Recombinant virus, preparation method therefor and use thereof

Abstract

Provided are a recombinant virus, a preparation method therefor and the use thereof. A viral protein of the recombinant virus contains at least one of a peptide segment recognizable by midnolin, a peptide segment recognizable by heat shock protein 70 (HSC70), or a peptide segment recognizable by microtubule-associated protein 1A / 1B-light chain 3 (LC3). The recognizable peptide segments are introduced into the viral protein to control virus replication; since there are a plurality of protein degradation systems and corresponding recognizable peptide segments thereof, the present invention has high applicability and few limiting factors, and thus can be applied to the preparation of all virus vaccines and only requires simple virus vector construction and virus packaging, which involve mature techniques and have high success rates, thus facilitating promotion, achieving the preparation of vaccines with different extents of inactivation, achieving controllable safety, and endowing the prepared vaccines with good immunogenicity.

Description

A recombinant virus, its preparation method and application Technical Field

[0001] This application belongs to the field of biotechnology and relates to a recombinant virus, its preparation method, and its application. Background Technology

[0002] Vaccines are biological agents used to prevent infectious diseases. They prevent disease by stimulating the body's immune system to produce a protective response. Types of vaccines include live attenuated vaccines, inactivated vaccines, subunit vaccines, vector vaccines, and nucleic acid vaccines. Live attenuated vaccines: Made using attenuated or non-virulent but replicating microorganisms, they can multiply in the body and produce long-term immune protection, such as measles and polio vaccines. Inactivated vaccines: Made from killed pathogens, they cannot multiply in the body and therefore usually require multiple doses to maintain immunity, such as inactivated hepatitis A vaccine. Subunit and peptide vaccines: Contain specific parts of the pathogen, such as proteins or polysaccharides; they have weaker immunogenicity and may require adjuvants. Vector vaccines: Utilize harmless microorganisms as vectors to carry the pathogen's antigenic genes, inducing an immune response. Nucleic acid vaccines: Including DNA and mRNA vaccines, they stimulate an immune response by delivering genetic material.

[0003] Over the past few decades, viral vaccine technology has made significant progress; however, certain limitations remain. For example, viral inactivation processes can destroy the natural structure of the antigen, reducing the immunogenicity of the vaccine; insufficient viral attenuation can leave residual viral replication activity, posing safety risks; and the complexity of viral vaccine preparation techniques prevents its application to other viral vaccines. Therefore, breaking through traditional viral vaccine design concepts and developing a novel vaccine technology that is safe, efficient, simple, and universal is a crucial direction for the future development of ideal viral vaccines, possessing significant scientific and clinical value.

[0004] In conclusion, developing safer, more widely applicable, and more immunogenic methods for preparing viral vaccines is of great significance to the vaccine field. Summary of the Invention

[0005] This application provides a recombinant virus, its preparation method, and its application. By inserting at least one recognition peptide into at least one protein-coding gene of the virus, the prepared recombinant virus can be recognized by the protein degradation system in the host cell and degrade the viral protein. The virus's replication ability is weakened or even completely lost, resulting in a safer vaccine with broad application prospects.

[0006] In a first aspect, this application provides a recombinant virus, wherein the viral protein of the recombinant virus contains at least one of a peptide recognized by midnolin, a peptide recognized by heat shock protein 70 (HSC70), or a peptide recognized by microtubule-associated protein 1A / 1B-light chain 3 (MAP1LC3, LC3).

[0007] In this application, a peptide recognized by Midnolin, HSC70, or LC3 is inserted into a viral protein to obtain a recombinant virus. This can induce the viral protein to degrade in the cell's proteasome or lysosome, reducing the replication ability of the prepared virus and thus weakening its virulence. This increases the safety of the recombinant viral strain, which can be used as a vaccine. It can be efficiently replicated and mass-produced in specific cell lines (such as engineered cell lines overexpressing tobacco etch virus protease (TEVp) or engineered cell lines with protein degradation system defects). However, in normal cells, the virus's replication ability is weakened or even completely lost, contributing to the development of the vaccine field.

[0008] It is understandable that the types and numbers of viral proteins and recognition peptides can be freely designed according to needs, such as introducing multiple recognition peptides into multiple viral proteins.

[0009] Preferably, the peptide recognized by Midnolin is selected from at least one peptide whose amino acid sequence includes the peptides shown in SEQ ID NO.1 to SEQ ID NO.12.

[0010] Preferably, the peptide recognized by HSC70 contains at most two hydrophobic residues, at most two positively charged residues and one negatively charged residue, flanked by N- or having a C-terminus composed of a glutamine residue. Preferably, the peptide recognized by HSC70 is selected from at least one peptide whose amino acid sequence includes those shown in SEQ ID NO. 13 to SEQ ID NO. 26.

[0011] Preferably, the core formula of the peptide recognized by LC3 is [W / F / Y]xx[L / I / V], where [W / F / Y] represents one of tryptophan, phenylalanine, or tyrosine, "xx" represents any two amino acids, and [L / I / V] represents one of leucine, isoleucine, or valine. Preferably, the amino acid sequence of the peptide recognized by LC3 includes the sequence shown in SEQ ID NO.30.

[0012] Preferably, the originating virus of the recombinant virus includes influenza virus, respiratory syncytial virus, HIV, SARS-CoV-2, hand-foot-and-mouth disease virus, Coxsackie virus, hepatitis C virus, hepatitis B virus, hepatitis A virus, hepatitis D virus, hepatitis E virus, human herpesvirus, human papillomavirus, herpes simplex virus, cytomegalovirus, varicella-zoster virus, vesicular stomatitis virus, dengue virus, Ebola virus, Marburg virus, Zika virus, severe acute respiratory syndrome virus, Middle East respiratory syndrome virus, rotavirus, rabies virus, and measles virus. The virus may be any one of the following: adenovirus, poliovirus, echovirus, Japanese encephalitis virus, tick-borne encephalitis virus, Hantavirus, novel enterovirus, rubella virus, mumps virus, parainfluenza virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus, foot-and-mouth disease virus, parvovirus, prions, smallpox virus, tobacco mosaic virus, bacteriophage, herpesvirus, West Nile virus, norovirus, human bocavirus, or coronavirus, preferably any one of influenza virus, respiratory syncytial virus, HIV, or porcine reproductive and respiratory syndrome virus.

[0013] In this application, each recognition peptide can be introduced into different sites of different viral proteins.

[0014] Preferably, the viral protein may include at least one of the following: influenza virus protein, respiratory syncytial virus protein, HIV protein, and porcine reproductive and respiratory syndrome virus protein.

[0015] Preferably, the influenza virus protein includes at least one of PA protein, PB1 protein, PB2 protein, NP protein, HA protein, NA protein, M1 protein, M2 protein, NS1 protein, or NEP protein.

[0016] Preferably, the respiratory syncytial virus protein includes at least one of the following: N protein, P protein, M2-1 protein, M2-2 protein, NS1 protein, NS2 protein, G protein, F protein, SH protein, L protein, and M protein.

[0017] Preferably, the HIV protein includes at least one of Vpu protein, Vpr protein, Vif protein, Nef protein, Tat protein, Rev protein, Gag protein, Env protein, and Pol protein.

[0018] Preferably, the proteins of the porcine reproductive and respiratory syndrome virus include at least one of the following: NSP1α protein, NSP1β protein, NSP2 protein, NSP3 protein, NSP4 protein, NSP5 protein, NSP6 protein, NSP7 protein, NSP8 protein, NSP9 protein, NSP10 protein, NSP11 protein, NSP12 protein, GP2a protein, E protein, GP3 protein, GP4 protein, GP5 protein, GP5a protein, M protein, or N protein.

[0019] Preferably, specific sites may include influenza virus proteins PB2 (N-terminus, R70, I176, V457, N510, Y531, A623, D680, E700, C-terminus), PB1 (N-terminus, D70, D295, R327, R430, F490, T566, N626, G710, C-terminus), PA (N-terminus, L268, D294, N350, E372, D396, L425, D426, H510, A553, E604, S624, C-terminus), NP (N-terminus, G126, N247, R317, V353, A366, Q409, E465, M481, C-terminus), and M1 (N-terminus, A33, V68, D89, R105, M135). The following viruses are identified: Q164, H222, A239 (C-terminal), M2 (C-terminal), NEP (C-terminal), NS1 (N-terminal, A76, A82, H101, A122, T151, L163 (C-terminal), etc.; Respiratory syncytial virus (RSV) NS1 (N-terminal, L16, T31, N52, D64, P81, P101, L104, K116 (C-terminal), NS2 (N-terminal, R19, K51, D100, H113 (C-terminal), N (N-terminal, M1, A57, H59, V95, N105, A117, E144, D175, T233 (C-terminal), P (N-terminal, T160, R163 (C-terminal), M (N-terminal, H9, K25, Q40, N54, P65, S76, T136) V153, D208, P218, E231, C-end); SH (N-end, E2, H51, F55, P58, V62, C-end); G (N-end, C-end); F (N-end, N67, L172, S182, P207, M264, N325, A346, P389, G418, K427, N444, V459, P480, C-end); M2-1 (N-end, I87, L120, S182, C-end); M2-2 (N-end, H9, K25, Q40, N54, P65, S76, T136, V153, D208, P218, E231, C-end); L (N-end, Y94, K256, P503, G754, N762) L823, D1026, L1031, D1047, E1190, Y1232, T1236, V1248, T1259, L1421, C-end, etc.;HIV Gag (A45, A146, A224, G340, N124, P279, Q308, R22, S129, T70, V135, V143, V218), Pol (E70, P91, K189, G273, K160, K241, Q215), Nef (E179, K94, P75, P129, E179, E154, H199), Rev (E11, P29), Tat (A21, P6, S16, Y47), Vif (F112, G71, K92, N3, P162, R41, R127, R132), Vpr (D52, E13, P35, Q8), etc.; Porcine reproductive and respiratory syndrome virus NSP9 (A31, A61, V82, G131, A196, L206, I310, L345, I380, A420, G455, P490, T530, A610, A680), NSP1α (Y60, N100, V110, I140), NSP1β (L50, G77, T100, V130, L170), NSP11 (D20, L50, Y60, F90, E100, L160), etc.

[0020] In this application, when the recognition peptide is introduced to the N-terminus or C-terminus of the viral protein, the recognition peptide can be directly linked to the viral protein, or the recognition peptide can be linked to the viral protein through a linker peptide that can be cleaved by TEVp (such as ENLYFQG (SEQ ID NO. 45)). When the recognition peptide is introduced to other sites on the viral protein, the peptide can be directly linked to the viral protein.

[0021] Secondly, this application provides a nucleic acid molecule that encodes the recombinant virus described in the first aspect.

[0022] Thirdly, this application provides a recombinant vector containing the nucleic acid molecules described in the second aspect.

[0023] Fourthly, this application provides an engineered cell that can support the preparation of the recombinant virus described in the first aspect, specifically including an engineered cell line overexpressing tobacco etch virus protease (TEVp) and an engineered cell line with a protein degradation system defect.

[0024] Fifthly, this application provides a preparation system for the recombinant virus described in the first aspect, the preparation system comprising the engineered cells described in the fourth aspect, the nucleic acid molecules described in the second aspect, and the virus rescue plasmid.

[0025] In this application, since protein degradation systems are widely distributed in host cells, to prevent the recombinant virus from being degraded by the intracellular protein degradation system during preparation, thus reducing production efficiency, this application provides an engineered cell line overexpressing tobacco etch virus protease (TEVp) and an engineered cell line deficient in the protein degradation system. In this artificially modified engineered cell line, the recognition peptide is not recognized, and the viral protein is not degraded by the protein degradation system and is thus preserved. Therefore, the recombinant virus can be efficiently replicated and produced in large quantities in this specific artificially modified engineered cell line. In normal cells, the protein degradation system recognizes the recognition peptide fused with the viral protein, thereby degrading the viral protein. The viral replication ability is weakened or even completely lost, thus the recombinant virus has extremely high safety.

[0026] Sixthly, this application provides a method for preparing the recombinant virus described in the first aspect, the method comprising:

[0027] Construct the engineered cells described in the fourth aspect;

[0028] The coding nucleic acid of the recombinant virus is inserted into the expression vector to obtain the recombinant vector;

[0029] The recombinant vector was introduced into the engineered cells and packaged to obtain the recombinant virus.

[0030] In a seventh aspect, this application provides a vaccine containing the recombinant virus described in the first aspect.

[0031] Preferably, the vaccine includes any one of a live attenuated vaccine, a live attenuated vaccine, a live attenuated vaccine with controllable replication, or an oncolytic virus vaccine.

