relevant viral protein, (2) the proteolytic target molecule introduced into the viral protein specific site can be inhibited in a specific virus production system, or selectively cut through the linking chain and separated from the viral protein, thereby avoiding or reducing the degradation of the viral protein by the ubiquitin-proteasome pathway, and (3) the proteolytic target molecule introduced into the viral protein specific site cannot be inhibited in normal host cells, or the linking chain connecting the proteolytic target molecule and the viral protein cannot be cut in normal host cells. Therefore, the prepared virus can be recognized and degraded by the ubiquitin-proteasome pathway in host cells such as animals and humans, but has a reduced replicative ability, or even completely loses the replicative and proliferative ability, thereby increasing the safety of the virus. [Means for solving the problem]

[0017] According to one aspect, the present invention provides a proteolytically targeted virus, in which one or more sites on one or more different viral proteins contain one or more proteolytic target molecules recognizable by the ubiquitin-proteasome system, and the viral proteins and the proteolytic target molecules are linked via one or more selectively cleavable linker chains.

[0018] Preferably, said sites are the C-terminus and / or N-terminus of a protein, ie the C-terminus and / or N-terminus of said viral gene segment.

[0019] According to the proteolytic target virus described in the present invention, the multiple proteolytic target molecules may be the same proteolytic target molecule or a combination of different proteolytic target molecules, and the multiple linking chains may be the same linking chain or a combination of different linking chains.

[0020] Preferably, said proteolysis target molecule is selected from the amino acid sequence shown in any one of SEQ ID NOs: 1-110. JPEG0007654625000001.jpg126168 JPEG0007654625000002.jpg87169 JPEG0007654625000003.jpg88167 JPEG0007654625000004.jpg114167 JPEG0007654625000005.jpg91168 JPEG0007654625000006.jpg88168 JPEG0007654625000007.jpg89169 JPEG0007654625000008.jpg86163 JPEG0007654625000009.jpg88169 JPEG0007654625000010.jpg176169 JPEG0007654625000011.jpg171150

[0021] Preferably, the linking chain is selected from any selectively cleavable molecule, such as a molecule selectively cleavable by TEV protease (Tobacco etch virus protease, TEVp), a cleavable molecule sensitive to thrombin (cleavage site is LVPR * GS, e.g., SEQ ID NO:111 LEAGCKNFFPRSFTSCGSLE), a molecule cleavable by clotting factor Xa (the cleavage site is IDGR *), a molecule cleavable by enterokinase (the cleavage site is DDDDK * ), a molecule cleavable by 3C protease (the cleavage site is ETLFQ * Preferred are SUMO protease cleavable molecules, sequences, and bacterial gelatinase cleavable molecules (e.g., GPLGV), and self-cleaving linking chains. Preferably, the self-cleaving linking chain is selected from 2A short peptides, such as P2A from porcine teschovirus-1, E2A from equine rhinitis A virus, F2A from foot and mouth disease virus, and self-cleavable T2A.

[0022] More preferably, the linker comprises a sequence EX that can be recognized and cleaved by any TEVp. aa -X aa -YX aa -Q-(G / S / M) (the cleavage occurs between Q and G, or between Q and S, or between Q and M, and X aa represents any amino acid. Herein, this sequence is referred to as a TEV protease cleavage sequence, and the cleavage site is referred to as a TEV protease cleavage site, Tobacco etch virus protease cleavage site, TEVcs), specifically selected from the amino acid sequences set forth in any one of SEQ ID NOs: 112-137. JPEG0007654625000012.jpg117168

[0023] Preferably, a flexible linker is further included between the proteolysis target molecule and the linking chain, More preferably, the proteolysis target molecule, the linking chain and the flexible linker have a linking scheme of flexible linker-linking chain-flexible linker-proteolysis target molecule, More preferably, the flexible linker-linker-flexible linker-proteolysis targeting molecule is selected from the amino acid sequences set forth in any one of SEQ ID NOs:138-149, 167 and 168. JPEG0007654625000013.jpg128169 JPEG0007654625000014.jpg122169

[0024] Preferably, the virus is influenza virus, AIDS virus, hand, foot and mouth virus, coxsackie virus, 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, giant cell virus, varicella-zoster virus, vesicular stomatitis virus, respiratory syncytial virus RSV, dengue virus, Ebola virus, Marburg virus, Zika virus Zika, SARS, Middle East respiratory syndrome virus, rotavirus, rabies virus, measles virus, adenovirus, poliovirus. The virus is selected from the group consisting of influenza virus, echovirus, Japanese encephalitis virus, tick-borne encephalitis virus, hantavirus, new enteric virus, rubella virus, parotitis virus, parainfluenza virus, blue ear virus, cholera virus, foot and mouth disease virus, minute virus, prion, smallpox virus, tobacco mosaic virus, adeno-associated virus, bacterial virus, herpes virus, West Nile virus, Norovirus, human bocavirus, coronavirus and the novel coronavirus SARS-CoV-2, more preferably the virus is influenza virus or the novel coronavirus SARS-CoV-2.

[0025] Preferably, the virus is a modified virus that contains a combination of multiple insertion modifications or modifications.

[0026] According to a specific embodiment of the present invention, there is provided a proteolytically targeted influenza virus (PROTAC influenza virus), the influenza virus comprising one or more different viral protein sites that contain one or more proteolytic target molecules that can be recognized by the ubiquitin-proteasome system, and the viral protein and the proteolytic target molecule are linked via a linker, wherein the linker is an EX that can be specifically recognized and cleaved by Tobacco etch virus protease (TEVp). aa -X aa -YX aa -Q-(G / S).

[0027] Preferably, one or more of the influenza virus PA, PB1, PB2, NP, HA, NA, M1, M2, NS1, NEP proteins comprise a proteolytic target molecule and a linking chain, i.e., one or more of the influenza virus genome PA, PB1, PB2, NP, HA, NA, M1, M2, NS1, NEP gene segments comprise nucleoside sequences encoding the proteolytic target molecule and a linking chain, i.e., the proteolytic target molecule and the linking chain are site-specifically modified at corresponding sites in one or more of the influenza virus PA, PB1, PB2, NP, HA, NA, M1, M2, NS1, NEP proteins.

[0028] Preferably, influenza virus PA and PB2 each contain one or more proteolytic target molecules and linking chains; Both influenza virus PA and PB1 proteins contain one or more proteolytic target molecules and linking chains, Both influenza virus PB2 and PB1 proteins contain one or more proteolytic target molecules and linking chains, The influenza virus PA protein, PB2 protein, and PB1 protein all contain one or more proteolytic target molecules and linking chains, The influenza virus PA protein, PB2 protein, PB1 protein, and M1 protein each contain one or more proteolytic target molecules and linking chains; The influenza virus PA protein, PB2 protein, PB1 protein, M1 protein, and NP protein each contain one or more proteolytic target molecules and linking chains; The PB2 protein, PB1 protein, and M1 protein of influenza virus each contain one or more proteolytic target molecules and linking chains; Both the influenza virus PA protein and M1 protein contain one or more proteolytic target molecules and linking chains, The PB1 and M1 proteins of influenza viruses both contain one or more proteolytic target molecules and linking chains, The PB2 and M1 proteins of influenza viruses both contain one or more proteolytic target molecules and linking chains, The PB2 protein, PB1 protein, M1 protein, and NS1 protein of influenza virus each contain one or more proteolytic target molecules and linking chains; The influenza virus PB2 protein, PB1 protein, M1 protein, and NEP protein each contain one or more linking chains to a proteolytic target molecule, or Both the NS1 and NEP proteins of influenza viruses contain one or more proteolytic target molecules and linking chains.

[0029] Preferably, the proteolytically targeted virus is a proteolytically targeted coronavirus, which comprises one or more proteolytically targeted molecules that can be recognized by the ubiquitin-proteasome system at one or more different viral protein sites, and the viral protein and the proteolytically targeted molecule are linked via a linker, wherein the linker is an EX that can be specifically recognized and cleaved by TEV protease. aa -X aa -YX aa -Q-(G / S / M).

[0030] Preferably, the virus is the novel coronavirus SARS-CoV-2.

