WO2023160550A1

WO2023160550A1 - Proteolysis targeting influenza virus, method for preparing same and use thereof

Info

Publication number: WO2023160550A1
Application number: PCT/CN2023/077449
Authority: WO
Inventors: 司龙龙; 李静; 陈丽; 李乐; 申权; 沈金影; 肖雪
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2022-02-22
Filing date: 2023-02-21
Publication date: 2023-08-31
Also published as: CN116676273A

Abstract

The present application provides a proteolysis targeting influenza virus, a method for preparing same and use thereof. The proteolysis targeting influenza virus comprises a hydrolysis targeting M1 protein, and the hydrolysis targeting M1 protein comprises a TEVp recognition site and a protein hydrolysis targeting molecule recognized by the ubiquitin-proteasome system which are sequentially inserted into the C-terminus. The protein hydrolysis targeting molecule recognized by the ubiquitin-proteasome system comprises the amino acid sequences set forth in SEQ ID Nos. 1-353. The hydrolysis targeting M1 protein can be recognized by the ubiquitin-proteasome system and thus hydrolyzed, such that the replicative capacity of the virus is reduced; therefore, the safety of the virus is relatively high. The proteolysis targeting influenza virus provided by the present application has many subtypes, which play an important role in the research and development of vaccines against influenza viruses.

Description

A kind of proteolytic targeting influenza virus and its preparation method and application

technical field

The application belongs to the field of biotechnology, and in particular relates to a proteolysis targeting influenza virus and its preparation method and application.

Background technique

Influenza viruses are divided into three types: A (A), B (B) and C (C), among which the outbreak of influenza A virus is the most frequent and has a wide impact. The genome of influenza A virus consists of 8 independent single-stranded RNA segments, encoding 10 proteins, including hemagglutinin protein (HA), matrix protein (M), neuraminidase (NA), nucleocapsid protein (NP ), nonstructural protein (NS) and three polymerases, PB1, PB2 and PA. Among them, matrix proteins include M1 and M2, and nonstructural proteins include NS1 and NEP. Among them, matrix proteins M1 and M2 play an important role in maintaining the shape of virus particles and the pathogenicity of viruses.

Influenza A virus can infect humans and poultry seasonally, and large-scale epidemics can cause extremely high morbidity and mortality, seriously threatening human health. At present, vaccination is the most important means of preventing influenza and controlling the spread of influenza.

CN102899294A discloses a H1N1 type swine influenza virus vaccine strain and its application. The H1N1 type swine influenza virus vaccine strain is an inactivated vaccine for preventing swine influenza, and can provide good protection to pigs challenged with homologous strong viruses. It has good immunogenicity, and can effectively prevent H1N1 swine flu regardless of single vaccine or combined vaccine.

CN106075424A discloses a kind of avian influenza virus vaccine, the antigen of described avian influenza virus vaccine is inactivated H9 subtype avian influenza virus, the HA of the new strain of H9 subtype avian influenza virus FJ11 strain that described avian influenza virus vaccine uses And the EID50 titer is high, the immunogenicity is good, and it can resist the attack of H9 subtype avian influenza virus that is prevalent and isolated in various places. The safety of the vaccine is good. After analyzing the data of traits, safety test and efficacy test in the storage period test, the analysis results are compared with similar products, and all indicators are stable and effective, and the H9 subtype avian influenza is inactivated. Vaccines produce antibodies quickly.

However, during the inactivation process of inactivated vaccines, the effective antigenic components may be destroyed or changed, affecting the immune effect. The immune effect of inactivated vaccines lasts for a short period of time, and multiple inoculations are required to strengthen the general cost. At the same time, the above-mentioned influenza virus Vaccines protect against only a few subtypes of the virus. Therefore, the development of an influenza virus library that is safe and controllable, targeted for degradation, and rich in subtypes plays an important role in the development of influenza virus vaccines.

Contents of the invention

The application provides a proteolysis-targeted influenza virus and its preparation method and application. The proteolysis-targeted influenza virus contains a hydrolysis-targeted M1 protein, which can be recognized by the ubiquitin-proteasome system and can be recognized by the tobacco plaque virus protease (Tobacco etch virus protease, TEVp) Cleavage, which can be produced in large quantities in cell lines overexpressing TEVp, while in normal cell lines, the ubiquitin-proteasome system will recognize proteolytic targeting molecules fused with viral proteins, thereby degrading viral proteins and virus replication The ability is weakened, or even completely loses the ability to replicate, and the resulting proteolytically targeted influenza virus has high safety. The proteolysis-targeted influenza virus can also be used as a live vaccine or an attenuated vaccine for the prevention of influenza; the proteolysis-targeted influenza virus can also be used in the treatment of tumors and used as an oncolytic virus. The subtypes of the proteolytically targeted influenza virus provided in the present application are rich in types, and play an important role in the research and development of influenza virus vaccines.