[0032] In this application, the dependence of the recombinant virus on a specific virus production system allows for the large-scale preparation of the recombinant virus within that system. Because normal cells in humans and animals possess protein degradation systems that can recognize recognition peptides fused with viral proteins, thereby degrading the viral proteins, the prepared recombinant virus exhibits reduced or no replication ability in animals and humans, increasing viral safety and thus enabling the recombinant virus to become an attenuated live virus vaccine.

[0033] Preferably, the vaccine further includes adjuvants and excipients.

[0034] Preferably, the excipients include any one or a combination of at least two of the following pharmaceutically acceptable carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

[0035] Eighthly, this application provides a drug containing the recombinant virus of the first aspect.

[0036] Preferably, the drug further includes any one or a combination of at least two of pharmaceutically acceptable carriers, diluents or excipients.

[0037] Compared with the prior art, this application has the following beneficial effects:

[0038] This application designs a novel recombinant virus that controls viral replication by introducing recognition peptides into viral proteins. Due to the existence of multiple protein degradation systems and their corresponding recognition peptides, it has greater applicability and fewer limiting factors. It can be applied to the preparation of all viral vaccines, and only requires simple viral vector construction and viral packaging. The technology is mature, has a high success rate, and is easy to promote. It can prepare vaccines with different degrees of inactivation, with controllable safety, and the prepared vaccines have good immunogenicity. Attached Figure Description

[0039] Figure 1 shows the efficiency of recombinant influenza virus preparation containing peptides that can be recognized by Midnolin in this application.

[0040] Figure 2 shows the safety results of the recombinant influenza virus containing a peptide that can be recognized by Midnolin at the cellular level in this application.

[0041] Figure 3 shows the results of verifying that viral proteins containing peptides that can be recognized by Midnolin can be recognized and bound by Midnolin.

[0042] Figures 4A and 4B show the results of verifying the attenuation mechanism of recombinant influenza virus containing peptides that can be recognized by Midnolin infecting cells, which can be enriched by proteasome inhibitors.

[0043] Figure 5 shows the design results demonstrating that this application conforms to the design of recombinant viral proteins being degraded via the proteasome without relying on ubiquitination modification.

[0044] Figure 6A shows the recombinant influenza virus (MIDTAR) containing a peptide that can be recognized by Midnolin, as described in this application. MTD1+2 The results demonstrate good safety at the animal level.

[0045] Figure 6B shows the recombinant influenza virus (MIDTAR) containing a peptide that can be recognized by Midnolin, as described in this application. dual MTDs The results demonstrate good safety at the animal level.

[0046] Figure 6C shows the recombinant influenza virus (MIDTAR) containing a peptide that can be recognized by Midnolin in this invention. MTD1+2The results show good safety in ferrets. Figure a shows the titer of the grinding fluid in the lungs of ferrets, and Figure b shows the titer of the grinding fluid in the nasal wash of ferrets.

[0047] Figure 7A shows an influenza virus (MIDTAR) containing a peptide that can be recognized by Midnolin, as described in this application. MTD1+2 The results of the vaccine demonstrating good immunogenicity at the animal level are shown in the following figures: Figure a shows neutralizing antibodies (NT) in serum; Figure b shows hemagglutination inhibition antibodies (HI); Figure c shows anti-HA IgG; Figure d shows anti-NP IgG; Figure e shows mucosal antibody anti-virus IgA; Figure f shows the T-cell immune response specific to influenza virus NP antigen (left image in the lung, right image in the spleen); Figure g shows the T-cell immune response specific to influenza virus PA antigen (left image in the lung, right image in the spleen).

[0048] Figure 7B shows an influenza virus (MIDTAR) containing a peptide that can be recognized by Midnolin, as described in this application. dual MTDs The results of the vaccine demonstrating good immunogenicity at the animal level are shown in the following figures: Figure a shows neutralizing antibodies (NT) in serum, Figure b shows hemagglutination inhibition antibodies (HI), Figure c shows anti-HA IgG, Figure d shows anti-NP IgG, Figure e shows mucosal antibody anti-virus IgA, and Figure f shows the T-cell immune response specific to influenza virus NP antigen, with the left figure showing the lung and the right figure showing the spleen.

[0049] Figure 8 shows the influenza virus (MIDTAR) containing a peptide that can be recognized by Midnolin in this invention. MTD1+2 ) as a vaccine (10 3 TCID 50 10 4 TCID 50 10 5 TCID 50The results of the immunogenicity of the immunization dose at the animal level are shown in the following figures: Figure 8A shows the neutralizing antibody (NT) in serum; Figure 8B shows the hemagglutination inhibition antibody (HI); Figure 8C shows the anti-HA IgG; Figure 8D shows the dilution gradient and absorbance of anti-HA IgG; Figure 8E shows the anti-NP IgG; Figure 8F shows the dilution gradient and absorbance of anti-NP IgG; Figure 8G shows the mucosal antibody anti-virus IgA; Figure 8H shows the T-cell immune response in the lungs specifically against influenza virus PA, NS1, NS2, and NP antigens; Figure 8I shows the T-cell immune response in the spleen specifically against influenza virus PA, NS1, NS2, and NP antigens; Figure 8J shows the intracellular kinase staining reaction in the lungs specifically against influenza virus PA, NS1, NS2, and NP antigens; Figure 8K shows the intracellular kinase staining reaction in the spleen specifically against influenza virus PA, NS1, NS2, and NP antigens.

[0050] Figure 9A shows an influenza virus (MIDTAR) containing a peptide that can be recognized by Midnolin, as described in this application. MTD1+2 The results of the vaccine providing good cross-immune protection in animals are shown in the figure. Figure a shows the viral titer in the lungs of mice after immunization with the homologous strain, Figure b shows the changes in mouse body weight and survival rate, Figure c shows the viral titer in the lungs of mice after immunization with the heterologous strain, and Figure d shows the changes in mouse body weight and survival rate.

[0051] Figure 9B shows an influenza virus (MIDTAR) containing a peptide that can be recognized by Midnolin, as described in this application. dual MTDs The results of the vaccine providing good cross-immune protection in animals are shown in the figure. Figure a shows the viral titer in the lungs of mice after immunization with the homologous strain, Figure b shows the changes in mouse body weight and survival rate, Figure c shows the viral titer in the lungs of mice after immunization with the heterologous strain, and Figure d shows the changes in mouse body weight and survival rate.

[0052] Figure 9C shows the influenza virus (MIDTAR) containing a peptide that can be recognized by Midnolin, as described in this invention. MTD1+2 ) as a vaccine (10 3 TCID 50 10 4 TCID 50 10 5 TCID 50 The results of the immunization dose providing good cross-immune protection at the animal level are shown in the figure. Figure a shows the viral titer in the lungs of mice after protection with the homologous strain, Figure b shows the changes in body weight and survival rate of mice after immunization with the homologous strain, Figure c shows the viral titer in the lungs of mice after immunization with the heterologous strain, and Figure d shows the changes in body weight and survival rate of mice after immunization with the heterologous strain.

[0053] Figure 10 shows the efficiency of recombinant influenza virus preparation containing peptides that can be recognized by HSC70 in this application.

[0054] Figure 11 shows the safety results of the recombinant influenza virus containing peptides that can be recognized by HSC70 at the cellular level in this application.

[0055] Figure 12 shows the results of verifying that viral proteins containing HSC70-recognized peptides can be recognized, bound, and degraded by HSC70. Figure a demonstrates that this application conforms to the design of introducing HSC70-recognized peptides into viral proteins to reduce viral protein levels. Figure b demonstrates the degradation of recombinant viral proteins containing HSC70-recognized peptides by lysosomes. Figure c demonstrates the interaction between recombinant viral proteins containing HSC70-recognized peptides and HSC70. Figure d uses overexpression plasmids to verify the degradation of viral proteins containing HSC70-recognized peptides by lysosomes. Figure e shows the degradation of viral proteins containing HSC70-recognized peptides in HEK293T wild-type cells. The results of verifying the attenuation mechanism of recombinant influenza virus infection containing a peptide recognized by HSC70 in A549 wild-type cells and LAMP2A knockout cells are shown in Figure f. Figure g shows the immunofluorescence staining of M1 protein of recombinant influenza virus and mutant virus in HEK293T wild-type cells and LAMP2A knockout cells. Figure h shows the immunofluorescence staining of M1 protein of recombinant influenza virus and mutant virus in A549 wild-type cells and LAMP2A knockout cells.

[0056] Figure 13 shows the CMATAR 2.0 generated by the recombination of the recombinant influenza virus in this application. dual CTMs The results show that recombinant influenza virus containing CMATAR 2.0 dual triCTMs (CMATAR 2.0) can still be recognized and bound by HSC70. Figure a demonstrates that recombinant viral proteins containing HSC70-recognizable peptides are degraded by lysosomes. Figure b uses an overexpression plasmid to verify the degradation of viral proteins containing HSC70-recognizable peptides by lysosomes. Figure c shows the results of attenuation mechanism of recombinant influenza virus infection containing HSC70-recognizable peptides in HEK293T wild-type cells and LAMP2A knockout cells. Figure d shows the results of attenuation mechanism of recombinant influenza virus infection containing HSC70-recognizable peptides in A549 wild-type cells and LAMP2A knockout cells. This verifies that viral proteins containing HSC70-recognizable peptides can be recognized and bound by HSC70.

[0057] Figure 14 shows the results of verification that viral proteins containing peptides recognizable by HSC70 in CMATAR 2.0 can be recognized, bound, and degraded by HSC70. Figure a shows the results of wild-type influenza virus and recombinant influenza virus CMA. Figure 2.0 shows the multi-cycle viral replication kinetics curves. Figure b demonstrates that this application conforms to the design of introducing HSC70-recognized peptides into viral proteins to reduce viral protein levels. Figure c demonstrates the degradation of recombinant viral proteins containing HSC70-recognized peptides by lysosomes. Figure d demonstrates the interaction between recombinant viral proteins containing HSC70-recognized peptides and HSC70. Figure e shows the results of verifying the attenuation mechanism of recombinant influenza virus infection containing HSC70-recognized peptides in HEK293T wild-type cells and LAMP2A knockout cells. Figure f shows the results of verifying the attenuation mechanism of recombinant influenza virus infection containing HSC70-recognized peptides in A549 wild-type cells and LAMP2A knockout cells. Figure g shows the immunofluorescence staining of M1 protein of recombinant influenza virus and mutant virus in HEK293T wild-type cells and LAMP2A knockout cells. Figure h shows the immunofluorescence staining of M1 protein of recombinant influenza virus and mutant virus in A549 wild-type cells and LAMP2A knockout cells.

[0058] Figure 15 shows the safety results of CMATAR 1.0 and CMATAR 2.0 recombinant influenza virus vaccines at the animal level. Figure a shows the changes in body weight and survival rate of CMATAR 1.0 recombinant influenza virus at the animal level; Figure b shows the good safety of CMATAR 1.0 recombinant influenza virus as a vaccine at the animal level; Figure c shows the changes in body weight and survival rate of CMATAR 2.0 recombinant influenza virus at the animal level; and Figure d shows the good safety of CMATAR 2.0 recombinant influenza virus as a vaccine at the animal level.

[0059] Figure 16 shows the immunogenicity results of the CMATAR 1.0 and CMATAR 2.0 recombinant influenza virus vaccines in this application at the animal level. Among them, af represents CMATAR 1.0, a represents neutralizing antibodies (NT) in serum, b represents hemagglutination inhibition antibodies (HI), c represents anti-HA IgG, d represents anti-NP IgG, e represents mucosal antibody anti-whole virus IgA, f represents T-cell immune response against recombinant influenza virus (left image in lung, right image in spleen), gl represents CMATAR 2.0, g represents neutralizing antibodies (NT) in serum, h represents hemagglutination inhibition antibodies (HI), i represents anti-HA IgG, j represents anti-NP IgG, k represents mucosal antibody anti-virus IgA, and l represents T-cell immune response against recombinant influenza virus (left image in lung, right image in spleen).

[0060] Figure 17 shows the results of cross-immunoprotection between CMATAR 1.0 and CMATAR 2.0 at the animal level. Figure a shows the viral titer in the lungs of mice after immunization with the same strain, Figure b shows the changes in mouse body weight and survival rate, Figure c shows the viral titer in the lungs of mice after immunization with the different strain, and Figure d shows the changes in mouse body weight and survival rate.

[0061] Figure 18A shows the efficiency of recombinant influenza virus preparation containing peptides that can be recognized by LC3 in this application.

[0062] Figure 18B shows the safety results at the cellular level for the recombinant influenza virus containing peptides that can be recognized by LC3 in this application.