[0031] More preferably, the coronavirus spike protein (S protein), envelope glycoprotein (E glycoprotein), membrane glycoprotein (M glycoprotein), nucleocapsid protein (N protein), nonstructural protein 1 (nsp1), nonstructural protein 2, nonstructural protein 3, nonstructural protein 4, nonstructural protein 5, nonstructural protein 6, nonstructural protein 7, nonstructural protein 8, nonstructural protein 9, nonstructural protein 10, nonstructural protein 11, nonstructural protein 12, nonstructural protein 13, nonstructural protein 14, nonstructural protein 15, nonstructural protein 16, 3a protein, 3b protein, 6 protein, 7a protein, 7b protein, 8a protein, 8b protein, 9b protein, 3C-like proteinase, leader protein, One or more of the following may comprise a proteolytic target molecule and one or more linking chains: an ORF1a polyprotein, an ORF10 protein, an ORF8 protein, an ORF7a protein, an ORF6 protein, an ORF3a protein, an ORF4 protein, an ORF5 protein, an ORF6 protein, an ORF7a protein, an ORF8 protein, an ORF9 protein, an ORF10 protein, an ORF11 protein, an ORF12 protein, an ORF13 protein, an ORF14 protein, an ORF15 protein, an ORF16 protein, an ORF17 protein, an ORF18 protein, an ORF19 protein, an ORF20 protein, an ORF21 protein, an ORF22 protein, an ORF23 protein, an ORF24 protein, an ORF25 protein, an ORF26 protein, an ORF27 protein, an ORF28 protein, an ORF29 protein, an ORF29 protein, an ORF29 protein, an ORF28 protein, an ORF29 protein, an ORF29 protein, an ORF29 protein, an ORF20 protein, an ORF21 protein, an ORF22 protein, an ORF23 protein, an ORF24 protein, an ORF25 protein, an ORF26 protein, an ORF27 protein, an ORF28 protein, an ORF29 ...

[0032] Preferably, the proteolytically targeted virus is a proteolytically targeted HIV virus, the HIV virus comprising one or more different viral proteins at a site thereof, the one or more proteolytically targeted molecules being recognizable by the ubiquitin-proteasome system, and the viral proteins and the proteolytically targeted molecules being linked via a linking chain, the linking chain being an EX that can be specifically recognized and cleaved by the TEV protease. aa -X aa -YX aa -Q-(G / S / M).

[0033] Preferably, the virus is the HIV virus.

[0034] Also preferred are Gag polyprotein, pol polyprotein, gp160, HIV trans-activator of transcription (Tat), regulator of expression of virion proteins (Rev), negative factor (Nef), lentivirus protein R (Vpr), viral infectivity factor (Vif), viral protein U (Vpu), matrix protein (MA, matrix protein, p17), capsid protein (CA, capsid protein, p24), spacer peptide 1 (SP1, spacer peptide 1, p2), nucleocapsid protein (NC, nucleocapsid protein, p7), spacer peptide 2 (SP2, spacer peptide 2, p1), P6, reverse transcriptase (RT, reverse One or more of the proteins ribonuclease H (RNase H), integrase (IN), HIV protease (PR), gp120, and gp41 contain one or more proteolytic target molecules and linking chains.

[0035] In one embodiment of the present invention, using reverse genetics technology, the inventors can replace any gene of an existing influenza virus model with a gene of another subtype or virulent strain, or replace an existing influenza virus model with another subtype or virulent strain to generate influenza viruses of other subtypes or virulent strains, making the method applicable to influenza viruses of any subtype or virulent strain, and the generated viruses are replicated in large quantities in a cell line that can selectively cleave proteolytic target molecules, but are recognized and degraded by the ubiquitin-proteasome system in normal host cells. It should be noted that this method may be applied to other subtypes or virulent strains, including 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, H7 N2, 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. In addition, reverse genetics technology can also be used to create multivalent influenza viruses, such as multivalent influenza viruses containing H1N1, H3N2, and influenza B virus surface antigens. More importantly, the mutant virulence and multivalent viruses created have very high safety and efficacy.

[0036] The present invention further provides a nucleic acid molecule encoding said proteolytic targeted virus.

[0037] The present invention further provides a nucleic acid vector expressing said proteolytic targeted virus.

[0038] According to another aspect, the present invention provides a method for producing a proteolytic targeted virus, said method comprising the steps of:

[0039] Step 1) Construction of a cell line: A cell line capable of stably expressing a protease having selective cleavage activity against the junctional chain of a protein hydrolysis target virus is constructed.

[0040] Preferably, said cell line is a mammalian cell line.

[0041] More preferably, said 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-bl cells, Sup-T1 cells, MRC-5 cells and VMK cells, LLC-MK2 cells, HCT-8 cells, Huh-7 cells, Caco2 cells, Even more preferably, said cell line is selected from the HEK293T cell line or the MDCK cell line, Preferably, said cell line is any ubiquitin-proteasome system deficient cell line, more preferably said cell line is an E3 ligase knockout or knockdown cell line.

[0042] Step 2) Site selection: By statistically analyzing the expression distribution of the ubiquitin-proteasome system in the host body, bioinformatics and protein structure prediction are performed on the virus to determine the type of proteolytic target molecule to be introduced, and the viral protein and site to which the proteolytic target molecule and the linking chain will be introduced.

[0043] Step 3), gene mutation: Using genetic engineering methods, a nucleoside sequence encoding a proteolytic target molecule and a linker chain is introduced into the coding gene of the determined viral protein and selected site.

[0044] Step 4), construction of an expression vector: the coding nucleotide sequence of the viral protein after gene mutation obtained in step 3) is operably linked to a vector to obtain an expression vector.

[0045] Preferably, the expression vector is a plasmid.

[0046] In step 5), using reverse genetics techniques, the expression vector in step 4) and another expression vector for influenza virus rescue are co-transfected into a cell line having selective cleavage at the linker chain of the proteolytic target virus constructed in step 1) to obtain a proteolytic target virus.

[0047] Optionally, in step 6), the proteolytic targeted virus is replicated in the cell line obtained in step 1) in order to produce said proteolytic targeted virus on a large scale.

[0048] Preferably, a proteasome inhibitor is added during the virus production process, and more preferably, the proteasome inhibitor is MG132, MG-341 or lactacystin.

[0049] Alternatively, the method includes the following steps.

[0050] Step [1], site selection: By statistically analyzing the expression distribution of the ubiquitin-proteasome system in the host body, bioinformatics and protein structure prediction are performed on the virus to determine the type of proteolytic target molecule to be introduced, and the viral protein and site to which the proteolytic target molecule and the linking chain will be introduced.

[0051] Step [2], gene mutation: Using genetic engineering methods, a nucleoside sequence encoding the proteolytic target molecule and the linker chain is introduced into the coding gene of the determined viral protein and selected site.

[0052] Step [3]: Construction of an expression vector for the mutated sequence: The coding nucleotide sequence for the viral protein after gene mutation obtained in step 3) is operably linked to a vector to obtain an expression vector.

[0053] Preferably, the expression vector is a plasmid.

[0054] Step [4]: ​​constructing an overexpression vector of a protease that selectively cuts the junction of a protein hydrolysis target virus; A cell line capable of stably expressing a protease that has selective cleavage activity against the junctional chain of a target virus for protein hydrolysis is constructed.

[0055] In step [5], using reverse genetics techniques, the expression vector obtained in step [3], other expression vectors required for viral rescue, and the expression vector obtained in step [4] are co-transfected into host cells, and the successfully transfected host cells are cultured in culture medium to obtain a protein hydrolysis target virus.

[0056] According to the above method of the present invention, the method further comprises step 7): Testing: Determine whether the modification of the proteolytic target virus is successful by measuring the replication ability of the proteolytic target virus obtained in step 5) in the cell line obtained in step 1) and in unmodified normal host cells, wherein the proteolytic target virus that replicates in the cell line obtained in step 1) and has reduced replication ability or cannot replicate in unmodified normal host cells is the successfully modified proteolytic target virus.

[0057] Optionally, the method further comprises step 8), Using the successfully modified proteolytically targeted viral vector, steps 2) to 5) are repeated to introduce a proteolytic target molecule and a linking chain onto each of multiple viral proteins of the proteolytically targeted virus, or to introduce multiple proteolytic target molecules and linking chains onto any one viral protein of the proteolytically targeted virus.

[0058] Optionally, the successful modification of the proteolytic targeted virus is determined by measuring the replication ability of the obtained proteolytic targeted virus in the cell line obtained in step 1) and in unmodified normal host cells, wherein the proteolytic targeted virus that replicates in the cell line obtained in step 1) and has reduced or no replication ability in unmodified normal host cells is a successfully modified proteolytic targeted virus.

[0059] According to a specific embodiment of the present invention, there is provided a method for the preparation and large-scale production of a proteolytically targeted influenza virus, the method comprising the steps of:

[0060] Step 1), Construction of a cell line: The TEV protease TEVp is stably transduced into a mammalian cell line to construct a cell line capable of stably expressing the TEV protease TEVp.