In the first aspect, the present application provides a proteolysis-targeted influenza virus, the proteolysis-targeted influenza virus contains a hydrolysis-targeted M1 protein;

Wherein, the C-terminus of the hydrolysis-targeted M1 protein is sequentially inserted into the TEVp recognition site and the proteolysis targeting molecule recognized by the ubiquitin-proteasome system; the proteolysis target recognized by the ubiquitin-proteasome system Include the SEQ ID to the molecule Amino acid sequences shown in No. 1-353.

In this application, the design principle of the proteolytic targeting virus is as follows:

(1) The proteolytic targeting molecule introduced into a specific site of the viral protein can be recognized by the ubiquitin-proteasome system in normal host cells, thereby degrading the corresponding viral protein and inactivating the virus;

(2) The proteolytic targeting molecules introduced into specific sites of viral proteins can be inhibited in specific viral production systems, or separated from viral proteins by selective cleavage of connecting chains, thereby avoiding or reducing the degradation of viral proteins. Degradation by the ubiquitin-proteasome system; and

(3) The proteolysis targeting molecule introduced into a specific site of the viral protein cannot be inhibited in normal host cells, or the connecting chain connecting the proteolysis targeting molecule and the viral protein cannot be cleaved in normal host cells. Therefore, the prepared virus can be recognized and degraded by the ubiquitin-proteasome system in host cells such as animals and humans, thereby reducing the replication ability, or even completely losing the replication and reproduction ability, which increases the safety of the virus.

In this application, 353 proteolytic targeting molecules that can be conditionally cleaved are introduced into the C-terminus of the hydrolysis-targeted M1 protein. Due to the ubiquitin-proteasome system in normal cells of humans and animals, it can recognize The proteolysis targeting molecule expressed in fusion with the viral protein can degrade the viral M1 protein through the ubiquitin-proteasome system, thereby degrading the viral protein. The hydrolysis-targeted influenza virus containing the hydrolysis-targeted M1 protein has weakened replication in animals and humans or even cannot replicate and reproduce, which increases the safety of the virus, and the obtained hydrolysis-targeted influenza virus can be prepared into a live influenza virus vaccine.

The amino acid sequence of the proteolysis targeting molecule is as follows, wherein SEQ ID No.1-4 and SEQ ID No.146-167 correspond to Keap1-Cul3 E3 ubiquitin ligase; SEQ ID No.5-7 correspond to SOCS2 E3 Ubiquitin ligase; SEQ ID No.8 corresponds to SOCS3 E3 ubiquitin ligase; SEQ ID No.9 corresponds to SOCS6 E3 ubiquitin ligase; SEQ ID No.10 corresponds to KLHL-12 E3 ubiquitin ligase; SEQ ID No. 11 corresponds to C-terminal Degrons-Cullin-RING E3 ubiquitin ligase; SEQ ID No.12-17 corresponds to KLHDC3 E3 ubiquitin ligase; SEQ ID No.18-26 corresponds to KLHDC2 E3 ubiquitin ligase; SEQ ID No. 27 corresponds to APPBP2 E3 ubiquitin ligase; SEQ ID No.28-31, SEQ ID No.53-60, SEQ ID No.168-174 corresponds to SPOP E3 ubiquitin ligase; SEQ ID No.32-34, SEQ ID No.175-176 corresponds to KLHL3 E3 ubiquitin ligase; SEQ ID No.35-38 corresponds to KLHL20 E3 ubiquitin ligase; SEQ ID No.39-40 corresponds to SPSB E3 ubiquitin ligase; SEQ ID No.41-43 Corresponding to FBXO31 E3 ubiquitin ligase; SEQ ID No.44-46, SEQ ID No.126-130, SEQ ID No.311-326 corresponding to β-TrCP1 E3 ubiquitin ligase; SEQ ID No.47-50, SEQ ID No.47-50, SEQ ID No.311-326 ID No.114-120, SEQ ID No.303-305 correspond to SCF ^FBW7 E3 ubiquitin ligase; SEQ ID No.51-52 correspond to ITCH E3 ubiquitin ligase; SEQ ID No.61-64, SEQ ID No. 177-182 correspond to MDM2 E3 ubiquitin ligase; SEQ ID No.65-67 correspond to CRBN E3 ubiquitin ligase; SEQ ID No.68-75, SEQ ID No.183-206 correspond to SEQ ID No.183 E3 ubiquitin Ligase; SEQ ID No.76-85, SEQ ID No.207-260 correspond to APC/C-Dbox E3 ubiquitin ligase; SEQ ID No.86-90, SEQ ID No.136-140, SEQ ID No. 261-278, SEQ ID No.331-345 correspond to APC/C-KENbox E3 ubiquitin ligase; SEQ ID No.91-95, SEQ ID No.294-298 correspond to COP1 E3 ubiquitin ligase; SEQ ID No. 96-100, SEQ ID No.299-300 correspond to CRL4_CDT2_1 E3 ubiquitin ligase; SEQ ID No.101-105, SEQ ID No.301-302 correspond to N-end UBR-box E3 ubiquitin ligase; SEQ ID No. .106-113 correspond to VHL E3 ubiquitin ligase; SEQ ID No.121-123 correspond to SCF ^Skp2-Cks1 E3 ubiquitin ligase; SEQ ID No.124-125, SEQ ID No.306-310 correspond to SCF ^TIR1 E3 ubiquitin SIAH-1 E3 ubiquitin ligase; SEQ ID No.131-135; SEQ ID No.327-330 corresponds to SIAH-1 E3 ubiquitin ligase; SEQ ID No.141-145, SEQ ID No.346-347 corresponds to APC/C-ABBA E3 ubiquitin ligase; SEQ ID No.279-293 corresponds to APC/C-TPR1 E3 ubiquitin ligase:

SEQ ID No. 352: LARKASLARFLEKRKERV; or

Preferably, the TEVp recognition site includes the amino acid sequence shown in SEQ ID No.354;

Preferably, the C-terminus of the M1 protein is connected to the TEVp recognition site through a flexible linker 1 . Preferably, the flexible linker 1 includes the amino acid sequence shown in SEQ ID No.355;

Preferably, the TEVp recognition site and the proteolysis targeting molecule are connected by a flexible linker 2 . Preferably, the flexible linker 2 includes the amino acid sequence of GSG;

Preferably, the proteolytically targeted influenza viruses are type A, B, and C viruses.

Preferably, the subtypes of the influenza virus include H1N1, H1N2, H1N3, H1N8, H1N9, H2N2, H2N3, H2N8, H3N1, H3N2, H3N8, H4N2, H4N4, H4N6, H4N8, H5N1, H5N2, H5N3, H5N6, H5N8 , H5N9, H6N1, H6N2, H6N4, H6N5, H6N6, H6N8, H7N1, H7N2, H7N3, H7N7, H7N8, H7N9, H8N4, H9N1, H9N2, H9N5, H9N8, H10N3, H10N4, H10N7, H10N8, H10N9, H11N2, H11N6 , H11N9, H12N1, H12N3, H12N5, H13N6, H13N8, H14N5, H15N2, H15N8, H16N3, H17N10 or H18N11 any one or a combination of at least two.

In the present application, the inventors found that by introducing the nucleotide sequence of the TEVp recognition site and the proteolytic targeting molecule into the genome of the influenza virus, in the cell line overexpressing TEVp, the influenza virus containing the proteolytic targeting molecule The viral genome can be replicated along with the replication of the viral genome, and can be fused and expressed in the viral protein along with the translation of the viral protein, thereby obtaining a virus modified by a proteolytic targeting molecule, that is, a proteolytic targeting virus (Proteolysis-targeting virus). Targeting chimeric virus, PROTAC virus).

In normal cells, the ubiquitin-proteasome system will recognize the proteolytic targeting molecule fused with the viral M1 protein, thereby degrading the viral protein, and the replication ability of the virus is weakened or even completely lost. Therefore, the PROTAC virus has a high security. At the same time, since the PROTAC virus contains the specific recognition sequence and site of the ubiquitin-proteasome system, the PROTAC virus can transform a cold tumor into a hot tumor in the treatment of tumors and play an oncolytic role.

In the present application, the PROTAC virus can be efficiently replicated and mass-produced in a specific artificially modified cell line. In addition, the PROTAC virus can be further modified, such as introducing an immune enhancer to a specific region or specific amino acid of the virus protein, so as to obtain a virus with improved performance, thereby obtaining a PROTAC virus with enhanced immunogenicity.

In this application, the used flexible linker and TEVp recognition site (connecting chain) and the amino acid sequence structure of the flexible linker and proteolysis targeting molecule can also be applied to type B influenza or other subtypes of influenza virus , can also be used for the transformation of other types of viruses, such as any one or a combination of at least two of HIV, SARS-CoV-2, hand-foot-mouth virus, hepatitis D virus or hepatitis E virus.

In a second aspect, the present application provides a nucleic acid molecule encoding the hydrolysis-targeted M1 protein described in the first aspect.