[0063] Figure 19 shows the results of verifying that viral proteins containing peptides that can be recognized by LC3 can be recognized and bound by LC3, leading to degradation. In Figure A, it is shown that the recombinant viral protein containing peptides that can be recognized by LC3 is degraded via the lysosomal pathway. Figure B shows the results of verifying the degradation of viral proteins containing peptides that can be recognized by LC3 via the lysosomal pathway using overexpression plasmids. Figure C shows the results of verifying the attenuation mechanism of recombinant influenza virus infection containing peptides that can be recognized by LC3 in HeLa wild-type cells and LC3 knockout cells.

[0064] Figure 20 shows the safety results of the recombinant influenza virus containing a peptide that can be recognized by LC3 in the animal level. Figure a shows the changes in body weight and survival rate of the recombinant influenza virus in the animal level, and Figure b shows that the recombinant influenza virus has good safety as a vaccine in the animal level.

[0065] Figure 21 shows the immunogenicity results of the influenza virus containing a peptide that can be recognized by LC3 as a vaccine at the animal level. In the figure, a is the neutralizing antibody (NT) in serum, b is the hemagglutination inhibition antibody (HI), c is the anti-HA IgG, d is the anti-NP IgG, e is the mucosal antibody anti-virus IgA, and f is the T cell immune response against recombinant influenza virus. The left figure is in the lung and the right figure is in the spleen.

[0066] Figure 22 shows the results of cross-immunoprotection of influenza virus containing peptides recognizable by LC3 in this application at the animal level. Figure a shows the viral titer in the lungs of mice after immunization with homologous strains, Figure b shows the changes in mouse body weight and survival rate, Figure c shows the viral titer in the lungs of mice after immunization with heterologous strains, and Figure d shows the changes in mouse body weight and survival rate.

[0067] Figure 23 shows the results of verifying the attenuation mechanism of recombinant respiratory syncytial virus.

[0068] Figure 24 shows the safety results of the recombinant RSV virus vaccine in this application at the animal level.

[0069] Figure 25 shows the immunogenicity results of the recombinant RSV virus vaccine at the animal level.

[0070] Figure 26 shows the cross-immunization effect of the recombinant RSV virus vaccine at the animal level. Detailed Implementation

[0071] The technical solution of this application will be further described below with reference to the accompanying drawings and specific embodiments. However, the examples below are merely simplified examples of this application and do not represent or limit the scope of protection of this application. The scope of protection of this application shall be determined by the claims.

[0072] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0073] Design of Category I viral vaccines (recombinant viruses containing peptides that can be recognized by Midnolin):

[0074] Introducing peptides that can be recognized by Midnolin at appropriate sites on viral proteins can induce viral protein degradation at the cellular proteasome, reducing the replication capacity of the prepared virus and thus weakening its virulence. This increases the safety of the recombinant viral strain, which can then be used as a vaccine. In specific embodiments, influenza virus and respiratory syncytial virus (RSV) were used as model viruses for design validation.

[0075] Example 1

[0076] This embodiment constructs a recombinant influenza virus vector containing a peptide that can be recognized by Midnolin.

[0077] (1) The genome of wild-type influenza virus was synthesized and ligated into a pHH21 or pHW2000 vector via the BsmBI restriction site. The influenza virus genomes used were stored in GenBank (CY034138.1, CY034139.1, CY034135.1, CY034134.1, X17336.1, HE802059.1, L25818.1, CY034136.1; CY147541.1, CY147540.1, CY147539.1, CY147534.1, CY147537.1, CY147536.1, CY147535.1, CY147538.1).

[0078] (2) Peptides that can be recognized by Midnolin were introduced into different sites of different proteins of influenza virus to construct recombinant viral vectors. Specifically, gene sequences expressing peptides that can be recognized by Midnolin were introduced into different sites of the viral genome. This work was entrusted to Sangon Biotech (Shanghai) Co., Ltd. for assistance, and the successful construction of the mutation was verified by sequencing.

[0079] In this embodiment, the peptides that can be recognized by Midnolin in Table 1 are used as representatives for the study. Other peptides with the same biological characteristics are theoretically also applicable.

[0080]

[0081] In this embodiment, the following influenza virus proteins and sites are used as representatives for research, but other sites are theoretically also applicable:

[0082] PB2 (N-end, R70, I176, V457, N510, Y531, A623, D680, E700, C-end);

[0083] PB1 (N-end, D70, D295, R327, R430, F490, T566, N626, G710, C-end);

[0084] PA (N-terminal, L268, D294, N350, E372, D396, L425, D426, H510, A553, E604, S624, C-terminal);

[0085] NP (N-terminal, G126, N247, R317, V353, A366, Q409, E465, M481, C-terminal);

[0086] M1 (N-end, A33, V68, D89, R105, M135, Q164, H222, A239, C-end);

[0087] M2 (C-end);

[0088] NEP (C-end);

[0089] NS1 (N terminal, A76, A82, H101, A122, T151, L163, C terminal).

[0090] When a peptide recognized by Midnolin is introduced to the N-terminus or C-terminus of a viral protein, the peptide can be directly linked to the viral protein, or it can be linked to the viral protein via the linker peptide ENLYFQG (SEQ ID NO. 45), which can be cleaved by TEVp. When a peptide recognized by Midnolin is introduced to other sites on a viral protein, the peptide can be directly linked to the viral protein.

[0091] Example 2

[0092] This embodiment prepares a recombinant influenza virus containing a peptide that can be recognized by Midnolin.

[0093] When a peptide recognizable by Midnolin is linked to the viral protein via ENLYFQG (SEQ ID NO.45), the corresponding virus is prepared using HEK293T cells and MDCK cells expressing TEVp; when a peptide recognizable by Midnolin is directly linked to the viral protein, the corresponding virus is prepared using Midnolin-knockout HEK293T cells and MDCK cells. The cell lines used were constructed using conventional lentiviral overexpression and Crispr-Cas9 knockout technology. The detailed steps for virus preparation are as follows:

[0094] (1) Seeding cells: HEK293T cells (200,000 cells / well) expressing TEVp or Midnolin knockout and MDCK cells (50,000 cells / well) were seeded together in a 6-well plate and cultured for 24 h.

[0095] (2) Plasmid transfection: Plasmids were transfected using the TransIT-X2 Dynamic Delivery System (Mirus, Cat# MIR6003) or PEI (YEASEN, Cat# 40816ES03), with each plasmid weighing 0.2 μg. Six hours after transfection, the culture medium was replaced with DMEM medium containing 0.5% FBS and 2 μg / mL TPCK-trypsin.

[0096] (3) Viral supernatant collection and viral amplification: 3-4 days after transfection, the supernatant was collected and used to infect new MDCK cells expressing TEVp or Midnolin knockout. The culture medium was DMEM containing 0.5% FBS and 2 μg / mL TPCK-trypsin. The supernatant was collected 3-4 days after infection. Half-maximal tissue culture infection dose (TCID50) was used. 50 The titer of the prepared virus was determined by a assay.

[0097] The experimental results are shown in Table 2 below. The titers of some influenza viruses containing peptides that can be recognized by Midnolin are compared with the titers of wild-type influenza viruses (10). 7 -10 9 TCID 50 The ratio ( / mL) is comparable. Furthermore, the same strain can contain multiple peptides that can be recognized by Midnolin.

[0098]

[0099]

[0100]

[0101]

[0102] Example 3

[0103] This embodiment evaluates the efficiency of recombinant influenza virus preparation containing peptides that can be recognized by Midnolin and its safety at the cellular level.

[0104] This embodiment uses PA-C MTD1 NS1-N MTD1 NS1-N MTD2 MIDTAR MTD1+2 MIDTAR dual MTDs PA-N350 MTD4 +NS1-L163 MTD5 PA-D294 MTD3 -N350 MTD5 +NS1-A76 MTD4 -L163 MTD5 To evaluate the preparation efficiency and safety at the cellular level of recombinant influenza virus, this study aims to use a representative example.

[0105] (1) Efficiency of recombinant influenza virus preparation

[0106] Recombinant influenza virus and wild-type (WT) influenza virus were used to infect MDCK cells stably expressing TEVp protein or Midnolin knockout MDCK cells (MOI=0.001), respectively. The culture medium used was DMEM containing 0.5% FBS and 2 μg / mL TPCK-trypsin. On days 1, 2, 3, and 4 post-infection, the culture supernatant was collected and analyzed using TCID50. 50 The viral titer was detected experimentally. The results are shown in Figure 1. In MDCK cells expressing TEVp protein or MDCK cells with Midnolin knockout, the growth curve of the recombinant influenza virus was comparable to that of the wild-type influenza virus, indicating that the recombinant influenza virus had high preparation efficiency.

[0107] (2) Safety of recombinant influenza virus at the cellular level

[0108] Normal MDCK cells were infected with recombinant influenza virus and wild-type (WT) influenza virus at an MOI ratio of 0.001. TCID was administered on days 1, 2, 3, and 4 post-infection. 50 The viral titer in the culture supernatant was measured experimentally. The results, shown in Figure 2, indicate that the replication ability of recombinant influenza virus was significantly weakened in normal MDCK cells compared to wild-type influenza virus; the growth curve of recombinant influenza virus containing multiple peptides recognized by Midnolin was particularly significantly reduced. These results suggest that recombinant influenza virus is safe at the cellular level, and introducing multiple peptides recognized by Midnolin into the virus can further enhance its safety.

[0109] Example 4

[0110] This embodiment verifies the attenuation mechanism of recombinant influenza virus.

[0111] Immunoprecipitation was used to detect whether recombinant viral proteins could interact with Midnolin. In HEK293T cells expressing Midnolin, overexpression of wild-type viral proteins (such as PA and NS1) and recombinant viral proteins (such as PA-C) was performed. MTD1 NS-N MTD1 NS1-N MTD2 ), or introduced viral proteins with disordered peptide sequences (such as PA-C) MTD1 Scrambled NS-N MTD1 Scrambled NS1-N MTD2 mutant Compared to recombinant viral proteins, the difference lies in the disordered peptides at the same site (which are no longer recognized by Midnolin). The preparation procedure can be the same as for recombinant viral proteins. After 48 h of protein expression, cell lysates were collected for immunoprecipitation. As shown in Figure 3, Midnolin can co-precipitate with recombinant viral proteins, but not with wild-type viral proteins or viral proteins with introduced disordered peptide mutations. This result confirms the interaction between recombinant viral proteins and Midnolin, consistent with the experimental design.

[0112] In addition, MDCK.2 cell lines were infected with recombinant influenza virus and wild-type (WT) influenza virus (MOI = 0.01 or 0.1), and the culture medium was supplemented with 0, 50, and 100 nM proteasome inhibitor MG-132, respectively. Cells were collected 48 h after infection, and viral protein levels were detected by Western blotting. As shown in Figure 4, MG-132 treatment increased the level of recombinant viral protein but had no effect on the level of wild-type (WT) viral protein. This result indicates that recombinant viral proteins are degraded by the proteasome through binding to Midnolin, consistent with the experimental design.

[0113] In addition, the MDCK.2 cell line was infected with recombinant influenza virus (MOI = 0.01 or 0.1), and the culture medium was supplemented with 0, 25, and 50 nM of TAK-243, an inhibitor of ubiquitin-activating enzyme E1. Cells were collected 48 h after infection, and viral protein levels were detected by Western blotting. As shown in Figure 5, TAK-243 had no effect on the level of recombinant viral proteins. This result indicates that the recombinant viral proteins we designed were degraded by the proteasome via binding to Midnolin without ubiquitination modification, which is consistent with our experimental design.

[0114] Example 5

[0115] This embodiment evaluates the safety of recombinant influenza virus at the animal level.

[0116] The safety of recombinant influenza virus at the animal level was evaluated using C57BL / 6J mice. MIDTAR mice were selected. MTD1+2 and MIDTAR dual MTDs Using the representative strain as an example, each strain underwent the same procedures as described below. The experimental steps are as follows:

[0117] (a) Thirty female C57BL / 6J mice (Vitalliwa) aged 6-8 weeks were divided into 3 groups of 10 mice each;

[0118] (b) Each mouse in the first group was given 10 drops of irrigated intranasally. 5 TCID 50 Wild-type (WT) influenza virus, 10 mg / mice in group 2 were administered via intranasal drip. 5 TCID 50 MIDTAR MTD1+2 or MIDTAR dual MTDs The recombinant strain was administered via intranasal inoculation to each mouse in the third group with DMEM (Vehicle).

[0119] (c) Three days after inoculation, five mice from each group were taken, and their lung tissue was taken to detect the viral titer.

[0120] (d) Continue to observe and monitor the weight and mortality of the remaining 5 mice in each group for 14 days.