[0061] Preferably, said mammalian cell lines are the HEK293T cell line and the MDCK cell line; The constructed stable cell lines are HEK293T-TEVp and MDCK-TEVp. In this embodiment, the research target is preferably TEV protease (Tobacco etch virus protease, TEVp) and its corresponding cleavable sequence, but as will be understood by those skilled in the art, the principle of the technology of the present invention may be extended to any other molecule (including proteins, polypeptides, nucleic acids, and chemical molecules) that has a cleavage function.

[0062] Step 2), site selection: To determine the gene fragment and site into which the proteolytic target molecule and the linking chain are introduced, bioinformatics and protein structure prediction are performed on the influenza virus through statistical analysis of the expression distribution of the ubiquitin-proteasome system in the host body, and the protein structure of the influenza virus after the proteolytic target molecule and the linking chain, the TEV protease cleavage sequence, are introduced into each protein of the influenza virus is predicted.

[0063] Preferably, one or more insertion sites are selected from gene segments encoding different influenza viruses proteins.

[0064] Step 3), gene mutation: Using genetic engineering methods, a nucleoside sequence encoding a proteolytic target molecule and a linker chain is introduced into the coding gene of the determined influenza virus protein and selected site.

[0065] Step 4) Plasmid construction: The coding nucleotide sequence of the viral protein after gene mutation obtained in step 3) is operably linked to a plasmid to obtain a coding plasmid.

[0066] In step 5), using reverse genetics techniques, the plasmid obtained in step 4) and other plasmids for influenza virus rescue are co-transfected into stable cells stably expressing the TEVp obtained in step 1) to obtain a protein hydrolysis-targeted influenza virus.

[0067] Here, the coding nucleotide sequence of the proteolytic target molecule and the TEV protease cleavage sequence are introduced into the genome of the proteolytic target influenza virus, thereby introducing the proteolytic target molecule and the TEV protease cleavage sequence into the corresponding influenza virus protein to obtain an influenza virus modified by the proteolytic target molecule (PROTAC influenza virus).

[0068] Preferably, the method further comprises culturing the successfully transfected host cells in DMEM medium containing 0.5% FBS and 2 μg / mL TPCK-trypsin; In summary, the resulting mutant sequence expression vector, other plasmids required for viral rescue, and a plasmid overexpressing TEVp are co-transfected into host cells, and successfully transfected host cells are cultured in DMEM medium containing 0.5% FBS and 2 μg / mL TPCK-trypsin.

[0069] Optionally, in step 6), the proteolytically targeted influenza virus is produced on a large scale in a stable cell line stably expressing TEVp.

[0070] Preferably, about 4 days after transfection or after the packaged virus has completely or 90% or more of the host cells in 4) have been infected, the supernatant is collected and used to infect new MDCK-TEVp cells, the medium being DMEM medium containing 0.5% FBS and 2 μg / mL TPCK-trypsin, and the supernatant is collected 4 days after infection or after the infected virus has completely infected the host cells in 4).

[0071] Preferably, the method further comprises step 7), Testing: Determine whether the modification of the proteolytic target influenza virus obtained in step 5) is successful by measuring the dependency of the proteolytic target influenza virus on TEVp and the dependency of the inactivation of its packaging product on the proteasome pathway.

[0072] Specifically, the supernatant is centrifuged and passed through a 0.45 μm filter to remove cell debris, and the TEVp dependency of the packaging product and the dependency of the inactivation of the packaging product on the proteasome pathway are examined, and mutants that maintain the TEVp dependency are retained and set as candidates for successful modification. The TEVp dependency of the packaging product means that the virus packaged using the above method can replicate and grow in a cell line that highly expresses TEVp, but has reduced or no replication ability in normal cells that do not express TEVp.

[0073] Optionally, the method further comprises step 8), Using the successfully modified proteolytically targeted influenza virus vector, steps 2) to 5) are repeated to introduce the proteolytic target molecule and the linking chain onto all of the multiple viral proteins of the proteolytically targeted influenza virus, or to introduce multiple proteolytic target molecules and linking chains onto any one viral protein of the proteolytically targeted influenza virus.

[0074] Preferably, the successful modification of the resulting proteolytically targeted influenza virus is determined by measuring its dependency on TEVp and on the proteasome pathway for inactivation of its packaging product, and proteolytically targeted viruses that still maintain their dependency on TEVp after long-term passage are retained as candidates for successful modification.

[0075] Optionally, the method further comprises: 9) selecting successfully modified candidates and purifying the products; The method further includes a step of conducting safety or immunogenicity testing on the proteolytic target influenza virus in step 9), wherein a safe influenza virus is an influenza virus that has been successfully modified compared to a wild-type virus.

[0076] In one specific embodiment of the present invention, the proteolytic target influenza virus gene can be inserted into a vector (e.g., pHH21 plasmid) and the vector, together with other vectors required for influenza virus rescue, can be transfected into a stable cell line capable of selectively cleaving the proteolytic target molecule, preferably HEK293T-TEVp and MDCK-TEVp, to obtain a PROTAC influenza virus, which is then mass-produced in a stable cell line capable of selectively cleaving the proteolytic target molecule, preferably HEK293T-TEVp and / or MDCK-TEVp.

[0077] In one embodiment of the present invention, in order to improve the production efficiency of influenza virus and realize future industrialized production, the inventors have constructed a stable cell line (preferably HEK293T-TEVp and MDCK-TEVp) capable of stably expressing TEV protease TEVp. This mammalian stable cell line can also solve the drawback of using conventional chicken embryo propagated viruses, which are prone to side effects such as human allergies. In order to further improve the production efficiency, an appropriate amount of a proteasome inhibitor may be added to the virus culture medium to suppress the degradation of viral proteins by the ubiquitin-proteasome system, or a ubiquitin-proteasome system-deficient cell line, such as an E3 ubiquitin ligase knockout or knockdown cell line, may be used.

[0078] The present invention provides mutant influenza viruses, i.e. PROTAC viruses, which can be produced in mammalian cells and introduce conditionally cleavable proteolytic targeting molecules into the genome and corresponding proteins, respectively, preferably H1N1, H5N1, H7N9, H3N2 or influenza B viruses.

[0079] The above viruses may be produced from mammalian stable cell lines (e.g., 293T cells, MDCK cells, CHO cells, Vero cells, etc.) that stably express TEVp. To further improve production efficiency, an appropriate amount of a proteasome inhibitor may be added to the virus culture medium to suppress the degradation of viral proteins by the ubiquitin-proteasome system, or a ubiquitin-proteasome system-deficient cell line, such as an E3 ubiquitin ligase knockout or knockdown cell line, may be used. The produced mutant influenza viruses have very good safety and immune activity.

[0080] In order to construct a mammalian cell line stably expressing TEVp, the inventors constructed a lentivirus overexpression vector carrying a puromycin-resistant TEVp expression gene, transduced HEK293T cells and MDCK cells via the virus respectively, and obtained stable cell lines stably expressing TEVp through puromycin screening. These stable cell lines were monocloned, and monoclonal cultured, and the stable cell lines with the highest TEVp expression were selected using Western blotting and RT-qPCR, and the final stable cell lines were HEK293T cells and MDCK cells.

[0081] The mutant PROTAC influenza virus can be obtained after purification by hollow fiber column and gel chromatography method or sucrose gradient density centrifugation method. In vitro and in vivo experiments have initially demonstrated that the PROTAC influenza virus has excellent safety and genetic stability, and has better immune effect than the inactivated virus.

[0082] The present invention further provides methods for producing live attenuated viruses, live replication-incompetent viruses, replication-controllable viruses, and related vaccines and drugs for preventing and treating viral infections, said methods comprising utilizing the above-mentioned proteolytically targeted viruses or using the above-mentioned methods to produce the proteolytically targeted viruses.

[0083] The present invention further provides a vaccine or pharmaceutical composition comprising the above-mentioned proteolytic target virus, Preferably, the vaccine is a live attenuated, live replication-incompetent or live replication-regulatable vaccine.

[0084] The present invention further provides a system for producing the above-mentioned proteolytic target virus, the system includes a cell line stably expressing a protease having selective cleavage action on the linkage chain of the proteolytic target virus, preferably the cell line stably expressing TEV protease TEVp, more preferably the cell line is a HEK293T cell line or an MDCK cell line stably expressing TEV protease TEVp. In order to further improve the production efficiency, an appropriate amount of a proteasome inhibitor may be added to the virus culture medium to suppress the degradation of viral proteins by the ubiquitin-proteasome system, or a cell line with a deficiency in the ubiquitin-proteasome system, such as an E3 ubiquitin ligase knockout or knockdown cell line, may be used.

[0085] More preferably, said system further comprises a nucleic acid vector expressing said proteolytic targeted virus.

[0086] According to yet another aspect, the present invention provides the use of the proteolytic targeted viruses of the present invention in the production of live attenuated, live replication-incompetent and live replication-regulatable vaccines and related drugs for the prevention and treatment of viral infections.