In a third aspect, the present application provides a recombinant vector containing at least one copy of the nucleic acid molecule described in the second aspect.

In a fourth aspect, the present application provides a method for preparing the proteolytically targeted influenza virus described in the first aspect, the preparation method comprising the following steps:

(1) Constructing an expression vector for preparing proteolytic targeting influenza virus;

(2) Using the expression vector obtained in step (1) to replace the plasmid expressing the influenza virus M gene in the influenza virus rescue system, and co-transfecting the cell line to obtain the proteolytically targeted influenza virus.

This application provides 353 proteolytic targeting viruses, the C-terminus of the M1 protein of the proteolytic targeting virus contains a proteolytic targeting molecule recognized by the ubiquitin-proteasome system, the M1 protein and the proteolytic targeting molecule The TEVp recognition site can be selectively cleaved by two flexible molecules between the M1 protein and the TEVp recognition site and between the TEVp recognition site and the proteolytic targeting molecule. The method is connected, and the operation of transforming the virus through the method is simple, and the obtained proteolysis-targeted influenza virus is safe and reliable, and has high immunity.

Preferably, in step (1), the expression vector includes the coding sequence of the hydrolysis-targeted M1 protein.

Preferably, in step (2), the cell line is an artificial cell line overexpressing TEVp.

Preferably, in step (2), the influenza virus rescue system includes a 12-plasmid rescue system for WSN influenza virus.

Preferably, the preparation method also includes targeting the proteolysis to the artificial modification of influenza virus overexpressing TEVp The steps for large-scale production are replicated in cell lines.

In the present application, the PROTAC virus can replicate only in the cell line overexpressing TEVp, and using the dependence of the PROTAC virus on the cell line overexpressing TEVp, it can be performed in the cell line overexpressing TEVp Bulk preparation of influenza virus.

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

(1) Construct the expression vector for preparing proteolytically targeted influenza virus, use the method of genetic engineering to transform the plasmid expressing influenza virus M gene in the 12 plasmid rescue system of WSN influenza virus, in the gene sequence of M1 protein introducing an insert sequence before the C-terminus and the stop codon to obtain an expression vector, the expression vector comprising the coding sequence of the hydrolysis-targeted M1 protein;

(2) Replace the plasmid expressing the influenza virus M gene in the influenza virus rescue system with the expression vector obtained in step (1), and co-transfect the influenza virus rescue system after replacement with an artificial cell line overexpressing TEVp to obtain the protein Hydrolysis targeting influenza virus;

The proteolytically targeted virus is replicated in an engineered cell line overexpressing TEVp to produce the proteolytically targeted virus on a large scale.

In a fifth aspect, the present application provides an influenza virus vaccine, which comprises the proteolytic targeting influenza virus described in the first aspect.

Preferably, the influenza virus vaccine is any one of an attenuated vaccine, a replication-deficient live virus vaccine or a replication-controllable live virus vaccine.

In the sixth aspect, the present application provides the proteolytic targeting influenza virus described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, and the proteolytic targeting influenza virus described in the fourth aspect The preparation method or the application of any one or a combination of at least two of the influenza virus vaccines described in the fifth aspect in the preparation of drugs for treating influenza and/or oncolytic drugs.

The numerical ranges described in this application not only include the above-mentioned point values, but also include any point values between the above-mentioned numerical ranges that are not listed. Due to space limitations and for the sake of brevity, this application no longer exhaustively lists the described ranges. The specific pip value to include.

Compared with the prior art, the present application has the following beneficial effects:

(1) This application uses the hydrolysis of the ubiquitin-proteasome system and the cleavage of TEVp to prepare a large number of proteolytically targeted influenza viruses in cell lines overexpressing TEVp, while in normal cell lines, ubiquitin-protease The body system will recognize the proteolytic targeting molecule fused with the viral protein, thereby degrading the viral protein, weakening the replication ability of the virus, or even completely losing the ability to replicate. Therefore, the proteolysis-targeted influenza virus has high safety, and the proteolysis-targeted influenza virus is an attenuated, replication-deficient and replication-controllable live virus.

(2) The preparation method of the proteolysis-targeted influenza virus is simple, safe and reliable; the proteolysis-targeted influenza virus in this application is produced by a mammalian stable cell line, which overcomes the shortcomings of the traditional use of chicken embryos to propagate viruses (use Chicken embryo reproduction virus is easy to cause adverse reactions such as human allergies), and the obtained proteolytic targeting influenza virus has high immunogenicity, and can be used for the preparation of vaccines and the development of drugs related to the treatment of viral infections, and has strong research and application value .