[0121] Figures 6A and 6B show that wild-type (WT) influenza virus replicates extensively in the lungs of mice, causing significant weight loss and death. In contrast, recombinant influenza virus titers in mouse lungs are below the detection limit and do not cause weight loss or death. Therefore, the recombinant influenza virus vaccine demonstrates good safety.

[0122] The safety of recombinant influenza virus at the animal level was evaluated using ferrets. MIDTAR was selected. MTD1+2 Using the representative strain as an example, each strain underwent the same procedures as described below. The experimental steps are as follows:

[0123] (a) Ten female ferrets aged 4-6 months were divided into two groups of three;

[0124] (b) Each ferret in the first group was given 10 drops of intranasal inoculation. 6 TCID 50 Wild-type (WT) influenza virus, group 2, each ferret was administered 10 doses via nasal drops. 6 TCID 50 MIDTAR MTD1+2 Recombinant strain.

[0125] (c) Three days after vaccination, three ferrets from each group were taken, and their lung tissue and nasal washing fluid were collected to test the viral titer.

[0126] As shown in Figure 6C, the results indicate that wild-type (WT) influenza virus can replicate highly in the lungs (a) and nasal washings (b) of ferrets, while the titer of recombinant influenza virus in the lungs and nasal washings of mice is below the detection limit. Therefore, the recombinant influenza virus vaccine has good safety.

[0127] Example 6

[0128] This embodiment evaluates the immunogenicity of a recombinant influenza virus vaccine at the animal level.

[0129] The most widely used inactivated influenza vaccine (IIV) (the inactivated influenza virus vaccine was prepared using homologous influenza virus particles according to the method provided in the Chinese Pharmacopoeia) and the clinically approved cold-adapted attenuated vaccine (CAIV) were used as controls, with MIDTAR as the control. MTD1+2 or MIDTAR dual MTDs Using a representative strain, the immunogenicity of the recombinant influenza virus vaccine was evaluated. The same procedures were performed on each strain. The experimental steps are shown below:

[0130] (1) Forty female C57BL / 6J mice aged 6-8 weeks were divided into 4 groups of 10 mice each;

[0131] (2) Each mouse in the first group was given 10 drops of intranasal inoculation. 5 TCID 50 MIDTAR MTD1+2 or MIDTAR dual MTDs The second group of mice was injected intranasally with 10 strains of the virus. 5 TCID 50 The CAIV influenza vaccine was administered via intramuscular injection to each mouse in the third group. 5 TCID 50 The fourth group of mice received DMEM via intranasal drip in the form of the IIV influenza vaccine.

[0132] (3) One week after inoculation, five mice from each group were taken, and their lung tissue and spleen were taken to detect the immune response of T cells.

[0133] (4) Three weeks after inoculation, five mice were taken from each group, and blood and lung lavage fluid were collected for hemagglutination inhibition (HI) test, neutralization (NT) antibody detection and ELISA test, respectively, to detect the antibody immune response.

[0134] As shown in Figures 7A and 7B, MIDTAR MTD1+2 and MIDTAR dual MTDs The strain can induce high levels of humoral immune responses in animals, including neutralizing antibodies (NT), hemagglutination inhibition antibodies (HI), anti-HA IgG, and anti-NP IgG in serum; MIDTAR MTD1+2 and MIDTAR dual MTDs The strain can induce high levels of respiratory mucosal immune responses in animals, such as anti-virus IgA in bronchoalveolar lavage fluid; MIDTAR MTD1+2 and MIDTAR dual MTDs The strain can induce high levels of T-cell immune responses in animals, including T-cell immune responses specific to influenza virus NP antigens in the lungs of mice and T-cell immune responses specific to influenza virus NP antigens in the spleen of mice, as well as MIDTAR. MTD1+2 T-cell immune responses specific to the influenza virus PA antigen were observed in the lungs and spleen of mice. Of particular importance, the recombinant strain prepared in this application induced significantly higher levels of immune responses than clinically used influenza vaccines CAIV and IIV.

[0135] The most widely used inactivated influenza vaccine (IIV) (the inactivated influenza virus vaccine is prepared using homologous influenza virus particles according to the method provided in the Chinese Pharmacopoeia), the clinically approved cold-adapted attenuated vaccine (CAIV), and the laboratory-prepared attenuated vaccine (M1-PTD) were used as controls, with MIDTAR as the control. MTD1+2 Using a single strain as a representative, the immunogenicity of the recombinant influenza virus vaccine was comprehensively evaluated. The same procedures were performed on each strain. The experimental steps are shown below:

[0136] (a) T cell response

[0137] (1) Twenty female C57BL / 6J mice aged 6-8 weeks were divided into 4 groups of 5 mice each;

[0138] (2) Each mouse in the first group was given 10 drops of intranasal inoculation. 5 TCID 50 MIDTAR MTD1+2 In the second group, each mouse was injected intranasally with 10 [units of something]. 5 TCID 50 The CAIV influenza vaccine was administered via intramuscular injection to each mouse in the third group. 5 TCID 50 The fourth group of mice received DMEM (Vehicle) via intranasal drip in the IV influenza vaccine.

[0139] (3) One week after inoculation, five mice were taken from each group, and their lung tissue and spleen were taken to detect the immune response of T cells (enzyme-linked immunospot assay and intracellular factor staining).

[0140] (ii) Humoral and mucosal immune responses

[0141] (1) Forty-five female C57BL / 6J mice aged 6-8 weeks were divided into 5 groups;

[0142] (2) The first group consisted of 15 mice, with 5 mice in each group. Each mouse was injected intranasally with 10 mg of ... 3 TCID 50 10 4 TCID 50 10 5 TCID 50 MIDTAR MTD1+2 The second group consisted of 15 mice, with 5 mice in each group. Each mouse was injected intranasally with 10 mg of the vaccine. 3 TCID 50 10 4 TCID 50 10 5 TCID5 CAIV influenza vaccine; Group 3, 5 mice, each injected intramuscularly with 10 5TCID 50 IIV influenza vaccine; Group 4, 5 mice, each mouse was given 10 doses via intranasal injection. 5 TCID 50 The M1-PTD influenza vaccine; Group 5 mice, each mouse was given DMEM (Vehicle) via intranasal drip.

[0143] (3) Three weeks after inoculation, five mice were taken from each group, and blood and lung lavage fluid were collected for hemagglutination inhibition (HI) test, neutralization (NT) antibody detection and ELISA test, respectively, to detect the antibody immune response.

[0144] As shown in Figure 8, 10 3 TCID 50 10 4 TCID 50 10 5 TCID 50 MIDTAR at immune dose MTD1+2 The strain can induce higher levels of humoral immune responses in animals, including neutralizing antibodies (NT), hemagglutination inhibition antibodies (HI), anti-HA IgG, and anti-NP IgG in serum; 10 3 TCID 50 10 4 TCID 50 10 5 TCID 50 MIDTAR MTD1+2 The strain can induce higher levels of mucosal immune responses in animals, such as anti-virus IgA antibodies in bronchoalveolar lavage fluid; 10 5 TCID 50 MIDTAR MTD1+2 The strain can induce higher levels of T-cell immune responses in animals, including T-cell immune responses and intracellular kinase responses (IFN-γ, granzyme B, perforin, IL-2, TNF-α) in the lungs and spleen of mice against influenza virus NP antigens, PA antigens, NS1 antigens, and NS2 antigens. Of particular importance, the recombinant strain prepared in this invention induces immune responses at levels of cellular immunity, humoral immunity, and mucosal immunity that are significantly superior to those induced by clinically used influenza vaccines CAIV and IIV.

[0145] Example 7

[0146] This embodiment evaluates the cross-immunoprotective effect of recombinant influenza virus vaccine at the animal level.

[0147] Using the most widely used inactivated influenza vaccine (IIV) in clinical practice (the inactivated influenza virus vaccine was prepared in this application using homologous influenza virus particles according to the method provided in the Chinese Pharmacopoeia) as a control, MIDTAR was used. MTD1+2 or MIDTAR dual MTDs Using a representative strain, the cross-immunization efficacy of the recombinant influenza virus vaccine was evaluated. The same procedures were performed on each strain. The experimental steps are shown below:

[0148] (1) Thirty female C57BL / 6J mice aged 6-8 weeks were divided into 3 groups of 10 mice each;

[0149] (2) Each mouse in the first group was given DMEM via intranasal drip, and each mouse in the second group was given 10 mg of DMEM via intranasal drip. 5 TCID 50 recombinant strain MIDTAR MTD1+2 or MIDTAR dual MTDs In the third group, each mouse was injected intramuscularly with 10 5 TCID 50 IIV;

[0150] (3) Three weeks after inoculation, each group of mice was challenged with 10 drops of the virus via intranasal drip. 5 TICD 50 Wild-type H1N1 influenza virus (homologous strain) or 10 3 TCID 50 Wild-type H3N2 influenza virus (heterogeneous strain A / X-31 (H3N2));

[0151] (4) Three days after inoculation with wild-type influenza virus, five mice from each group were taken, and their lung tissue was taken to detect the viral titer.

[0152] (5) Continue to observe and monitor the weight and mortality of the remaining 5 mice in each group for 14 days.

[0153] As shown in Figures 9A and 9B, when challenged with H1N1, the recombinant strain MIDTAR prepared in this application... MTD1+2 and MIDTAR dual MTDs It provides complete protection, including viral titers in mouse lungs below the detection limit (as shown in Figure a), with all mice surviving and maintaining their body weight (as shown in Figure b); while the same dose of inactivated vaccine failed to provide immune protection. When challenged with A / X-31 (H3N2), the recombinant strain MIDTAR prepared in this application... MTD1+2 and MIDTAR dual MTDsIt provides complete protection, including viral titers in mouse lungs below the detection limit (as shown in Figure c), with all mice surviving and maintaining their body weight (as shown in Figure d); while the same dose of inactivated vaccine failed to provide immune protection. These results indicate that the recombinant influenza virus vaccine prepared in this application has superior cross-immunological protection, significantly better than the most widely used influenza vaccine in clinical practice.

[0154] The most widely used inactivated influenza vaccine (IIV) (the inactivated influenza virus vaccine prepared in this invention using homologous influenza virus particles according to the method provided in the Chinese Pharmacopoeia) and the clinically approved cold-adapted attenuated vaccine (CAIV) were used as controls, with MIDTAR as the control. MTD1+2 Using the recombinant influenza virus strain as a representative, a comprehensive evaluation of the cross-immunization protection efficacy of the recombinant influenza virus vaccine was conducted. The same procedures were performed on each strain. The experimental steps are shown below:

[0155] (1) Eighty female C57BL / 6J mice aged 6-8 weeks were divided into 4 groups;

[0156] (2) Group 1 consisted of 10 mice, each of which was intranasally inoculated with DMEM; Group 2 consisted of 30 mice, with 10 mice per group, each of which was intranasally inoculated with 10 DMEM. 3 TCID 50 10 4 TCID 50 10 5 TCID 50 recombinant strain MIDTAR MTD1+2 The third group consisted of 30 mice, with 10 mice in each group. Each mouse was injected intranasally with 10 [units of something]. 3 TCID 50 10 4 TCID 50 10 5 TCID 50 CAIV influenza vaccine; Group 4, 10 mice, each mouse was injected intramuscularly with 10 5 TCID 50 IIV;

[0157] (3) Three weeks after inoculation, each group of mice was challenged with 10 drops of the virus via intranasal drip. 5 TCD 50 Wild-type H1N1 influenza virus (homologous strain) or 10 3 TCID 50 Wild-type H3N2 influenza virus (heterogeneous strain influenza A / Hong Kong / 8 / 68 (H3N2));

[0158] (4) Three days after inoculation with wild-type influenza virus, five mice from each group were taken, and their lung tissue was taken to detect the viral titer.

[0159] (5) Continue to observe and monitor the weight and mortality of the remaining 5 mice in each group for 14 days.

[0160] As shown in Figure 9C, when challenged with H1N1, the recombinant strain MIDTAR prepared in this invention... MTD1+2 In 10 3 TCID 50 10 4 TCID 50 10 5 TCID 50 At the immunization dose, complete protection is provided, including viral titers in mouse lungs below the detection limit (as shown in Figure a), with all mice surviving and maintaining their body weight (as shown in Figure b); while the same dose of inactivated vaccine and 10 3 TCID 50 Immunization doses of the CAIV influenza vaccine failed to provide complete protection. When challenged with the heterologous influenza strain A / Hong Kong / 8 / 68 (H3N2), the recombinant strain MIDTAR prepared in this invention provided protection. MTD1+2 In 10 3 TCID 50 10 4 TCID 50 10 5 TCID 50 At the same immunization dose, the viral titer in the lungs of mice was significantly reduced (as shown in Figure c), and all mice survived with no change in body weight (as shown in Figure d); while the same dose of inactivated vaccine and 10 3 TCID 50 Immunization doses of the CAIV influenza vaccine failed to provide any protection, and 10 4 TCID 50 and 10 5 The CAIV influenza vaccine at a TCID5 immunization dose failed to provide complete protection against body weight gain in mice. These results indicate that the recombinant influenza virus vaccine prepared in this invention has superior cross-immunoprotective effects, significantly better than the most widely used influenza vaccines in clinical practice.