[0087] The present invention further provides a vaccine, wherein the composition comprises the PROTAC influenza virus, the PROTAC novel coronavirus or the PROTAC HIV virus.

[0088] Preferably, the vaccine contains adjuvants and other auxiliary materials.

[0089] The present invention further provides a pharmaceutical composition comprising said PROTAC influenza virus, PROTAC novel coronavirus or PROTAC HIV virus, and a pharma-ceutical compatible vector, diluent or excipient.

[0090] The present invention further provides the use of said PROTAC influenza virus, PROTAC novel coronavirus or PROTAC HIV virus in producing live attenuated vaccines, live replication-incompetent vaccines, live replication-controllable vaccines, and related drugs for preventing and treating viral infections.

[0091] The present invention further provides a lentiviral vector carrying a Tobacco etch virus protease (TEVp) gene, the sequence of which is shown below. After packaging into a virus, the vector can be transduced into cells and the Tobacco etch virus protease (TEVp) can be integrated into host cells using puromycin screening. The amino acid sequence of the TEVp is shown in SEQ ID NO:150. SEQ ID NO:150: MGESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQ TKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMN

[0092] The present invention further provides mammalian stable cell lines, HEK293T-TEVp and MDCK-TEVp, which stably express TEVp.

[0093] Here, the stable cell lines MDCK-TEVp and HEK293T-TEVp obtained by lentiviral transduction carry the Tobacco etch virus protease (TEVp) gene, and these stable cell lines can be used to introduce a protein hydrolysis target molecule with a TEVp cleavage sequence as a linker into a specific site of any target protein of the virus, thereby realizing large-scale production of PROTAC viruses.

[0094] The above compositions, pharmaceutical compositions and vaccines may be prepared by conventional techniques in the art based on the production of the site-directed mutagenesis modified PROTAC influenza viruses of the present invention, and they can be used for the prevention or treatment of influenza virus infections, including influenza virus infections in humans and animals.

[0095] According to a further aspect, the present invention provides a site-specific mutation, site-specific modification, and replication-controllable influenza virus live vaccine, in which the site-specific mutation and site-specific modification are the introduction of a peptide, amino acid sequence, or other molecule that may induce proteolysis (hydrolysis) into any protein of an influenza virus, and the introduced peptide, amino acid sequence, or other molecule that may induce proteolysis (hydrolysis) can be selectively cleaved, suppressed, or inactivated during the virus production process. The present invention further relates to the creation of an influenza virus in which a peptide, amino acid sequence, or other molecule that may induce proteolysis (hydrolysis) is introduced into multiple gene fragments by combining sites modified by site-specific mutation.

[0096] The present invention further provides a method for site-specific mutation and modification of influenza virus, which includes introducing a proteolysis-targeting molecule (including a polypeptide, amino acid sequence, protein, or other molecule) that can induce proteolysis (hydrolysis) into a gene (protein) of influenza virus via zero, one, or more selectively cleavable linkers (including a polypeptide, amino acid sequence, protein, or other molecule), so that the mutated influenza virus protein can be degraded (hydrolyzed) in a normal host (cell), reducing the viral replication ability (activity), or even completely losing the replication ability.

[0097] The present invention further provides a method for selectively inhibiting viral protein degradation (hydrolysis), the method comprising selectively inhibiting or inactivating molecules that may induce protein degradation (hydrolysis) and selectively cleaving the linker between the protein degradation molecule and the viral protein, so as to ensure high production efficiency in the process of virus production. In order to further improve the production efficiency of PROTAC virus, an appropriate amount of proteasome inhibitor (e.g., MG132, MG-341, lactacystin, etc.) may be added in the process of PROTAC virus production to inhibit proteasome-mediated protein degradation, or a ubiquitin-proteasome system-deficient cell, such as a cell line with a corresponding E3 ligase knockout or knockdown, may be used to produce PROTAC virus.

[0098] The present invention further provides applications of the site-specific mutation-modified or multi-site-conjugated influenza viruses, such as live influenza virus vaccines, attenuated vaccines, replication-incompetent vaccines, influenza virus virulence safety models, etc. The technology of the present invention can be widely applied to any virus modification, production, and / or application thereof, such as live vaccines, attenuated vaccines, inactivated vaccines, virulence safety models, etc. Effect of the Invention

[0099] Compared with the prior art, the present invention has the following advantages: 1) Versatility: All viruses have proteins, and by simply introducing a "cleavable protein hydrolysis target molecule" into the viral protein to be degraded, it is possible to control viral replication. Therefore, this technology can be used to create vaccines for all viruses.

[0100] 2) There are thousands of selectable proteolytic target molecules that can be developed and used to create this virus vaccine, thus providing a rich pool of selectable molecules.

[0101] 3) Easy to operate: only simple viral vector construction and virus production techniques are required, and producers do not need much knowledge of virus biology to make such vaccines.

[0102] 4) Experiments have demonstrated that this technology can be used to design vaccines with different degrees of inactivation and has the potential to be safely controlled.

[0103] 5) Experiments have demonstrated that these vaccines have good immunogenicity. [Brief description of the drawings]

[0104] Next, embodiments of the present invention will be described in detail in conjunction with the drawings. [Figure 1] FIG. 1 is a chromatogram of a TEVp overexpressing lentiviral vector. [Figure 2A] FIG. 2A shows an electropherogram of RT-PCR identification of potential HEK293T-TEVp cells (the cells indicated by the arrows are those with relatively high mRNA expression levels of the target gene TEVp). [Figure 2B] Figure 2B shows the electropherogram of RT-PCR identification for potential MDCK-TEVp cells (O (old) represents the first batch of MDCK cells, N (new) represents the second batch of MDCK cells, and the arrows indicate cells with relatively high mRNA expression levels of the target gene TEVp). [Diagram 3] FIG. 3 shows a graph of protein expression detection of the target protein TEVp in HEK293T-TEVp and MDCK-TEVp cell lines prepared by the method of the present invention, and a graph of virus packaging efficiency. [Figure 4] FIG. 4 is a histogram of the replication titers of proteolytically targeted influenza viruses (PROTAC influenza viruses) produced by the methods of the present invention in MDCK-TEVp cell lines and normal cell lines two days after infection. [Diagram 5]FIG. 5 is a histogram of replication titers following infection of MDCK-TEVp cell lines and normal cell lines with proteolytically targeted influenza viruses (PROTAC influenza viruses) containing multiple proteolytic molecules generated by the methods of the present invention. [Figure 6] FIG. 6 is a histogram comparing the replication abilities of proteolytically targeted influenza viruses (PROTAC influenza viruses) generated by the methods of the invention in MDCK-TEVp cells and normal cell lines. [Figure 7] FIG. 7 examines the genetic stability of the proteolytically targeted influenza virus (PROTAC influenza virus) prepared by the method of the present invention, and shows that the prepared PROTAC influenza virus remains stable after multiple passages. [Figure 8] FIG. 8 is a diagram showing a Western blot experiment used to evaluate whether the PROTAC influenza virus according to the present invention can be degraded by the proteasome in normal cells. [Figure 9] FIG. 9 is a fluorescent immunodetection diagram using immunofluorescence experiments to evaluate the replication of the PROTAC influenza virus according to the present invention in the MDCK-TEVp cell line and normal cells. [Figure 10] FIG. 10 shows animal-level immunogenicity and protection results of the PROTAC virus according to the present invention. [Figure 11] FIG. 11 is a flow chart of a method for producing a proteolytic targeted virus using the method of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0105] In order to better understand the present invention, the inventors will describe and explain specific tests in conjunction with examples, where the examples are used for illustration only and do not limit the scope of protection of the present invention. Any equivalent modifications or embodiments of the present invention are also included in the present invention. EXAMPLES

[0106] Example 1: Construction of mammalian stable cell lines HEK293-TEVp and MDCK-TEVp capable of stably expressing TEVp This mammalian cell line stably expressing TEVp was developed with the cooperation of Beijing CorreGene.

[0107] (1) Construction of TEVp overexpression lentiviral vector: The TEVp overexpression lentiviral vector chromatogram is shown in Figure 1. The lentiviral vector has puromycin resistance.

[0108] (2) Packaging, purification, and quantification of lentivirus [1] Day 0 cell inoculation: Inoculate 293T cells into 10cm / 15cm culture dishes (the number of inoculations is determined by the expected amount of virus, 1 × 10 8 The inoculation density was controlled, and the cells grew to 80% confluence the next day.

[0109] [2] Day 1 Plasmid transfection: 293T cells were co-transfected with the TEVp overexpression lentiviral vector described in (1), psPAX2 plasmid (this plasmid is available from Addgene), and pVSVG plasmid (this plasmid is available from Addgene).