(3) The proteolytic targeting influenza virus can be further modified, for example, introducing an immune enhancer to a specific region or specific amino acid of the viral protein to obtain a virus with improved performance, thereby obtaining a proteolytic target with enhanced immunogenicity to the flu virus.

(4) The proteolytically targeted influenza virus can activate the tumor microenvironment, change cold tumors into hot tumors, and increase the therapeutic effect of tumors.

Description of drawings

FIG. 1 is a schematic diagram of the preparation process of the proteolytically targeted influenza virus in Example 1.

Figure 2 is the growth curve of proteolytically targeted influenza virus in MDCK-TEVp (+TEVp) cells and normal MDCK (-TEVp) cells. Each E3 ubiquitin ligase selects a strain as a representative for data display, so each strain in the figure uses its corresponding E3 ubiquitin ligase to rename M1-PTD ^KLHDC2 (M1-PTD22), M1-PTD ^KLHDC3 (M1-PTD43), M1-PTD ^APPBP2 (M1-PTD71), M1-PTD ^KLHL20 (M1-PTD84), M1-PTD ^FBXO31 (M1-PTD91), M1-PTD ^β-TrCP (M1-PTD94), M1 -PTD ^SOCS2 (M1-PTD57), M1-PTD ^FEM1C (M1-PTD63), M1-PTD ^ITCH (M1-PTD101), M1-PTD ^SPOP (M1-PTD109), M1-PTD ^MDM2 (M1-PTD110), M1 -PTD ^CRBN (M1-PTD116), M1-PTD ^APC/C-ABBA (M1-PTD205), M1-PTD ^CBL (M1-PTD119), M1-PTD ^APC/C-KENbox (M1-PTD138), M1-PTD ^COP1 (M1-PTD148), M1-PTD ^{N-end UBR-box} (M1-PTD165), M1-PTD ^FBW7 (M1-PTD174), M1-PTD ^Skp2-Cks1 (M1-PTD183), M1-PTD ^SIAH-1 (M1-PTD194), M1-PTD ^APC/C-TPR1 (M1-PTD143), M1-PTD ^APC/C-Dbox (M1-PTD132).

Figure 3 is the safety evaluation of proteolytically targeted influenza virus in the mouse model, where *** indicates that there is a significant difference compared with WT. M1-PTD ^KLHDC2 (M1-PTD22), M1-PTD ^KLHDC3 (M1-PTD43), M1-PTD ^APPBP2 (M1-PTD71), M1-PTD ^KLHL20 (M1-PTD84), M1-PTD ^FBXO31 (M1-PTD91), M1-PTD ^β-TrCP (M1-PTD94) is used as a representative for data display.

Figure 4 is the verification of the mechanism of proteolysis targeting influenza virus. One strain is selected for each E3 ubiquitin ligase as a representative for data display, so each strain in the figure is renamed using its corresponding E3 ubiquitin ligase.

Fig. 5 is the immunogenicity evaluation of proteolytic targeting influenza virus in mouse model; Among them, (a) neutralizing (NT) antibody and hemagglutination inhibitory (HI) antibody level; (b) Anti-HA IgG, Anti -NP IgG antibody level; (c) Anti-NP IgA antibody level; (d) T cell immune response against influenza virus M1 antigenic peptide (MGLIYNRM) in mouse lung tissue; (e) T cell immune response against influenza virus in mouse spleen tissue T cell immune response levels of NP antigenic peptide (ASNENMETME) (left panel) and M1 antigenic peptide (MGLIYNRM) (right panel); (f) Antigen presentation of proteolytically targeted influenza virus in macrophage Raw264.7 cells level. *, **, *** indicate significant differences compared with wild-type virus (WT); #, ##, ### indicate significant differences compared with inactivated influenza vaccine (IIV); +, + +, +++ indicates that there is a significant difference compared with cold-adapted attenuated influenza vaccine (CAIV). M1-PTD ^KLHDC2 (M1-PTD22), M1-PTD ^KLHDC3 (M1-PTD43), M1-PTD ^APPBP2 (M1-PTD71), M1-PTD ^KLHL20 (M1-PTD84), M1-PTD ^FBXO31 (M1-PTD91), M1-PTD ^β-TrCP (M1-PTD94) is used as a representative for data display.

Figure 6 is the evaluation of the cross-immune protection effect of proteolytic targeting influenza virus vaccine in adult (7 weeks old) mouse model; wherein, (a) three days after mice were infected with wild-type WSN influenza virus, the wild-type WSN influenza virus titer; (b) mouse body weight and survival rate within two weeks after mice were infected with wild-type influenza virus A/X-31(H3N2); (c) three days after mice were infected with wild-type influenza virus A/X-31(H3N2), lung Titers of wild-type influenza virus A/X-31 (H3N2) in tissues; (b) body weight and survival rate of mice infected with wild-type influenza virus A/X-31 (H3N2) within two weeks. M1-PTD ^β-TrCP (M1-PTD94) is used as a representative for data display.