[0161] In summary, this application introduces a peptide that can be recognized by Midnolin into the viral protein, resulting in a recombinant virus whose replication ability is significantly weakened compared to the wild-type virus. The recombinant virus, when formulated into corresponding vaccines or drugs, exhibits excellent safety and significantly superior immunogenicity compared to clinically approved vaccines, showing broad application prospects. Furthermore, the preparation method of the recombinant virus is highly adaptable and simple to operate, promoting the use and promotion of the product.

[0162] Design of Category II viral vaccines (recombinant viruses containing peptides that can be recognized by HSC70):

[0163] Introducing peptides that can be recognized by HSC70 at appropriate sites on viral proteins can induce the degradation of viral proteins in lysosomes within cells, reducing the replication capacity of the prepared virus and thus weakening its virulence. This increases the safety of the recombinant viral strain, which can then be used as a vaccine. Influenza virus was used as a model virus for design validation.

[0164] Example 8

[0165] This embodiment constructs a recombinant influenza virus vector containing peptides that can be recognized by HSC70.

[0166] Recombinant viral vectors were constructed by introducing peptides recognizable by HSC70 into different sites on different proteins of the influenza virus. Specifically, gene sequences expressing peptides recognizable by HSC70 were introduced into different sites on the viral genome. This work was assisted by Sangon Biotech (Shanghai) Co., Ltd., and the successful construction of the mutations was verified by sequencing.

[0167] Peptides that can be recognized by HSC70 have the following characteristics: containing at most two hydrophobic residues [isoleucine (I), phenylalanine (F), leucine (L) or valine (V)], at most two positively charged residues [arginine (R) or lysine (K)] and one negatively charged residue [glutamate (E) or aspartic acid (D)], with N- on both sides or a C-terminus consisting of a glutamine (Q) residue. Specifically, it includes KFERQ (SEQ ID NO.13), KFEKQ (SEQ ID NO.14), KFEEQ (SEQ ID NO.15), KFDEQ (SEQ ID NO.16), RFDEQ (SEQ ID NO.17), KFDRQ (SEQ ID NO.18), RFDRQ (SEQ ID NO.19), RFEDQ (SEQ ID NO.20), KFDKQ (SEQ ID NO.21), RFDKQ (SEQ ID NO.22), RFEKQ (SEQ ID NO.23), QFERK (SEQ ID NO.24), VKKDQ (SEQ ID NO.25), KILDQ (SEQ ID NO.26), and combinations thereof.

[0168] In this embodiment, the following influenza virus proteins and sites are used as representatives for research, but other sites are theoretically also applicable:

[0169] PB2 (N-end, R70, I176, V457, N510, Y531, A623, D680, E700, C-end);

[0170] PB1 (N-end, D70, D295, R327, R430, F490, T566, N626, G710, C-end);

[0171] PA (N-terminal, D294, N350, E372, L425, H510, A553, E604, S624, C-terminal);

[0172] NP (N-terminal, G126, N247, R317, V353, A366, Q409, E465, M481, C-terminal);

[0173] M1 (N-end, A33, V68, D89, R105, M135, Q164, H222, A239, C-end);

[0174] M2 (C-end);

[0175] NEP (C-end);

[0176] NS1 (N terminal, A76, A82, H101, A122, T151, L163, C terminal).

[0177] When a peptide recognizable by HSC70 is introduced to the N-terminus or C-terminus of a viral protein, the peptide can be directly linked to the viral protein, or it can be linked to the viral protein via the linker peptide ENLYFQG (SEQ ID NO. 45), which can be cleaved by TEVp. When a peptide recognizable by HSC70 is introduced to other sites on a viral protein, the peptide can be directly linked to the viral protein.

[0178] Example 9

[0179] This embodiment prepares a recombinant influenza virus containing peptides that can be recognized by HSC70.

[0180] When HSC70-recognizable peptides are linked to viral proteins via ENLYFQG (SEQ ID NO. 45), the corresponding viruses are prepared using HEK293T cells and MDCK cells expressing TEVp. When HSC70-recognizable peptides are directly linked to viral proteins, the corresponding viruses are prepared using HSC70-knockout or LAMP2A-knockout HEK293T cells and MDCK cells. The cell lines used are constructed using conventional lentiviral overexpression and CRISPR-Cas9 knockout technology. The detailed steps for virus preparation are as described in Example 2, except that the plasmid used for virus preparation is replaced with a plasmid containing a recombinant influenza virus containing a peptide recognizable by HSC70.

[0181] The experimental results are shown in Table 3. The titers of some influenza viruses containing peptides that can be recognized by HSC70 are compared with the titers of wild-type influenza viruses (10... 7 -10 9 TCID 50 The ratio ( / mL) is comparable. Furthermore, the same strain can contain multiple peptides that can be recognized by HSC70.

[0182]

[0183] Example 10

[0184] This embodiment evaluates the preparation efficiency and safety at the cellular level of recombinant influenza virus containing peptides that can be recognized by HSC70.

[0185] This embodiment uses NS-N CTM , PB-Y531, PA-N350, PA-E372, NP-G126, NP-Q409, NS1-H101, NS1-A122, CMATAR 2.0 dual CTMs Using CMATAR 2.0 dual triCTMs as an example, we evaluated the preparation efficiency and safety of recombinant influenza virus at the cellular level.

[0186] (1) Efficiency of recombinant influenza virus preparation

[0187] Recombinant influenza virus and wild-type (WT) influenza virus were used to infect MDCK cells stably expressing TEVp protein or HSC70 knockout MDCK cells (MOI=0.001), respectively. The culture medium used was DMEM containing 0.5% FBS and 2 μg / mL TPCK-trypsin. On days 1, 2, 3, and 4 post-infection, the culture supernatant was collected and analyzed using TCID50. 50The viral titer was detected experimentally. The results are shown in Figure 10. In MDCK cells expressing TEVp protein or MDCK cells with HSC70 knockout, the growth curves of most recombinant influenza virus strains were comparable to those of wild-type influenza virus, indicating that most recombinant influenza viruses had high preparation efficiency.

[0188] (2) Safety of recombinant influenza virus at the cellular level

[0189] Normal MDCK cells were infected with recombinant influenza virus and wild-type (WT) influenza virus at an MOI ratio of 0.001. TCID was administered on days 1, 2, 3, and 4 post-infection. 50 The viral titer in the culture supernatant was measured experimentally. The results, shown in Figure 11, indicate that in normal MDCK cells, the replication ability of most recombinant influenza viruses was significantly reduced compared to wild-type influenza viruses; the growth curve of recombinant influenza viruses containing multiple HSC70-recognizable peptides was particularly significantly reduced. These results demonstrate that recombinant influenza viruses are safe at the cellular level, and introducing multiple HSC70-recognizable peptides into the virus can further enhance its safety.

[0190] Example 11

[0191] This embodiment verifies the attenuation mechanism of recombinant influenza virus.

[0192] 1. First, use NS-N CTM The study used the virus strain as a representative example, and specific experiments included:

[0193] (1) It was demonstrated that the introduction of peptides that can be recognized by HSC70 into viral proteins reduces viral protein levels:

[0194] NS-N CTM The NS1 gene fragment was constructed into a eukaryotic overexpression vector, and a eukaryotic overexpression vector was also constructed by mutating the VKKDQKFERQ (SEQ ID NO.42) peptide to the VKKAAKFEAA (SEQ ID NO.44) peptide. 300 ng of the overexpression plasmid was transfected into 293T cells (6-well plates). After 24 h, NS1 protein levels and mRNA expression levels were detected by Western blotting. The results showed (Figure 12a) that the NS1 protein (NS1-N) with VKKDQKFERQ (SEQ ID NO.42) introduced at the N-terminus... CTM The level was lower than that of the NS1 protein (NS1-N) with VKKAAKFEAA (SEQ ID NO.44) introduced at the N-terminus. CTM mutantThe results showed that the VKKDQKFERQ (SEQ ID NO.42) peptide mediated a decrease in viral protein levels; specifically, the mutation of key amino acids DQ and RQ in the peptide to AA increased the protein level of NS1.

[0195] (2) Prove that the recombinant viral protein is degraded by lysosomes:

[0196] Expressing recombinant protein NS1-N CTM or NS1-N CTM mutant The plasmid (300 ng) was transfected into HEK293T cells (6-well plate). After 18 h, DMSO, MG132 (10 μM), 3-MA (10 μM), Baf.A1 (0.4 μM), or CQ (50 μM) were added, respectively. Cell samples were collected after 6 h, and the expression level of NS1 protein was detected by Western blotting. The results showed (Figure 12b) that only after adding the lysosomal inhibitors Baf.A1 and CQ could the expression level of NS1-N be reduced. CTM The significantly increased expression level of the protein indicates that the recombinant viral protein is degraded via the lysosomal pathway.

[0197] Recombinant protein NS1-N was transfected into HEK293T wild-type and HSC70 or LAMP2A knockout cells. CTM or NS1-N CTM mutant The plasmid (300 ng) was used, and cell samples were collected 24 h later. Western blotting was used to detect the expression level of viral proteins. The results showed (Figure 12c) that in HSC70 or LAMP2A knockout cells, the expression level of recombinant viral protein NS1-N was significantly increased. CTM The significantly increased levels indicate that the recombinant viral protein is degraded via a molecular chaperone-mediated lysosomal pathway.

[0198] (3) Demonstrate the interaction between recombinant viral proteins and HSC70:

[0199] Recombinant protein NS1-N was transfected into HSC70-expressing HEK293T cells (in 6-well plates). CTM or NS1-N CTM mutant The plasmid (300 ng) was used, and cell samples were collected 24 h later for immunoprecipitation experiments. The results showed (Figure 12, d) that NS1-N CTM There is an interaction with HSC70, and the recombinant protein NS1-N with key amino acid mutations... CTM mutant It does not interact with HSC70.

[0200] (4) At the viral level, it has been demonstrated that the introduction of peptides that can be recognized by HSC70 into viral proteins reduces viral protein levels:

[0201] Recombinant virus NS-N CTM and mutant control virus NS-N CTM mutant (MOI=0.01) HEK293T wild-type (WT) and LAMP2A knockout HEK293T (KO) cells, wild-type A549 cells (WT) and LAMP2A knockout A549 cells (KO) were infected, respectively. Cell samples and supernatants were collected after 48 h. Western blotting was used to detect the expression level of NS1 protein in the cell samples (Figures e and f in Figure 12), and the viral titer in the supernatant was measured (Figures g and h in Figure 12). The results showed that the expression of NS1 protein of recombinant influenza virus was increased in LAMP2A knockout cells, while NS-N... CTM mutant The expression of the NS1 protein of recombinant influenza virus remained unchanged in LAMP2A knockout cells. This result further demonstrates that the introduction of peptides recognized by HSC70 into viral proteins reduces viral protein levels and weakens viral replication.

[0202] 2. To verify the mechanism of introducing the HSC70 recognition peptide into the intermediate viral protein to prepare recombinant virus, specific experiments included:

[0203] (1) Prove that the recombinant viral protein is degraded by lysosomes:

[0204] Taking the recombinant viral protein in Figure 13a as an example, the plasmid expressing the recombinant protein (300 ng) was transfected into HEK293T cells (6-well plate). After 18 h, DMSO or Baf.A1 (0.4 μM) was added, respectively. Cell samples were collected after 6 h, and the expression level of the recombinant viral protein was detected by Western blotting. The results showed that the expression level of the recombinant viral protein increased significantly after the addition of the lysosomal inhibitor Baf.A1, indicating that the recombinant viral protein is degraded through the lysosomal pathway.

[0205] As shown in Figure 13b, plasmids expressing the recombinant protein shown in the figure were transfected into LAMP2A knockout cells (300 ng). Cell samples were collected 24 h later, and the expression level of the recombinant viral protein was detected by Western blotting. The results showed that the level of recombinant viral protein was significantly increased in LAMP2A knockout cells, indicating that the recombinant viral protein was degraded via the molecular chaperone-mediated lysosomal pathway.