[0110] [3] Day 2 Fluid Change: 18 hours after transfection, the fluid was changed (the transfected system is toxic), the medium was completely aspirated, and 20 mL of fresh complete medium (15 cm culture dish) was carefully added.

[0111] [4] Pretreatment of viral supernatant: After collecting the viral supernatant on days 3, 4, and 5, the culture supernatant was transferred to a 50 mL centrifuge tube and centrifuged at maximum rotation 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.

[0112] [5] Virus purification by high-speed centrifugation: Using a 20 mL syringe, 10% sucrose solution was slowly injected into the bottom of the centrifuge bottle to maintain a volume ratio of 4:1 (4 parts virus supernatant, 1 part sucrose solution), and centrifuged at 4°C and 14,000 rpm for 2 hours.

[0113] [6] Virus resuspension: Discard the supernatant, aspirate thoroughly, add 100ul to 1000ul of PBS, aspirate, and blow to mix evenly.

[0114] [7] The viral supernatant was dispensed and frozen at -80°C. After dispensing, approximately 30 ul of the remaining solution was frozen separately and used for titer measurement.

[0115] [8] The lentivirus titer was measured using a chemiluminescence assay (CMIA), and the titer of pLenti-CMV-puro-2A-TEVp-8534bp was 5.12E+7TU / mL, in a volume of 1000ul, and in a total amount of 5.12E+7TU, which confirmed that the transduction requirements were met.

[0116] (3) Construction of stable monoclonal cell lines [1] Day 0 Cell inoculation: HEK293T or MDCK were inoculated into a 12 / 6-well culture dish (the number of inoculations was determined by the expected amount of virus, 1 × 10 6 The inoculation density was controlled by culturing the cells in a 6-well plate culture dish, and the cells were grown to 20-30% confluence the next day.

[0117] [2] Day 1 Virus thawing: Remove the virus from the -80°C refrigerator and thaw it on ice. Infect cells with the appropriate moi (MOI=10).

[0118] [3] Day 2 viral transduction: Cells were infected with the appropriate moi for 24 hours and then replaced with 2μg / mL puro resistance medium for resistance screening.

[0119] [4] Resistance screening was continued for about 7 days (total killing of blank control strains).

[0120] [5] Viable cells were digested and counted, and 100 / 200 cells were evenly distributed into a 96-well plate (on average, each well had 1-2 cells).

[0121] [6] Clonal proliferation was observed within 3 to 5 days, and monoclonal cells were selected and expanded, then expanded in stages into 24-well plates and 12-well plates.

[0122] [7] Monoclonal Identification: Identification primers TEVp-F: SEQ ID NO:151 TCATTACAAACAAGCACTTG TEVp-R: SEQ ID NO:152 TAGGCATGCGAATAATTATC Fragment size: 144bp 293t-GAPDH-eF: SEQ ID NO:153 CCACATCGCTCAGACACCAT 293t-GAPDH-eR: SEQ ID NO:154 GGCAACAATATCCACTTTACCAGAGT Fragment size: 114bp

[0123] The desired monoclonal cells were digested, RNA was extracted, and RT-PCR was performed to verify whether the cells had TEVp integration and expression. The growth conditions were as shown in Table 1 below.

[0124] [Table 1] JPEG0007654625000015.jpg33165

[0125] The results are shown in Figure 2. Figure 2A is the electrophoretic diagram of RT-PCR identification of potential HEK293T-TEVp cells (the cells indicated by the arrow are cells with a relatively high mRNA expression level of the target gene TEVp). This validation work was completed with the cooperation of Beijing CorreGene. Figure 2B is the electrophoretic diagram of RT-PCR identification of potential MDCK-TEVp cells (O (old) represents the first batch of MDCK cells, N (new) represents the second batch of MDCK cells, and the cells indicated by the arrow are cells with a relatively high mRNA expression level of the target gene TEVp). This validation work was completed with the cooperation of Beijing CorreGene.

[0126] [8] Preferred monoclonal cell line growth cultures The cell lines with the highest TEVp expression efficiency were expanded and cryopreserved, and designated HEK293T-TEVp and MDCK-TEVp, respectively.

[0127] The results are shown in Figure 3. Anti-TEVp antibody was used to perform western blot analysis on HEK293T-TEVp and MDCK-TEVp cell lines. The results showed that TEV protease TEVp could be efficiently and stably expressed in HEK293T-TEVp and MDCK-TEVp stable cell lines. Furthermore, the modification of cells did not cause any change in virus production efficiency, demonstrating the compatibility of TEVp in cells. EXAMPLES

[0128] Example 2: Construction of a genetic vector of influenza virus WSN containing a cleavable proteolytic target molecule (1) Obtaining a plasmid to rescue wild-type influenza virus WSN: The genetic sequence of influenza virus A / WSN / 1933 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+NP, https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=WSN+M, Based on https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=WSN+NS, the genes of each gene fragment of this influenza virus were obtained through total gene synthesis. Then, they were connected to pHH21, pCDNA3(neo), and pcAAGGS / MCS vectors (obtained from Beijing Zhongke Yubo Biotechnology Co., Ltd.) respectively to obtain a plasmid for rescuing wild-type influenza virus WSN. The name and composition of the obtained plasmid are shown in Table 2.

[0129] [Table 2] JPEG0007654625000016.jpg149168

[0130] (2) Construction of a viral vector carrying a cleavable proteolytic target molecule The inventors introduced the gene sequences of proteolytic target molecules cleavable by TEVp into the C-terminus, before the stop codon, of the gene coding regions corresponding to each viral protein (PA, PB2, PB1, NP, HA, NA, M1, M2, NS1, NEP) of influenza virus WSN, and constructed the following viral vectors.

[0131] Specifically, the gene sequence for introducing the proteolytic target molecule cleavable by TEVp into any one of the viral proteins and the expressed amino acid sequence are as follows, but are not limited to the following sequences: The gene sequence corresponding to the amino acid sequence used was humanized and optimized and inserted at the C-terminus of the coding region of the target protein gene, before the stop codon. This work was completed with the cooperation of Beijing Guanke Biotechnology Co., Ltd., and the successful construction of the mutation was verified by sequence measurement. Herein, 1) The sequence shown in SEQ ID NO:138 was introduced into the C-terminus of the PA protein of influenza virus, i.e., the nucleoside sequence shown in SEQ ID NO:155 was introduced into the C-terminus of the PA protein gene coding region, before the stop codon, and this was named PA-TEVcs+PROTAC-1 (or PA-PTD1). SEQ ID NO:138 GSGGENLYFQGGSGALAPYIP, SEQ ID NO:155 GGTTCTGGTGGTGAGAAT CTGTAC TTC CAA GGTGGATCTGGAGCA TTG GCC CCC TAC ATTCCA,

[0132] 2) Similarly, the sequence shown in SEQ ID NO:138 was introduced into the C-terminus of the M1 protein of influenza virus, i.e., the nucleoside sequence shown in SEQ ID NO:155 was introduced into the C-terminus of the PA protein gene coding region, before the stop codon, and this was named M1-TEVcs+PROTAC-1 (or M1-PTD1).

[0133] 3) The sequence shown in SEQ ID NO:139 was introduced into the C-terminus of the PB1 protein of influenza virus, i.e., the nucleoside sequence shown in SEQ ID NO:156 was introduced into the C-terminus of the PB1 protein gene coding region, before the stop codon, and this was marked as PB1-TEVcs+PROTAC-2 (or named PB1-PTD2). SEQ ID NO:139 GSGGENLYFQGGSGDRHDSGLDSM SEQ ID NO:156 GGTTCTGGTGGTGAG AAT CTGTAC TTC CAA GGTGGATCTGGAGAT CGC CAC GAT TCA GGG CTC GAT TCC ATG

[0134] 4) The sequence shown in SEQ ID NO:139 was introduced into the C-terminus of the PB2 protein of influenza virus, i.e., the nucleoside sequence shown in SEQ ID NO:156 was introduced into the C-terminus of the PB2 protein gene coding region, before the stop codon, and this was marked as PB2-TEVcs+PROTAC-2 (or named PB2-PTD2).

[0135] 5) The sequence shown in SEQ ID NO:139 was introduced into the C-terminus of the M1 protein of influenza virus, i.e., the nucleoside sequence shown in SEQ ID NO:156 was introduced into the C-terminus of the M1 protein gene coding region, before the stop codon, and this was marked as M1-TEVcs+PROTAC-2 (or named M1-PTD2).