Figure 7 is the evaluation of the immune protection effect of proteolytic targeting influenza virus vaccine in aged (15 months old) mice and ferret models; wherein, (a) the neutralization induced by proteolytic targeting influenza virus vaccine in aged mice ( NT) antibody, hemagglutination inhibitory (HI) antibody, anti-HA IgG antibody, anti-NP-IgG antibody levels; (b) proteolytically targeted influenza virus vaccine in lung tissue (left panel) and spleen (right panel) of aged mice ) T cell immune response level induced against influenza virus NP antigen peptide (ASNENMETME); (c) Three days after immunization and non-immunization aged mice were infected with wild-type WSN influenza virus, the lung tissue wild-type WSN influenza virus titer; (d) body weight and survival rate of immunized and unimmunized old mice infected with wild-type WSN influenza virus within two weeks; (e) immunized and unimmunized ferrets infected with wild-type WSN Virus titers in nasal washes three days after influenza virus. M1-PTD ^β-TrCP (M1-PTD94) is used as a representative for data display.

Figure 8 shows the anti-tumor effect of proteolytically targeting influenza virus; among them, M1-PTD ^β-TrCP (M1-PTD94) is used as a representative for data display.

Detailed ways

The technical solutions of the present application will be further described below through specific implementation methods. It should be clear to those skilled in the art that the embodiments are only for helping to understand the present application, and should not be regarded as a specific limitation on the present application.

If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products commercially available through regular channels.

Example 1

This example provides a series of proteolysis-targeted influenza viruses. The proteolysis-targeted influenza virus contains a hydrolysis-targeted M1 protein, and the C-terminus of the hydrolysis-targeted M1 protein is sequentially inserted into the TEVp recognition site and The proteolysis targeting molecule recognized by the ubiquitin-proteasome system; the amino acid sequence of the proteolysis targeting molecule recognized by the ubiquitin-proteasome system is shown in SEQ ID No.1-353. The amino acid sequence of the TEVp recognition site is shown in SEQ ID No.354, the C-terminal of the M1 protein and the TEVp recognition site are connected by a flexible linker 1, and the amino acid sequence of the flexible linker 1 is shown in SEQ ID No. As shown in .355, the TEVp recognition site and the proteolysis targeting molecule are connected by a flexible linker 2, and the amino acid sequence of the flexible linker 2 is GSG; the amino acid sequence structure from the C-terminus of the M1 protein to the stop codon It is: C-terminal of M1 protein-flexible linker 1-TEVp recognition site-flexible linker 2-proteolysis targeting molecule.

The amino acid sequence of SEQ ID No.354 is ENLYFQG;

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

The amino acid sequences of the proteolysis targeting molecules recognized by the ubiquitin-proteasome system are shown in Table 1.

Table 1

The influenza viruses in the proteolytic targeting influenza virus are A, B, and C viruses, and the subtypes of the influenza viruses include H1N1, H1N2, H1N3, H1N8, H1N9, H2N2, H2N3, H2N8, H3N1, H3N2, H3N8 , H4N2, H4N4, H4N6, H4N8, H5N1, H5N2, H5N3, H5N6, H5N8, H5N9, H6N1, H6N2, H6N4, H6N5, H6N6, H6N8, H7N1, H7N2, H7N3, H7N7, H7N8, H7N9, H8N4, H9N1, H9N2 , H9N5, H9N8, H10N3, H10N4, H10N7, H10N8, H10N9, H11N2, H11N6, H11N9, H12N1, H12N3, H12N5, H13N6, H13N8, H14N5, H15N2, H15N8, H16N3, H17N10, and H 18N11.

The schematic diagram of the preparation process of the proteolytic targeting influenza virus is shown in Figure 1, and the proteolytic targeting influenza virus The preparation method comprises the steps:

(1) Construction of an expression vector for preparing proteolytic targeting influenza virus:

Using the plasmid expressing the influenza virus M gene in the influenza virus rescue system as a template, insert an expressible "flexible linker 1 (SEQ ID No. 355 : GSGG)-TEVp recognition site (SEQ ID No.354: ENLYFQG)-flexible linker 2: GSG)-the gene sequence of the amino acid sequence of the proteolytic targeting molecule". When the amino acid sequence of the proteolysis targeting molecule is PTD3, the constructed vector is named M1-PTD3; when the amino acid sequence of the proteolysis targeting molecule is PTD4, the constructed vector is named M1-PTD4, Carrier naming rules, and so on. The obtained target vector was verified by sequencing, and the expression vector was successfully constructed.