[0206] (2) At the viral level, it has been demonstrated that the introduction of peptides recognized by HSC70 into viral proteins reduces viral protein levels:

[0207] As shown in Figures 13c and 13d, wild-type HEK293T cells or A549 cells and LAMP2A knockout HEK293T cells or A549 cells were infected with recombinant virus (MOI=0.01), respectively. Cell samples and supernatants were collected after 48 h. The expression level of recombinant viral proteins in the cell samples was detected by Western blotting. The results showed that the expression of recombinant influenza virus proteins was increased in LAMP2A knockout cells. This result further demonstrates that the introduction of peptides recognized by HSC70 into viral proteins reduces viral protein levels.

[0208] 3. Demonstrating the attenuation mechanism of recombinant viruses containing multiple peptides recognizable by HSC70:

[0209] A recombinant virus was prepared by simultaneously introducing peptides recognizable by HSC70 into the N350 site of influenza virus protein PA and the G126 site of NP. The recombinant virus with the insertion of a set of KFERQ (SEQ ID NO.13) motifs was named CMATAR 2.0. dual CTMs The recombinant virus with three KFERQ (SEQ ID NO.13) motifs inserted is named CMATAR 2.0 dual triCTMs (CMATAR 2.0). This application uses CMATAR 2.0 as a representative for verification.

[0210] As shown in Figures e and f of Figure 14, wild-type HEK293T cells or A549 cells and LAMP2A knockout HEK293T cells or A549 cells were infected with recombinant virus CMATAR 2.0 (MOI=0.01), respectively. Cell samples and supernatants were collected after 48 h. The expression levels of recombinant viral proteins in the cell samples were detected by Western blotting, and the viral titer in the supernatant was determined by immunofluorescence staining (Figures g and h of Figure 14). The results showed that the expression of PA and NP proteins of recombinant influenza virus increased in LAMP2A knockout cells, while the expression of PA and NP proteins of CMATAR 2.0 mutant recombinant influenza virus remained unchanged in LAMP2A knockout cells. This result further demonstrates that for recombinant viruses containing multiple peptides that can be recognized by HSC70, the introduction of HSC70-recognized peptides into the viral proteins reduces viral protein levels and weakens viral replication.

[0211] The above experimental results show that the recombinant virus prepared in this application can be recognized by HSC70 and degraded in lysosomes, thereby weakening the virus's replication ability and achieving an attenuation effect.

[0212] Example 12

[0213] This embodiment evaluates the safety of recombinant influenza virus at the animal level.

[0214] The safety of recombinant influenza virus at the animal level was evaluated using C57BL / 6J mice. NS-N was selected. CTM The virus strain was used as a representative example. The experimental steps are as follows:

[0215] (a) Thirty female C57BL / 6J mice aged 6-8 weeks were divided into 3 groups of 10 mice each;

[0216] (b) Each mouse in the first group was given 10 drops of irrigated intranasally. 5 TCID 50 Wild-type (WT) influenza virus, 10 mg / mice in group 2 were administered via intranasal drip. 5 TCID 50 NS-N CTM The recombinant strain was administered via intranasal inoculation to each mouse in the third group with DMEM (Vehicle).

[0217] (c) Three days after inoculation, five mice from each group were taken, and their lung tissue was taken to detect the viral titer.

[0218] (d) Continue to observe and monitor the weight and mortality of the remaining 5 mice in each group for 14 days.

[0219] Figure 15 shows that wild-type (WT) influenza virus replicates extensively in the lungs of mice, causing significant weight loss and death. In contrast, recombinant influenza virus titers in mouse lungs are close to the detection limit and do not cause weight loss or death. Therefore, the recombinant influenza virus vaccine demonstrates good safety.

[0220] Example 13

[0221] This embodiment evaluates the immunogenicity of a recombinant influenza virus vaccine at the animal level.

[0222] The most widely used inactivated influenza vaccine (IIV) (the inactivated influenza virus vaccine was prepared using homologous influenza virus particles according to the method provided in the Chinese Pharmacopoeia) and the clinically approved cold-adapted attenuated vaccine (CAIV) were used as controls, with NS-N CTM Using a representative strain, the immunogenicity of a recombinant influenza virus vaccine was evaluated. The experimental steps are as follows:

[0223] (1) Forty female C57BL / 6J mice aged 6-8 weeks were divided into 4 groups of 10 mice each;

[0224] (2) Each mouse in the first group was given 10 drops of intranasal inoculation. 5 TCID 50 NS-N CTM The second group of mice was injected intranasally with 10 strains of the virus.5 TCID 50 The CAIV influenza vaccine was administered via intramuscular injection to each mouse in the third group. 5 TCID 50 The fourth group of mice received DMEM via intranasal drip in the form of the IIV influenza vaccine.

[0225] (3) One week after inoculation, five mice from each group were taken, and their lung tissue and spleen were taken to detect the immune response of T cells.

[0226] (4) Three weeks after inoculation, five mice were taken from each group, and blood and lung lavage fluid were collected for hemagglutination inhibition (HI) test, neutralization (NT) antibody detection and ELISA test, respectively, to detect the antibody immune response.

[0227] As shown in Figure 16, NS-N CTM The strain can induce high levels of humoral immune responses in animals, including neutralizing antibodies (NT) (Figure a), hemagglutination inhibition antibodies (HI) (Figure b), anti-HA IgG (Figure c), and anti-NP IgG (Figure d); NS-N CTM The strain can induce high levels of respiratory mucosal immune responses in animals, such as anti-virus IgA in bronchoalveolar lavage fluid (Figure e); NS-N CTM The strain can induce high levels of T-cell immune responses in animals, including T-cell immune responses specific to the influenza virus NP antigen in the mouse lungs (f, left) and T-cell immune responses specific to the influenza virus NP antigen in the mouse spleen (f, right). Of particular importance, the recombinant strain prepared in this application induces immune responses significantly superior to those induced by clinically used influenza vaccine IIV.

[0228] Example 14

[0229] Evaluation of the cross-immunization efficacy of recombinant influenza virus vaccines in animals:

[0230] Using the most widely used inactivated influenza vaccine (IIV) in clinical practice (the inactivated influenza virus vaccine was prepared in this application using homologous influenza virus particles according to the method provided in the Chinese Pharmacopoeia) as a control, and NS-N CTM Using a representative strain, the cross-immunization efficacy of a recombinant influenza virus vaccine was evaluated. The experimental steps are as follows:

[0231] (1) Thirty female C57BL / 6J mice aged 6-8 weeks were divided into 3 groups of 10 mice each;

[0232] (2) Each mouse in the first group was given DMEM via intranasal drip, and each mouse in the second group was given 10 mg of DMEM via intranasal drip. 5 TCID50 Recombinant strain NS-N CTM In the third group, each mouse was injected intramuscularly with 10 5 TCID 50 IIV;

[0233] (3) Three weeks after inoculation, each group of mice was challenged with 10 drops of the virus via intranasal drip. 5 TICD 50 Wild-type H1N1 influenza virus (homologous strain) or 10 3 TCID 50 Wild-type H3N2 influenza virus (heterogeneous strain);

[0234] (4) Three days after inoculation with wild-type influenza virus, five mice from each group were taken, and their lung tissue was taken to detect the viral titer.

[0235] (5) Continue to observe and monitor the weight and mortality of the remaining 5 mice in each group for 14 days.

[0236] As shown in Figure 17, when challenged with H1N1, the recombinant strain NS-N prepared in this application... CTM It can provide complete protection, including viral titers in mouse lungs below the detection limit (as shown in Figure a), with all mice surviving and maintaining their body weight (Figure b); while the same dose of inactivated vaccine failed to provide immune protection (Figures a and b). When challenged with H3N2, the recombinant strain NS-N prepared in this application... CTM It provides complete protection, including viral titers in mouse lungs falling below the detection limit (as shown in Figure c), with all mice surviving and maintaining their body weight (as shown in Figure d); while the same dose of inactivated vaccine failed to provide immune protection (as shown in Figures c and d). These results indicate that the recombinant influenza virus vaccine prepared in this application has superior cross-immunoprotective effects, significantly better than the most widely used influenza vaccines in clinical practice.

[0237] In summary, this application introduces a peptide that can be recognized by HSC70 into the viral protein, resulting in a recombinant virus whose replication ability is significantly weakened compared to the wild-type virus. The recombinant virus, when formulated into corresponding vaccines or drugs, exhibits excellent safety and significantly superior immunogenicity compared to clinically approved vaccines, showing broad application prospects. Furthermore, the preparation method of the recombinant virus is highly adaptable and simple to operate, promoting the use and promotion of the product.

[0238] Design of Category III viral vaccines (recombinant viruses containing peptides that can be recognized by microtubule-associated protein 1A / 1B-light chain 3 (LC3)):

[0239] Introducing peptides that can be recognized by LC3 at appropriate sites on viral proteins can induce the degradation of viral proteins in lysosomes within cells, reducing the replication capacity of the prepared virus and thus weakening its virulence. This increases the safety of the recombinant viral strain, which can then be used as a vaccine. Influenza virus and respiratory syncytial virus (RSV) were used as model viruses for design validation.

[0240] Example 15

[0241] This embodiment constructs a recombinant influenza virus vector containing peptides that can be recognized by LC3.

[0242] Recombinant viral vectors were constructed by introducing peptides recognizable by LC3 into different sites on different proteins of the influenza virus. Specifically, gene sequences expressing peptides recognizable by LC3 were introduced into different sites on the viral genome. This work was assisted by Sangon Biotech (Shanghai) Co., Ltd., and the successful construction of the mutations was verified by sequencing.

[0243] Peptides recognizable by HSC70 have the following characteristics: they typically contain a short amino acid sequence, with the core sequence being "[W / F / Y]xx[L / I / V]"; here, "[W / F / Y]" represents one of tryptophan (W), phenylalanine (F), or tyrosine (Y), "xx" represents any two amino acids, and "[L / I / V]" represents one of leucine (L), isoleucine (I), or valine (V). This example uses EDYIIILPE (SEQ ID NO. 30) as an example for investigation.

[0244] In this embodiment, the following influenza virus proteins and sites are used as representatives for research, but other sites are theoretically also applicable:

[0245] PB2 (N-end, R70, I176, V457, N510, Y531, A623, D680, E700, C-end);

[0246] PB1 (N-end, D70, D295, R327, R430, F490, T566, N626, G710, C-end);

[0247] PA (N-terminal, D294, N350, E372, L425, H510, A553, E604, S624, C-terminal);

[0248] NP (N-terminal, G126, N247, R317, V353, A366, Q409, E465, M481, C-terminal);

[0249] M1 (N-end, A33, V68, D89, R105, M135, Q164, H222, A239, C-end);

[0250] M2 (C-end);

[0251] NEP (C-end);

[0252] NS1 (N terminal, A76, A82, H101, A122, T151, L163, C terminal).

[0253] When a peptide recognized by LC3 is introduced to the N-terminus or C-terminus of a viral protein, the peptide can be directly linked to the viral protein, or it can be linked to the viral protein via the linker peptide ENLYFQG (SEQ ID NO. 45), which can be cleaved by TEVp. When a peptide recognized by LC3 is introduced to other sites on a viral protein, the peptide can be directly linked to the viral protein.

[0254] Example 16

[0255] This embodiment prepares a recombinant influenza virus containing peptides that can be recognized by LC3.

[0256] When LC3-recognizable peptides are linked to viral proteins via ENLYFQG (SEQ ID NO. 45), the corresponding viruses are prepared using HEK293T cells and MDCK cells expressing TEVp; when LC3-recognizable peptides are directly linked to viral proteins, the corresponding viruses are prepared using LC3-knockout HEK293T cells and MDCK cells. The cell lines used were constructed using conventional lentiviral overexpression and CRISPR-Cas9 knockout technology. Detailed steps for virus preparation are as described in Example 2, except that the plasmid used for virus preparation was replaced with a plasmid containing a recombinant influenza virus containing an LC3-recognizable peptide.

[0257] Experimental results show that influenza viruses containing peptides that can be recognized by LC3 (named NS-N in this application) ATM The titer of the virus was compared with that of the wild-type influenza virus (10). 7 -10 9 TCID 50 The strain contains the EDYIIILPEGSGGENLYFQGGSG (SEQ ID NO.46) peptide at the N-terminus of influenza virus NS1.

[0258] Example 17

[0259] This embodiment evaluates the recombinant influenza virus NS-N ATMThe preparation efficiency and safety at the cellular level.