[0136] 6) The sequence shown in SEQ ID NO:140 was introduced into the C-terminus of the PA protein of influenza virus, i.e., the nucleoside sequence shown in SEQ ID NO:157 was introduced into the C-terminus of the PA protein gene coding region, before the stop codon, and this was named PA-TEVcs+PROTAC-3 (or PA-PTD3). SEQ ID NO:140:GSGGENLYFQGGGGSSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV; SEQ ID NO:157 GGTTCTGGTGGTGAGAATCTGTACTTCCAAGGTGGAGGAGGATCCAGCCATGGCTTCCCGCCGGAGGTGGAGGAGCAGGATGATGGCACGCTGCCCATGTCTTGTGCCAGGAGACGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGGATCAATGTG

[0137] 7) PA-TEVcs+PROTAC-1 (PA-PTD1) and PB1-TEVcs+PROTAC-2 (PB1-PTD2) were combined to construct viruses carrying one cleavable proteolytic molecule in PA and one in PB1, respectively.

[0138] Furthermore, the inventors have constructed the following viral vectors:

[0139] [Table 3] JPEG0007654625000017.jpg137169 JPEG0007654625000018.jpg141170 JPEG0007654625000019.jpg235169 [Table 3] JPEG0007654625000020.jpg756 JPEG0007654625000021.jpg224168 JPEG0007654625000022.jpg47170 EXAMPLES

[0140] Example 3: Rescue of PROTAC influenza viruses after modification by site-directed mutagenesis According to the method of rescuing normal influenza virus, 12 plasmids used for rescuing influenza virus were co-transfected into stable cell line, and the plasmid modified by site-specific mutation in Example 2 was used to replace the corresponding plasmid in these 12 plasmids. 0.2μg of each plasmid was added to each well of the six-well plate. After transfection, the lesion status of cells was observed, and the insertion site, proteolytic target molecule, TEVp-cleavable linker, and their combinations that can rescue the virus and have dependency on TEVp were screened. The virulence strains screened were named according to the protein and the cleavable proteolytic target molecule introduced.

[0141] As an example, after introducing the cleavable proteolytic target molecule TEVcs+PROTAC-1 onto the Ben3 pPolI-WSN-PA plasmid, this plasmid and other plasmids that rescue influenza viruses were used: 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, Ben13 By co-transfecting pcAGGS / MCS-NP into the stable cell line constructed in Example 1, a mutant influenza virus in which TEVCs+PROTAC-1 was introduced onto the influenza virus PA gene fragment was rescued and named PA-TEVcs+PROTAC-1 (or PA-PTD1). After introducing the cleavable proteolytic target molecule TEVcs+PROTAC-1 into the C-terminus of the coding region of the M1 protein on the pPolI-WSN-M plasmid, this plasmid and other plasmids that rescue influenza viruses were used: 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, Ben13 By co-transfecting pcAGGS / MCS-NP into the stable cell line constructed in Example 1, a mutant influenza virus in which TEVCs+PROTAC-1 was introduced onto the influenza virus M1 gene fragment was rescued and named M1-TEVcs+PROTAC-1 (or M1-PTD1).

[0142] In a similar manner, mutant influenza viruses introducing cleavable proteolytic targeting molecules at other sites can be obtained and named according to the same rules.

[0143] All constructed PROTAC virus vectors were considered according to the criteria of whether they could induce cell lesions. If HEK293T-TEVp and / or MDCK-TEVp cell lesions could be induced, this PROTAC virus rescue was successful, and if HEK293T-TEVp and / or MDCK-TEVp cell lesions could not be induced, this PROTAC virus rescue was unsuccessful. The rescue status of some mutant influenza viruses is shown in Table 4 and Figure 4 as an example. Table 4 summarizes some of the PROTAC influenza virulence strains that can induce lesions in MDCK-TEVp cells. Figure 4 shows the virus titers in the cell supernatants detected the day after infection after some virulence strains were inoculated into MDCK-TEVp cells and normal MDCK cells (MOI = 0.01). The above results demonstrated that selectively cleavable proteolytic target molecules could be introduced into the eight gene fragments of influenza virus, where PA, PB2, PB1, M1, etc. could all rescue PROTAC influenza virus.

[0144] [Table 4] JPEG0007654625000023.jpg149169

[0145] From the above mutation sites and cleavable proteolytic target molecules, the inventors selected and combined mutation modifications that have high efficiency in rescuing influenza viruses, are genetically stable, and have a significantly reduced or even completely lost final replication ability in normal host cells to create influenza viruses containing multiple proteolytic target molecules, and further selected preferred toxin strains, which were named M1-TEVcs+PROTAC-1+PROTAC-2 (or M1-PTD6), M1-TEVcs+PROTAC-2+PROTAC-1 (or M1-PTD5), and PROTAC-V1 (Table 4 and Figure 5). The M1-TEVcs+PROTAC-1+PROTAC-2 was a PROTAC toxin strain containing two types of proteolytic target molecules, which was created by introducing PROTAC-1 and PROTAC-2 in tandem to the C-terminus of the viral M1 protein. M1-TEVcs+PROTAC-2+PROTAC-1 was a PROTAC venom strain containing two kinds of protein hydrolysis target molecules, which was prepared by introducing PROTAC-2 and PROTAC-1 in series into the C-terminus of the viral M1 protein. PROTAC-V1 was a PROTAC venom strain (Table 4) containing two kinds of protein hydrolysis target molecules, which was prepared by introducing TEVcs-PROTAC-1 into the C-terminus of the viral PA protein and TEVcs-PROTAC-2 into the C-terminus of the viral PB1 protein. Furthermore, Figure 11 provided a flow chart of preparing a protein hydrolysis target virus by the method of the present invention. EXAMPLES

[0146] Example 4: Expression and purification of site-directed mutant influenza viruses 1) Rescue of PROTAC viruses containing cleavable proteolytic target molecules The mutant influenza virus packaging plasmid obtained by the influenza virus rescue after modification by site-specific mutation in the step of Example 3 was co-transfected into the stable cell line of Example 1, and after 6 hours, the medium was replaced with a new medium containing 1% FBS and 2 μg / mL TPCK-trypsin, and normal cells were used as a control. The positive control used in 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 for the different plasmids used to rescue the virus. After the transfection was completed, the state of the cells was observed every day, and mutants that developed lesions in the TEVp stable cell line but did not develop lesions or had relatively few lesions in normal cells were considered positive mutants. Wild-type influenza virus developed lesions in both the TEVp stable cell line and the normal cell line.

[0147] 2) PROTAC influenza virus purification a. Step 1) When the stable cell line rescuing the mutant PROTAC influenza virus was completely infected or about 4 days after transfection, the cell supernatant was collected and centrifuged at 5000g for 10 minutes, and then expanded in large quantities using a new stable cell line. When the cell was completely infected or about 4 days after expansion, the cell supernatant was collected and filtered through a 0.45 μm filter membrane. b. Influenza virus was purified using sucrose gradient density centrifugation. The specific steps are as follows: 1) 10 ml of the virus solution was placed in a 50 mL centrifuge tube (high speed only). 5 The mixture was centrifuged at 4°C for 2 h and the pellet was resuspended in 1 mL of PBS. c. Sucrose was dissolved in NTE Buffer (100 mM NaCl, 10 mM Tris-Cl, pH 7.4, 1 mM EDTA) to prepare a 20% sucrose solution, which was then filtered through a 0.45 μm filter membrane. d. The sucrose from step 3) was added to a 50 mL or 15 mL centrifuge tube, and the PBS resuspension from step 2) was added dropwise to the sucrose solution. 4 g and centrifuged for 2 h. e. Add about 15 mL of NTE buffer to the precipitate and dissolve in 11 x 104 Centrifugation was continued at RT for 2 h. f. The precipitate from step 5) was resuspended in PBS. EXAMPLES

[0148] Example 5: Safety considerations at the cellular level of proteolytically targeted influenza viruses The inventors verified the safety of the PROTAC viruses by examining the dependency of the TEVp protein on the 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 that they had created. They examined the stability of the cleavable proteolytic target molecule in the mutant viruses by performing long-term subculture.

[0149] Specific experiment 1: The mutant PROTAC influenza virus prepared was infected into MDCK-TEVp cells and normal MDCK cells at an MOI of 0.01. After 3-4 days, the supernatant was taken and the virus titer was detected. The virus titer of the PROTAC virus in MDCK-TEVp cells was set as 100%. The relative virus titers of the PROTAC virus in MDCK-TEVp cells and normal MDCK cells were compared, revealing the difference in the replication ability of the virus in the two types of cells.

[0150] Specific experiment 2: The newly constructed mutant PROTAC influenza virus was inoculated into new medium at a ratio of MOI=0.01 to infect stable cells, and the medium contained 1% FBS and 2μg / mL TPCK-trypsin, and the normal cell line was used as a control. When the TEVp stable cell line was completely diseased, the supernatant was removed and filtered through a 0.45μm filter membrane, and inoculated into new medium at a ratio of MOI=0.01 to infect stable cells, and the normal cell line was also used as a control. This was repeated to perform long-term virus passage. Gene sequencing was used to detect whether mutations occurred in the introduced protein hydrolysis target molecule.