(2) Replace the plasmid expressing the influenza virus M gene in the influenza virus rescue system with the expression vector obtained in step (1), and co-transfect the influenza virus rescue system after replacement with the cell line to obtain the proteolytically targeted influenza virus .

WSN virus was rescued using the 12-plasmid rescue system for WSN influenza virus. When rescuing proteolytic hydrolysis targeting influenza virus, it is only necessary to replace the plasmid used to express the M gene in the 12 plasmid rescue system with the expression vector constructed in step (1), and to rescue the gained virus strain with step (1) ) to name the corresponding carrier.

Cells expressing TEVp were seeded in 6-well plates; the next day, the M1-PTD3 vector in step (1) was co-transfected with the other 11 plasmids in the WSN influenza virus rescue system into a cell line expressing TEVp protein (HEK293T cell line), corresponding to each well of a 6-well plate, add 0.2 μg of each plasmid. Six hours after transfection, the medium was replaced with a new DMEM medium containing 0.5% FBS, 1 μg/mL TPCK-trypsin and double antibodies. Afterwards, the lesion of the cells was observed every day, and when the lesion of the cells reached 80%, the virus supernatant was collected to obtain the proteolysis-targeted virus, which was named M1-PTD3.

According to the same method, other proteolytically targeted influenza viruses can be obtained and named according to the same rules. The obtained proteolytically targeted influenza viruses are shown in Table 2:

Table 2

The obtained virus supernatant was infected with a new cell line expressing TEVp protein, and the constructed proteolytic targeting virus was investigated according to the standard of whether it could cause cytopathic disease:

If it can cause pathological changes in cell lines expressing TEVp protein (such as HEK293T cells expressing TEVp and/or MDCK cells expressing TEVp), it means that the proteolytic targeting virus rescue is successful;

If the TEVp-expressing cells (such as TEVp-expressing HEK293T cells and/or TEVp-expressing MDCK cells) cannot be caused to become pathological, it means that the rescue of the proteolytic targeting virus fails.

The results showed that all proteolytically targeted influenza viruses were successfully rescued.

test case 1

Preparation efficiency and safety evaluation of the proteolytic targeting influenza virus:

(1) Preparation efficiency and safety evaluation of proteolysis-targeted influenza virus at the cellular level:

Investigate the cytopathic conditions and growth curves of M1-PTD3 to M1-PTD413 influenza virus strains in TEVp-expressing MDCK cell lines (MDCK-TEVp cell lines) and normal MDCK cell lines, and investigate the preparation efficiency and safety of the strains sex. Wild-type influenza virus was used as a control.

The criteria for judging the safety of the virus strain are as follows:

Based on the observation of cytopathy, it was determined that wild-type influenza virus could cause complete cytopathy (100%) in both MDCK-TEVp cells and MDCK cells. If the virus strain can cause obvious cytopathy in the MDCK-TEVp cell line (cytopathy reaches 50-100%), it shows that the preparation efficiency of the virus strain is higher; compared with the wild-type virus, if the virus strain in normal MDCK cells If the strain cannot cause cytopathy or causes less cytopathy (the cytopathy is lower than 100% cytopathy caused by the wild-type virus), it shows that the virus strain is safe.

Based on the investigation of the growth curve, it is determined that compared with the wild-type virus, if the virus strain can be highly replicated in the MDCK-TEVp cell line (the virus titer is higher than the wild-type virus, comparable to the wild-type virus, or not lower than the wild-type virus 1/1,000), indicating that the preparation efficiency of the strain is higher; compared with the wild-type virus, if the replication ability of the strain is weakened or even not replicated in the normal MDCK cell line (the virus titer is lower than that of the wild-type virus degree), indicating that the strain is safe.

(a) The steps of cytopathic detection are as follows:

The prepared proteolysis-targeted influenza virus and wild-type influenza virus were respectively infected at the ratio of MOI=0.01 to MDCK-TEVp cell line and normal MDCK cell line, and the pathological changes of the cells were observed and recorded every day for 4 days.

Test results showed that in the MDCK-TEVp cell line, all proteolytically targeted influenza virus and wild-type influenza Both viruses could cause significant cytopathic effects; whereas in normal MDCK cell lines, only wild-type influenza viruses could cause significant cytopathic effects, whereas proteolytically targeted influenza viruses caused reduced or no lesions. The results indicated that the prepared proteolysis-targeted influenza virus was safe at the cellular level.