[0260] (1) Efficiency of recombinant influenza virus preparation:

[0261] Recombinant influenza virus and wild-type (WT) influenza virus were used to infect MDCK cells stably expressing TEVp protein (MOI=0.001). The culture medium was DMEM containing 0.5% FBS and 2 μg / mL TPCK-trypsin. On days 1, 2, 3, and 4 post-infection, the culture supernatant was collected and analyzed with TCID50. 50 The viral titer was detected experimentally. The results are shown in Figure 18A. In MDCK cells expressing TEVp protein, the growth curve of the recombinant influenza virus was comparable to that of the wild-type influenza virus, indicating that the recombinant influenza virus had high preparation efficiency.

[0262] (2) Safety of recombinant influenza virus at the cellular level:

[0263] Normal MDCK cells were infected with recombinant influenza virus and wild-type (WT) influenza virus at an MOI ratio of 0.001. TCID was administered on days 1, 2, 3, and 4 post-infection. 50 The viral titer in the culture supernatant was measured experimentally. The results, shown in Figure 18B, indicate that the replication ability of the recombinant influenza virus was significantly reduced in normal MDCK cells compared to wild-type influenza virus. This result demonstrates the safety of the recombinant influenza virus at the cellular level.

[0264] Example 18

[0265] This embodiment verifies the attenuation mechanism of recombinant influenza virus.

[0266] 1. First, use NS-N ATM The study used the virus strain as a representative example, and specific experiments included:

[0267] (1) Prove that the recombinant viral protein is degraded by lysosomes:

[0268] Expressing recombinant protein NS1-N ATM or NS1-N ATM mutant A plasmid (300 ng) containing an amino acid mutation (alanine in the LC3 recognition sequence) was transfected into HEK293T cells (6-well plates). After 18 h, DMSO, Baf.A1 (0.4 μM), or CQ (50 μM) were added, respectively. Cell samples were collected after 6 h, and the expression level of NS1 protein was detected by Western blotting. The results showed (Figure 19, A) that after adding the lysosomal inhibitors Baf.A1 and CQ, the expression level of NS1-N... ATM The significantly increased expression level of the protein indicates that the recombinant viral protein is degraded via the lysosomal pathway.

[0269] Transfecting LC3 knockout cells with recombinant protein NS1-N ATM Alternatively, a plasmid containing the NS1 protein (300 ng) was used. Cell samples were collected 24 h later, and the expression level of the viral protein was detected by Western blotting. The results showed (Figure 19, B) that in LC3 knockout cells, the expression level of the recombinant viral protein NS1-N was significantly increased. ATM The significantly increased levels indicate that the recombinant viral protein is degraded via the LC3-mediated autophagy-lysosomal pathway.

[0270] (2) At the viral level, it was demonstrated that the introduction of peptides that can be recognized by LC3 into viral proteins reduces viral protein levels:

[0271] Recombinant virus NS-N ATM and mutant control virus NS-N ATM mutant Wild-type HeLa cells (LC3 KO-) and LC3 knockout HeLa cells (LC3 KO+) were infected with the virus (MOI=0.01), and cell samples were collected after 48 h. The expression level of NS1 protein in the cell samples was detected using Western blotting. The results showed (Figure 19, C) that the expression of recombinant influenza virus NS1 protein was increased in LC3 knockout cells, while LC3 knockout had no effect on the level of mutant NS1 protein. This result further demonstrates that the introduction of LC3-recognizable peptides into viral proteins reduces viral protein levels.

[0272] The above experimental results show that the recombinant virus prepared in this application can be recognized by LC3 and degraded through the autophagy-lysosome pathway, thereby weakening the virus's replication ability and achieving an attenuation effect.

[0273] Example 19

[0274] Safety evaluation of recombinant influenza virus at the animal level:

[0275] The safety of recombinant influenza virus at the animal level was evaluated using C57BL / 6J mice. NS-N was selected. ATM The virus strain was used as a representative example. The experimental steps are as follows:

[0276] (a) Thirty female C57BL / 6J mice aged 6-8 weeks were divided into 3 groups of 10 mice each;

[0277] (b) Each mouse in the first group was given 10 drops of irrigated intranasally. 5 TCID 50 Wild-type (WT) influenza virus, 10 mg / mice in group 2 were administered via intranasal drip. 5 TCID 50 NS-N ATMThe recombinant strain was administered via intranasal inoculation to each mouse in the third group with DMEM (Vehicle).

[0278] (c) Three days after inoculation, five mice from each group were taken, and their lung tissue was taken to detect the viral titer.

[0279] (d) Continue to observe and monitor the weight and mortality of the remaining 5 mice in each group for 14 days.

[0280] As shown in Figure 20, the results indicate that wild-type (WT) influenza virus can replicate extensively in the lungs of mice, causing significant weight loss and death. In contrast, the recombinant influenza virus titer in the mouse lungs is close to the detection limit, and it does not cause weight loss or death in mice. Therefore, the recombinant influenza virus vaccine demonstrates good safety.

[0281] Example 20

[0282] This embodiment evaluates the immunogenicity of a recombinant influenza virus vaccine at the animal level.

[0283] The most widely used inactivated influenza vaccine (IIV) (the inactivated influenza virus vaccine was prepared using homologous influenza virus particles according to the method provided in the Chinese Pharmacopoeia) and the clinically approved cold-adapted attenuated vaccine (CAIV) were used as controls, with NS-N ATM Using a representative strain, the immunogenicity of a recombinant influenza virus vaccine was evaluated. The experimental steps are as follows:

[0284] (1) Forty female C57BL / 6J mice aged 6-8 weeks were divided into 4 groups of 10 mice each;

[0285] (2) Each mouse in the first group was given 10 drops of intranasal inoculation. 5 TCID 50 NS-N ATM The second group of mice was injected intranasally with 10 strains of the virus. 5 TCID 50 The CAIV influenza vaccine was administered via intramuscular injection to each mouse in the third group. 5 TCID 50 The fourth group of mice received DMEM via intranasal drip in the form of the IIV influenza vaccine.

[0286] (3) One week after inoculation, five mice from each group were taken, and their lung tissue and spleen were taken to detect the immune response of T cells.

[0287] (4) Three weeks after inoculation, five mice were taken from each group, and blood and lung lavage fluid were collected for hemagglutination inhibition (HI) test, neutralization (NT) antibody detection and ELISA test, respectively, to detect the antibody immune response.

[0288] As shown in Figure 21, NS-N ATM The strain can induce high levels of humoral immune responses in animals, including neutralizing antibodies (NT) (Figure a), hemagglutination inhibition antibodies (HI) (Figure b), anti-HA IgG (Figure c), and anti-NP IgG (Figure d); NS-N ATM The strain can induce high levels of respiratory mucosal immune responses in animals, such as anti-virus IgA in bronchoalveolar lavage fluid (Figure e); NS-N ATM The strain can induce high levels of T-cell immune responses in animals, including T-cell immune responses specific to the influenza virus NP antigen in the mouse lungs (f, left) and T-cell immune responses specific to the influenza virus NP antigen in the mouse spleen (f, right). Of particular importance, the recombinant strain prepared in this application induces immune responses significantly superior to those induced by clinically used influenza vaccine IIV.

[0289] Example 21

[0290] This embodiment evaluates the cross-immunoprotective effect of recombinant influenza virus vaccine at the animal level.

[0291] Using the most widely used inactivated influenza vaccine (IIV) in clinical practice (the inactivated influenza virus vaccine was prepared in this application using homologous influenza virus particles according to the method provided in the Chinese Pharmacopoeia) as a control, and NS-N ATM Using a representative strain, the cross-immunization efficacy of a recombinant influenza virus vaccine was evaluated. The experimental steps are as follows:

[0292] (1) Thirty female C57BL / 6J mice aged 6-8 weeks were divided into 3 groups of 10 mice each;

[0293] (2) Each mouse in the first group was given DMEM via intranasal drip, and each mouse in the second group was given 10 mg of DMEM via intranasal drip. 5 TCID 50 Recombinant strain NS-N ATM In the third group, each mouse was injected intramuscularly with 10 5 TCID 50 IIV;

[0294] (3) Three weeks after inoculation, each group of mice was challenged with 10 drops of the virus via intranasal drip. 5 TICD 50 Wild-type H1N1 influenza virus (homologous strain) or 10 3 TCID 50 Wild-type H3N2 influenza virus (heterogeneous strain);

[0295] (4) Three days after inoculation with wild-type influenza virus, five mice from each group were taken, and their lung tissue was taken to detect the viral titer.

[0296] (5) Continue to observe and monitor the weight and mortality of the remaining 5 mice in each group for 14 days.

[0297] As shown in Figure 22, when challenged with H1N1, the recombinant strain NS-N prepared in this application... ATM It can provide complete protection, including viral titers in mouse lungs below the detection limit (as shown in Figure a), with all mice surviving and maintaining their body weight (Figure b); while the same dose of inactivated vaccine failed to provide immune protection (Figures a and b). When challenged with H3N2, the recombinant strain NS-N prepared in this application... ATM It provides complete protection, including viral titers in mouse lungs below the detection limit (as shown in Figure c), with all mice surviving and maintaining their body weight (Figure d); while the same dose of inactivated vaccine failed to provide immune protection (Figures c and d). These results indicate that the recombinant influenza virus vaccine prepared in this application has superior cross-immunoprotective efficacy, significantly better than the most widely used influenza vaccine in clinical practice.

[0298] In summary, this application introduces a peptide that can be recognized by LC3 into the viral protein, resulting in a recombinant virus whose replication ability is significantly weakened compared to the wild-type virus. The recombinant virus, when formulated into corresponding vaccines or drugs, exhibits excellent safety and significantly superior immunogenicity compared to clinically approved vaccines, showing broad application prospects. Furthermore, the preparation method of the recombinant virus is highly adaptable and simple to operate, promoting the use and promotion of the product.

[0299] The above examples primarily use influenza virus as the research subject. The following examples use respiratory syncytial virus (RSV) as the research subject. The design schemes of the aforementioned three types of vaccines are applied to the design of an RSV vaccine.

[0300] Example 22

[0301] This embodiment uses peptides from the aforementioned three types of vaccine design schemes to construct recombinant respiratory syncytial virus.

[0302] (1) The vectors used were provided by the reverse genetics systems pSM_RSV_A_0594 and pSM-RSV_B_9671 for respiratory syncytial virus isolates constructed in the article "Reverse genetics systems for contemporary isolates of respiratory syncytial virus enable rapid evaluation of antibody escape mutants". The respiratory syncytial virus genome used has been stored in GenBank (MW582528.1).

[0303] (2) The aforementioned peptides were introduced into different sites of different proteins of respiratory syncytial virus to construct recombinant viral vectors. Specifically, the gene sequences expressing the peptides were introduced into different sites of the viral genome. This work was entrusted to Sangon Biotech (Shanghai) Co., Ltd. and Qingke Biotechnology Co., Ltd. for assistance, and the successful construction of the mutation was verified by sequencing.

[0304] In this embodiment, the following respiratory syncytial virus proteins and sites are used as representatives for research, but other sites are theoretically also applicable:

[0305] NS1 (N-terminal, L16, T31, N52, D64, P81, P101, L104, K116, C-terminal);

[0306] NS2 (N-end, R19, K51, D100, H113, C-end);

[0307] N (N-terminal, M1, A57, H59, V95, N105, A117, E144, D175, T233, C-terminal);

[0308] P (N-end, T160, R163, C-end);

[0309] M (N-end, H9, K25, Q40, N54, P65, S76, T136, V153, D208, P218, E231, C-end);

[0310] SH (N-terminal, E2, H51, F55, P58, V62, C-terminal);

[0311] G (N-end, C-end);

[0312] F (N-end, N67, L172, S182, P207, M264, N325, A346, P389, G418, K427, N444, V459, P480, C-end);

[0313] M2-1 (N-end, I87, L120, S182, C-end);

[0314] M2-2 (N-end, H9, K25, Q40, N54, P65, S76, T136, V153, D208, P218, E231, C-end);

[0315] L (N-end, Y94, K256, P503, G754, N762, L823, D1026, L1031, D1047, E1190, Y1232, T1236, V1248, T1259, L1421, C-end);

[0316] When the target peptide is introduced to the N-terminus or C-terminus of a viral protein, the peptide can be directly linked to the viral protein, or it can be linked to the viral protein via the linker peptide ENLYFQG (SEQ ID NO. 45), which can be cleaved by TEVp. When the target peptide is introduced to other sites on a viral protein, the peptide can be directly linked to the viral protein.

[0317] Example 23

[0318] This embodiment prepares recombinant respiratory syncytial virus containing the target peptide.