[0151] As can be seen from Figure 6, the PROTAC virus can replicate in MDCK-TEVp cells, but its replication ability is significantly reduced in normal MDCK cells, and even replication is impossible. This explains the dependency on TEVp and the safety of this virus. As can be seen from Figure 7, after long-term passage, the proteolytic target molecule introduced into the mutant PROTAC virus virulence strain did not mutate. As can be seen from the above results, the cleavable proteolytic target molecule introduced into the influenza virus gene was stable and genetically stable. EXAMPLES

[0152] Example 6. Discuss whether attenuation of influenza virus replicative capacity by proteolysis is controlled by the cellular proteasome The inventors used M1-TEVcs+PROTAC-1 and M1-TEVcs+PROTAC-2 as representative virulent strains and considered whether the attenuation of the replication ability of the designed PROTAC influenza viruses in normal cells is mediated by the intracellular ubiquitin-proteasome system.

[0153] Specific experiment 1: The mutant PROTAC influenza virus or wild-type virus prepared was infected into normal MDCK cells (MOI=0.1), and the medium was supplemented with 100nM proteasome inhibitor MG-132 or DMSO at the same dilution ratio as a control. At 24 and 48 hours after infection, respectively, cell samples were collected and the viral M1 protein levels were detected by western blot.

[0154] Specific experiment 2: The mutant PROTAC influenza virus or wild-type virus prepared was infected into MDCK-TEVp cells and normal MDCK cells (MOI=0.01), and the medium was supplemented with different concentrations (0, 50, or 100 nM) of the proteasome inhibitor MG-132 or the same dilution ratio of DMSO as a control. 48 hours after infection, the cells were fixed with 4% PFA, and the viral NP protein level was detected using immunofluorescence experiments.

[0155] As can be seen from Figure 8, after the wild-type virus infects MDCK cells, it can replicate in large quantities and produce large amounts of viral protein. In contrast, after modifying the viral protein M1 of the PROTAC virus with the proteolytic targeting molecule TEVcs+PROTAC-1 or TEVcs+PROTAC-2, 48 hours after infection, the viral M1 protein is degraded, while the addition of the proteasome inhibitor MG-132 to the culture medium inhibits the degradation of the viral protein M1 mediated by the proteasome. As can be seen from this experimental result, the proteolytic targeting molecule introduced by the inventor can mediate the degradation of viral protein, and the reduction in the replication ability of the PROTAC virus in normal cells is due to the degradation of viral protein mediated by the proteasome, which is consistent with the inventor's principle.

[0156] As can be seen from the immunofluorescence experiment results in Figure 9, after the PROTAC virus infects MDCK-TEVp cells, it can replicate in large quantities and synthesize large amounts of viral proteins. Since the PROTAC virus cannot replicate in large quantities after infecting normal MDCK cells, relatively little signal of the viral protein NP was detected. Meanwhile, when the cellular proteasome system was inhibited, the signal of the viral protein NP increased, explaining that the inhibition of the proteasome system enhances the replication ability of the virus. This result is consistent with the results in Figure 8, and further proves that the introduction of protein hydrolysis targeting molecules can mediate the degradation of the cellular proteasome against the viral protein, which can further inhibit the replication ability of the virus, and the inhibition of the cellular proteasome system can restore the replication ability of the virus.

[0157] As can be seen from the above results, the reduction or loss of the replication ability of the PROTAC virus was mediated by the cellular ubiquitin-proteasome system, which was consistent with the inventors' design expectations for the PROTAC virus. EXAMPLES

[0158] Example 7. Immunogenicity and protection of proteolytically targeted influenza viruses (PROTAC influenza viruses) at the animal level In this study, we used ferrets (provided by Wuxi Cay Ferret Farm, Jiangsu, China) to evaluate the immunogenicity and efficacy of PROTAC viruses at the animal level. Inactivated influenza vaccine (IIV) (the inactivated influenza virus vaccine was produced by the inventors using homogeneous influenza virus particles according to the method provided by the Chinese Pharmacopoeia) was used as a control, and M1-TEVcs+PROTAC-1 was selected as a representative PROTAC virus to evaluate the immunogenicity and protective properties of the PROTAC virus.

[0159] Specific experiments: 1) Nine female ferrets aged 4 to 6 months were divided into three groups, with three animals in each group. 2) Viral vaccination: The first group received nasal PBS vaccination, and the second group received 10 5 One group was inoculated with PFU M1-TEVcs-PROTAC-1 by nasal drop, and the third group was inoculated with the same dose of inactivated virus. 3) Three weeks after vaccination, serum samples were collected from each group for hemoglobin inhibition (HI) tests and neutralizing (NT) antibody detection. 4) Three weeks after inoculation, 10 7 PFU of wild-type virus WSN was inoculated intranasally. 5) Three days after inoculation with the wild-type virus, lung tissues were harvested and the virus content was measured by plaque assay.

[0160] The results are shown in Figure 10, where the PROTAC virus can induce high levels of anticoagulant antibody titers (A) and neutralizing antibody titers (B) in animals. The anticoagulant and neutralizing antibody levels induced by the PROTAC virus are significantly higher than those induced by the inactivated vaccine. Vaccination with the PROTAC virus vaccine can significantly reduce the wild-type virus titers (C) in the lung tissue of animals, and the protection provided by the PROTAC virus vaccine is significantly better than that of the inactivated vaccine.

[0161] The above description is merely some embodiments of the present invention. Those skilled in the art may make various modifications and improvements without departing from the above principles of the present invention, and these modifications and improvements are also within the scope of protection of the present invention.

Claims

1. a proteolytically targeted influenza virus, wherein the site of one or more distinct viral proteins comprises one or more proteolytically targeted molecules recognizable by the ubiquitin-proteasome system, and the viral proteins and the proteolytically targeted molecules are linked via one or more linker chains selectively cleavable by TEV protease; the site is at the C-terminus and / or N-terminus of the viral protein; the viral protein is one or more of the following influenza virus proteins: PA, PB1, PB2, NP, HA, NA, M1, M2, NS1, NEP; The proteolytic target molecule is composed of an amino acid sequence shown in any one of SEQ ID NOs: 1-110; The linker is composed of an amino acid sequence shown in any one of SEQ ID NOs: 112-137; Proteolytic targeting of influenza virus.

2. The proteolytic target influenza virus described in claim 1, further comprising a flexible linker between the proteolytic target molecule and the connecting chain.

3. A proteolytically targeted influenza virus as described in claim 2, wherein the proteolytically targeted molecule, connecting chain and flexible linker have a connection method of flexible linker-connecting chain-flexible linker-proteolytically targeted molecule.

4. The proteolytic target influenza virus described in claim 3, wherein the flexible linker-linking chain-flexible linker-proteolytic target molecule is composed of an amino acid sequence shown in any one of SEQ ID NOs: 138-149, 167 and 168.

5. The protein hydrolysis targeted influenza virus of claim 1, wherein the virus is a modified virus.

6. The protein hydrolysis targeted influenza virus of claim 1, wherein the virus is an H1N1, H5N1, H7N9, H3N2 or influenza B virus.

7. Influenza virus PA and PB2 each contain one or more proteolytic target molecules and linking chains; Both the influenza virus PA protein and the PB1 protein contain one or more proteolytic target molecules and linking chains; Both the PB2 and PB1 proteins of influenza viruses contain one or more proteolytic target molecules and linking chains; The influenza virus PA protein, PB2 protein, and PB1 protein each contain one or more linking chains to a proteolytic target molecule; The influenza virus PA protein, PB2 protein, PB1 protein, and M1 protein each contain one or more linking chains to a proteolytic target molecule; The influenza virus PA protein, PB2 protein, PB1 protein, M1 protein, and NP protein each contain one or more linking chains to a proteolytic target molecule; The PB2 protein, PB1 protein, and M1 protein of influenza virus each contain one or more proteolytic target molecules and linking chains; The PB1 protein and M1 protein of influenza virus each contain one or more proteolytic target molecules and linking chains; The PB2 protein and M1 protein of influenza virus each contain one or more proteolytic target molecules and linking chains; The PB2 protein, PB1 protein, M1 protein, and NS1 protein of influenza virus each contain one or more linking chains to a proteolytic target molecule; The PB2 protein, the PB1 protein, the M1 protein, and the NEP protein of influenza virus each contain one or more linking chains to a proteolytic target molecule, or 2. The proteolytic target influenza virus according to claim 1, wherein the NS1 protein and NEP protein of the influenza virus each contain one or more linking chains to the proteolytic target molecule.