[0319] When the target peptide is linked to the viral protein via ENLYFQG (SEQ ID NO.45), the corresponding virus is prepared using HEP-2 cells or HeLa cells expressing TEVp; when the target peptide is directly linked to the viral protein, the corresponding virus is prepared using Midnolin-knockout HEP-2 cells or HeLa cells. The cell lines used were constructed using conventional lentiviral overexpression and CRISPR-Cas9 knockout technology. The detailed steps for virus preparation are as follows:

[0320] (1) Seeding cells: Seed HEP-2 cells (500,000 cells / well) or HeLa cells (500,000 cells / well) expressing TEVp or Midnolin knockout in 6-well plates and culture for 24 h.

[0321] (2) HEP-2 cells or HeLa cells expressing TEVp or Midnolin knockout were digested and counted, and prepared into 5×10⁻⁶ cells. 6 / mL cell suspension.

[0322] (3) Plasmid transfection: Plasmid transfection was performed using the Neon NxT electrotransfection system NEON1. 3.2 μg of the genomic plasmid and its helper plasmids (pCG vectors) expressing RSV-A-0594 N (1.6 μg), P (1.2 μg), M2-1 (0.8 μg) and L (0.4 μg) proteins were mixed with the cells, the electrotransfection program was run, and the mixture was incubated at 37°C and 5% CO2.

[0323] (4) Poxvirus incubation: After 8-12 h, inoculate MVA-T7 with MOI=2 and incubate for 1 h. After incubation, discard the poxvirus and wash twice with PBS.

[0324] (5) Replenish cells: After 24 hours of poxvirus incubation, observe the cell density under a microscope and replenish 500,000 to 1,000,000 corresponding cells according to the density to maintain the cell density above 90%.

[0325] (6) Monitor cells for fluorescent cell diffusion foci or syncytial formation. Generally, recombinant RSV virus is harvested 5 to 6 days after transfection, and cells are separated into the supernatant using a cell scraper. New HEP-2 cells (T25 flasks) expressing TEVp or Midnolin knockout are infected with 1 ml of the collected virus-cell suspension and stabilized (P0) after 3 to 4 days. The virus stockpile is harvested and infected using half-maximum tissue culture dose (TCID50). 50 The titer of the prepared virus was determined by a assay.

[0326] Representative experimental results are shown in Table 4.

[0327]

[0328] Example 24

[0329] This embodiment verifies the attenuation mechanism of recombinant respiratory syncytial virus (RSV) proteins.

[0330] The genes of each respiratory syncytial virus (RSV) were synthesized from the whole genome. These genes were then ligated into pCDNA3.1 and PLVX-IRES-mcherry vectors to obtain overexpression plasmids for each RSV protein. Target peptides were introduced into the viral proteins, and a 3×Flag tag sequence was introduced at the other end of the protein, which was then ligated to the plasmids to construct a series of recombinant plasmids. This work was completed with the assistance of Beijing Qingke Biotechnology Co., Ltd., and the successful mutation construction was verified by sequencing.

[0331] The recombinant viral protein shown in Figure 23 was used as a representative protein in this study. Plasmids containing recombinant respiratory syncytial virus (RSV) protein and wild-type RSV protein were overexpressed in 293T or HEP-2 cell lines, respectively. Twenty-four hours after transfection, 10 μM of the proteasome inhibitor MG-132 was added to the culture medium, with DMSO (at the same dilution as the virus) used as a control. Eight hours after treatment, cell samples were collected, and viral protein expression levels were detected by Western blotting. As shown in the figure below, MG-132 treatment increased the level of recombinant viral protein but had no effect on the level of wild-type (WT) viral protein. This result indicates that the target peptide mediated the degradation of viral protein by the proteasome, consistent with the experimental design.

[0332] Example 25

[0333] This embodiment evaluates the safety of the recombinant RSV virus vaccine at the animal level.

[0334] The safety of the recombinant RSV virus vaccine was evaluated at the animal level using cotton rats and BALB / c mice. A representative recombinant RSV virus vaccine strain, N-MTD, was selected. The experimental procedures are as follows:

[0335] (a) Ten 6-8 week old female cotton rats and ten 6-8 week old female BALB / c mice were divided into two groups of five each.

[0336] (b) Each cotton rat and BALB / c mouse in the first group was intranasally inoculated with 10 6 PFU wild-type (WT) RSV virus was administered intranasally to each cotton mouse and BALB / c mouse in the second group at a dose of 10. 6 PFU N-MTD vaccine strain;

[0337] (c) Five days after vaccination, lung tissue was taken and the viral titer was tested.

[0338] Figure 24 shows that wild-type (WT) RSV virus replicates extensively in the lungs of cotton rats and BALB / c mice, while the recombinant RSV virus vaccine titer in the lungs of cotton rats and BALB / c mice is below the detection limit. Therefore, the recombinant RSV virus vaccine has good safety.

[0339] Example 26

[0340] This embodiment evaluates the immunogenicity of the recombinant RSV virus vaccine at the animal level.

[0341] The immunogenicity of the recombinant RSV virus vaccine was evaluated using the N-MTD strain as a representative. The experimental steps are as follows:

[0342] (1) Ten female cotton rats aged 6-8 weeks and ten female BALB / c mice aged 6-8 weeks were divided into two groups of 5 mice each.

[0343] (2) Each cotton rat or BALB / c mouse in the first group was intranasally inoculated with 10 6 The N-MTD vaccine strain of PFU was administered intranasally to each cotton mouse and BALB / c mouse in the second group with the same volume of DMEM.

[0344] (3) Three weeks after vaccination, blood was collected from 5 cotton rats and BALB / c mice in each group to detect the serum neutralizing (NT) antibody titer.

[0345] As shown in Figure 25, the N-MTD vaccine strain can induce high levels of neutralizing antibodies (NT) in animals. Therefore, the recombinant RSV virus vaccine has good immunogenicity.

[0346] Example 27

[0347] This embodiment evaluates the protection against challenge with a recombinant RSV virus vaccine at the animal level.

[0348] Using the N-MTD strain as a representative, the challenge protection of the recombinant RSV virus vaccine was evaluated. The experimental steps are as follows:

[0349] (1) Ten female cotton rats aged 6-8 weeks and ten female BALB / c mice aged 6-8 weeks were divided into two groups of 5 mice each.

[0350] (2) Each cotton rat and BALB / c mouse in the first group was intranasally inoculated with 10 6 The N-MTD strain of PFU was administered intranasally to each cotton mouse or BALB / c mouse in the second group with the same volume of DMEM.

[0351] (3) Three weeks after inoculation, 5 cotton rats or BALB / c mice in each group were infected with 10 6 PFU wild-type RSV.

[0352] (4) Four days after infection, lung tissue was taken from each group of cotton rats or BALB / c mice, and the titer of lung grinding fluid was detected.

[0353] As shown in Figure 26, high levels of RSV titers were found in the lung tissues of unvaccinated mice and BALB / c mice; however, no RSV titers were detected in the lung tissues of mice vaccinated with the recombinant RSV virus vaccine. This indicates that vaccination with the recombinant RSV virus vaccine provides complete protection against challenge.

[0354] In summary, this application involves inserting a peptide recognized by Midnolin, HSC70, or LC3 into a viral protein to obtain a recombinant virus. This recombinant virus can induce degradation of the viral protein in the cell's proteasome or lysosome, reducing the replication capacity of the prepared virus and thus weakening its virulence. This increases the safety of the recombinant viral strain, which can be used as a vaccine. It can achieve efficient replication and large-scale production in specific cell lines (with deficiencies in the protein degradation system), but the virus's replication capacity is weakened or even completely lost in normal cells, contributing to the development of the vaccine field.

[0355] The applicant declares that the above description is only a specific implementation of this application, but the protection scope of this application is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application fall within the protection and disclosure scope of this application.

Claims

1. A recombinant virus, wherein, The viral protein of the recombinant virus contains at least one of the following: a peptide recognized by midbrain nucleolar protein, a peptide recognized by heat shock protein 70, or a peptide recognized by microtubule-associated protein 1A / 1B-light chain 3.

2. The recombinant virus according to claim 1, wherein, The peptide recognized by the midbrain nucleolar protein is selected from at least one of the peptides whose amino acid sequences include those shown in SEQ ID NO.1 to SEQ ID NO.

12.

3. The recombinant virus according to claim 1, wherein, The peptide recognized by heat shock protein 70 contains at most two hydrophobic residues, at most two positively charged residues and one negatively charged residue, flanked by N- or C-terminus composed of a glutamine residue.

4. The recombinant virus according to claim 1, wherein, The peptides recognized by HSC70 are selected from at least one of the peptides whose amino acid sequences include those shown in SEQ ID NO.13 to SEQ ID NO.

26.

5. The recombinant virus according to claim 1, wherein, The core general formula of the peptide segment recognized by microtubule-associated protein 1A / 1B-light chain 3 is [W / F / Y]xx[L / I / V], where [W / F / Y] represents one of tryptophan, phenylalanine or tyrosine, "xx" represents any two amino acids, and [L / I / V] represents one of leucine, isoleucine or valine. Preferably, the amino acid sequence of the peptide recognized by microtubule-associated protein 1A / 1B-light chain 3 includes the sequence shown in SEQ ID NO.

30.

6. The recombinant virus according to any one of claims 1-6, wherein, The recombinant viruses originating from the following viruses include influenza virus, respiratory syncytial virus, HIV, SARS-CoV-2, hand-foot-and-mouth disease virus, Coxsackie virus, hepatitis C virus, hepatitis B virus, hepatitis A virus, hepatitis D virus, hepatitis E virus, human herpesvirus, human papillomavirus, herpes simplex virus, cytomegalovirus, varicella-zoster virus, vesicular stomatitis virus, dengue virus, Ebola virus, Marburg virus, Zika virus, severe acute respiratory syndrome virus, Middle East respiratory syndrome virus, rotavirus, rabies virus, and measles virus. Any one of the following: adenovirus, poliovirus, echovirus, Japanese encephalitis virus, tick-borne encephalitis virus, Hantavirus, novel enterovirus, rubella virus, mumps virus, parainfluenza virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus, foot-and-mouth disease virus, parvovirus, prions, smallpox virus, tobacco mosaic virus, bacteriophage, herpesvirus, West Nile virus, norovirus, human bocavirus, or coronavirus, preferably any one of influenza virus, respiratory syncytial virus, HIV, or porcine reproductive and respiratory syndrome virus.

7. The recombinant virus according to claim 6, wherein, The viral proteins include at least one of the following: influenza virus proteins, respiratory syncytial virus proteins, HIV proteins, and porcine reproductive and respiratory syndrome virus proteins. Preferably, the influenza virus protein includes at least one of PA protein, PB1 protein, PB2 protein, NP protein, HA protein, NA protein, M1 protein, M2 protein, NS1 protein, or NEP protein. Preferably, the respiratory syncytial virus protein includes at least one of the following: N protein, P protein, M2-1 protein, M2-2 protein, NS1 protein, NS2 protein, G protein, F protein, SH protein, L protein, and M protein. Preferably, the HIV protein includes at least one of Vpu protein, Vpr protein, Vif protein, Nef protein, Tat protein, Rev protein, Gag protein, Env protein, and Pol protein; Preferably, the proteins of the porcine reproductive and respiratory syndrome virus include at least one of the following: NSP1α protein, NSP1β protein, NSP2 protein, NSP3 protein, NSP4 protein, NSP5 protein, NSP6 protein, NSP7 protein, NSP8 protein, NSP9 protein, NSP10 protein, NSP11 protein, NSP12 protein, GP2a protein, E protein, GP3 protein, GP4 protein, GP5 protein, GP5a protein, M protein, or N protein.

8. A nucleic acid molecule encoding a recombinant virus as described in any one of claims 1-7.

9. A recombinant vector containing the nucleic acid molecule as described in claim 8.

10. An engineered cell that supports the preparation of the recombinant virus according to any one of claims 1-7, specifically comprising an engineered cell line overexpressing tobacco etch virus protease (TEVp) and an engineered cell line with a defective protein degradation system.

11. A system for preparing a recombinant virus according to any one of claims 1-7, comprising the engineered cells of claim 10, the nucleic acid molecule of claim 8, and the virus rescue plasmid.

12. A method for preparing a recombinant virus according to any one of claims 1-7, wherein, The preparation method includes: Construct the engineered cell as described in claim 10; The coding nucleic acid of the recombinant virus is inserted into the expression vector to obtain the recombinant vector; The recombinant vector was introduced into the engineered cells and packaged to obtain the recombinant virus.

13. A vaccine comprising the recombinant virus according to any one of claims 1-7; Preferably, the vaccine comprises any one of a live attenuated vaccine, a live attenuated vaccine, a live attenuated vaccine with controllable replication, or an oncolytic virus vaccine; Preferably, the vaccine further includes adjuvants and excipients.

14. A drug comprising the recombinant virus according to any one of claims 1-7.

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