8. A nucleic acid molecule encoding the proteolytic target influenza virus according to any one of claims 1 to 7.

9. A nucleic acid vector expressing the proteolytic target influenza virus of any one of claims 1 to 7.

10. A method for producing a proteolytically targeted influenza virus according to any one of claims 1 to 7, comprising the steps of: Construction of a cell line: Step 1) of constructing a cell line capable of stably expressing a protease having a selective cleavage activity against the junction of a proteolytic target virus; Step 2) site selection: performing bioinformatics and protein structure prediction for the virus by statistically analyzing the expression distribution of the ubiquitin-proteasome system in the host body to determine the viral protein and site of the proteolytic target molecule and the linking chain to be introduced; 3) gene mutation: using genetic engineering methods to introduce nucleoside sequences encoding the proteolytic target molecule and the linker into the coding gene of the determined viral protein and the selected site; Construction of an expression vector: Step 4) operably linking the coding nucleotide sequence of the viral protein after gene mutation obtained in step 3) to a vector to obtain an expression vector; Step 5) using reverse genetics technology to co-transfect the expression vector in step 4) and another expression vector for influenza virus rescue into a cell line having selective cleavage to the linking chain of the proteolytic target virus constructed in step 1) to obtain a proteolytic target virus; Optionally, step 6) of replicating the proteolytic target virus in the cell line obtained in step 1) to produce said proteolytic target virus, Alternatively, the method may further comprise the steps of: Site selection: By statistically analyzing the expression distribution of the ubiquitin-proteasome system in the host body, bioinformatics and protein structure prediction are performed on the virus to determine the viral protein and site of the proteolytic target molecule and the linking chain to be introduced [1]; Genetic mutagenesis: using genetic engineering methods to introduce nucleoside sequences encoding the proteolytic target molecule and the linker into the coding gene of the determined viral protein and selected site [2]; Construction of an expression vector for the mutated sequence: operably linking the coding nucleotide sequence of the mutated viral protein obtained in step [2] to a vector to obtain an expression vector [3]; Construct an overexpression vector for a protease that selectively cuts the junction of a protein hydrolysis target virus; [4] constructing a cell line capable of stably expressing a protease having selective cleavage activity against the junction of a proteolytic target virus; and step [5] of co-transfecting the expression vector obtained in step [3], other expression vectors required for virus rescue, and the expression vector obtained in step [4] into a host cell using reverse genetics techniques, culturing the successfully transfected host cell in a medium, and obtaining a protein hydrolysis target virus.

11. The method of claim 10, wherein in step 1), the cell line is a mammalian cell line.

12. The method according to claim 10, wherein in step 1), 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-bl cells, Sup-T1 cells, MRC-5 cells, VMK cells, LLC-MK2 cells, HCT-8 cells, Huh-7 cells, and Caco2 cells.

13. The method described in claim 12, wherein the cell line is selected from a HEK293T cell line and an MDCK cell line.

14. The method of claim 10, wherein the cell line is any ubiquitin-proteasome system-deficient cell line.

15. The method described in claim 10, wherein the cell line is an E3 ligase knockout or knockdown cell line.

16. The method described in claim 10, wherein in step 4) or step [3], the expression vector is a plasmid.

17. The method described in claim 10, wherein a proteasome inhibitor is added during the virus production process.

18. The method described in claim 17, wherein the proteasome inhibitor is MG132, MG-341 or lactacystin.

19. The method comprises: Testing: determining whether the modification of the proteolytic target virus is successful by measuring the replicative ability of the proteolytic target virus obtained in step 5) in the cell line obtained in step 1) and in unmodified normal host cells, wherein the proteolytic target virus that replicates in the cell line obtained in step 1) and has reduced replicative ability or cannot replicate in unmodified normal host cells is the proteolytic target virus that has been successfully modified; Optionally, the method further comprises: Step 8) is further included in which the steps 2) to 5) are repeated using the successfully modified proteolytic target virus to introduce a proteolytic target molecule and a linking chain onto each of a plurality of viral proteins of the proteolytic target virus, or to introduce a plurality of proteolytic target molecules and linking chains onto any one viral protein of the proteolytic target virus; Optionally, the method according to claim 10, further comprising measuring the replication ability of the obtained proteolytic target virus in the cell line obtained in step 1) and in unmodified normal host cells to determine whether the modification of said proteolytic target virus has been successful, wherein said proteolytic target virus that replicates in the cell line obtained in step 1) and has reduced or no replication ability in unmodified normal host cells is a successfully modified proteolytic target virus.

20. Construction of a cell line: Step 1) of stably transducing a TEV protease TEVp into a mammalian cell line to construct a cell line capable of stably expressing the TEV protease TEVp; 2) site selection: performing bioinformatics and protein structure prediction for influenza virus through statistical analysis of the expression distribution of the ubiquitin-proteasome system in the host body, and predicting and analyzing the protein structure of influenza virus after introducing the proteolytic target molecule and the linking chain TEV protease cleavage sequence into each protein of influenza virus, so as to determine the gene fragment and the site for introducing the proteolytic target molecule and the linking chain; 3) gene mutation: using genetic engineering methods to introduce nucleoside sequences encoding the proteolytic target molecule and the linker into the coding gene of the determined influenza virus protein and the selected site; Construction of plasmid: the coding nucleotide sequence of the viral protein after gene mutation obtained in step 3) is operably linked to a plasmid to obtain a coding plasmid; step 4); Step 5) using reverse genetics technology to co-transfect the plasmid obtained in step 4) and other plasmids for influenza virus rescue into a cell line stably expressing the TEVp obtained in step 1) to obtain a proteolytic target influenza virus; 20. The method of claim 10 or 19, optionally comprising step 6) producing the proteolytic targeted influenza virus in a stable cell line stably expressing TEVp.

21. The method described in claim 20, wherein in step 1), the mammalian cell line is a HEK293T cell line or an MDCK cell line.

22. The method described in claim 20, wherein in step 2), one or more insertion sites are selected from gene fragments of different proteins encoded by influenza viruses.

23. The method according to claim 22, Testing: further comprising step 7) of determining whether the proteolytic target influenza virus obtained in step 5) is successfully modified by measuring its dependency on TEVp and its dependency on the proteasome pathway for inactivation of its packaging product; Optionally, the method further comprises: Step 8) is further included in which the proteolytic target influenza virus vector is used to introduce the proteolytic target molecule and the linking chain onto multiple viral proteins of the proteolytic target influenza virus, or steps 2) to 5) are repeated to introduce multiple proteolytic target molecules and linking chains onto any one viral protein of the proteolytic target influenza virus; Optionally, the method further comprises: 9) selecting successful modified candidates and purifying the products; The method of claim 20, further comprising: (a) a step of conducting safety or immunogenicity testing on the proteolytic target influenza virus in step 9), wherein compared with the wild-type virus, the safe influenza virus is the successfully modified influenza virus in step 10).

24. The method according to claim 23, wherein in step 8), the successful modification of the proteolysis targeted influenza virus is determined by measuring the dependency of the obtained proteolysis targeted influenza virus on TEVp and the dependency of inactivation of its packaging product on the proteasome pathway, and the proteolysis targeted virus that still maintains its dependency on TEVp after long-term passaging is retained as a candidate for successful modification.

25. Methods for producing live attenuated viruses, live replication incompetent viruses, replication regulatable viruses, and related vaccines and drugs for preventing and treating viral infections, comprising: A method comprising utilizing a proteolytically targeted influenza virus according to any one of claims 1 to 7 or producing a proteolytically targeted virus using a method according to any one of claims 10 to 24.

26. A vaccine or pharmaceutical composition comprising a proteolytic targeted virus according to any one of claims 1 to 7.

27. The vaccine or pharmaceutical composition of claim 26, wherein the vaccine is a live attenuated vaccine, a live replication-incompetent vaccine or a live replication-controllable vaccine.

28. A system for producing a proteolytic target influenza virus according to any one of claims 1 to 7, comprising: The system includes a cell line stably expressing a protease having selective cleavage activity against the junctional chain of a proteolytic target virus.

29. The system described in claim 28, wherein the cell line is a cell line that stably expresses TEV protease TEVp.

30. The system described in claim 28, wherein the cell line is a HEK293T cell line or an MDCK cell line stably expressing TEV protease TEVp.

31. The system described in claim 28, wherein the cell line is any ubiquitin-proteasome system-deficient cell line.

32. The system described in claim 28, wherein the cell line is an E3 ligase knockout or knockdown cell line.

33. The system described in claim 28, further comprising a nucleic acid vector expressing a protein hydrolysis targeted influenza virus described in any one of claims 1 to 7.

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