(b) The steps based on growth curve detection are as follows:

Infect MDCK-TEVp cell line and normal MDCK cell line with the prepared proteolysis-targeted influenza virus and wild-type influenza virus according to the ratio of MOI=0.001, and take the cell culture supernatant at 24h, 48h, 72h and 96h after infection , _TCID50 assay and plaque assay were used to detect the virus titer in the supernatant, so as to know the replication ability of the virus in the two cells.

The results shown in Figure 2 show that in the MDCK-TEVp cell line, all proteolytically targeted influenza viruses and the wild-type influenza virus have good replication ability; while in the normal MDCK cell line, only the wild-type influenza virus exhibits good replication ability. The replication ability of proteolytically targeted influenza virus is weakened or even replication deficient. The results indicated that the prepared proteolysis-targeted influenza virus was safe at the cellular level.

(2) Safety evaluation of proteolysis-targeted influenza virus at the animal level:

The safety of the proteolytically targeted influenza virus was evaluated at the animal level using BALB/c and C57BL/6J mice. Six proteolytically targeted influenza virus strains (M1-PTD ^KLHDC2 , M1-PTD ^KLHDC3 , M1-PTD ^APPBP2 , M1-PTD ^KLHL20 , M1-PTD ^FBXO31 , M1-PTD ^β-TrCP ) were selected as proteolytically targeted influenza strains. Virus representative, carry out virus safety evaluation.

The specific steps of safety evaluation are as follows:

(a) 80 7-week-old female BALB/c mice or C57BL/6J mice were divided into 8 groups, 10 in each group;

(b) Each mouse in the first group was inoculated with PBS (Vehicle) intranasally, each mouse in the second group was inoculated with 1×10 ⁵ TCID ₅₀ wild-type WSN influenza virus (WT), and the third to eighth groups were inoculated with M1-PTD ^KLHDC2 , M1-PTD ^KLHDC3 , M1-PTD ^APPBP2 , M1-PTD ^KLHL20 , M1-PTD ^FBXO31 , or M1-PTD ^β-TrCP were inoculated nasally with 1×10 ⁵ TCID ₅₀ ;

(c) Three days after the inoculation, 5 mice were taken from each group, and their lung tissues were taken to detect the virus titer therein;

(d) Continue to observe and monitor the body weight and death of the remaining 5 mice in each group for 14 days.

The results shown in Figure 3 show that the wild-type WSN influenza virus can highly replicate in the lungs of mice, and cause significant weight loss and death of mice. The proteolytically targeted influenza virus replicated weakly (below the limit of detection) in the lungs of mice, and did not cause weight loss or death in mice. Therefore, the proteolytic targeting virus vaccine has good safety.

test case 2

The replication ability of proteolytically targeted influenza virus was investigated:

(1) Use Western Blot to detect the M1 protein expression level of the proteolytic targeting influenza virus, select 5 strains of the proteolytic targeting influenza virus as representative strains, and investigate the replication of the proteolytic targeting influenza virus in normal cells ability.

The proteolytically targeted influenza virus and the wild-type influenza virus were respectively infected with normal MDCK cell lines (MOI=0.1), supplemented with 25nM, 50nM and 100nM proteasome inhibitor MG-132 in the culture medium, and DMSO (same as the virus Dilution ratio) mixed with normal MDCK cell line as a control. After 24h, 48h, and 72h of infection, cell samples were collected, and Western Blot was used to detect the expression level of virus M1 protein.

(2) Detecting the expression level of the proteolytically targeted influenza virus M1 protein by immunofluorescence experiments, and investigating the replication ability of the proteolytically targeted influenza virus.

Targeted proteolysis of influenza virus and wild-type influenza virus to infect MDCK-TEVp cell line and normal MDCK cell line (MOI=0.01) respectively, supplement 0nM, 25nM, 50nM and 100nM protease in the medium respectively Inhibitor MG-132, the mixed solution of DMSO (same dilution ratio as virus) and normal MDCK cell line was used as control. After 48 hours of infection, the cells were fixed with 4% PFA, and the expression level of the proteolytically targeted influenza virus M1 protein was detected by immunofluorescence experiment.

The experimental results shown in Figure 4 show that the proteolysis-targeted influenza virus can replicate in large quantities after infecting MDCK-TEVp cells, and synthesize a large amount of viral proteins. However, after the proteolytically targeted influenza virus infects normal MDCK cells, it cannot replicate in large quantities, so less viral protein M1 signals are detected; and when the proteasome system of the cells is inhibited, the viral protein M1 signal increases, indicating that when After the proteasome system is inhibited, the replication ability of the virus is enhanced. The detection results are consistent with the Western Blot detection results in Test Example 2, which further proves that the introduction of proteolytic targeting molecules can mediate the degradation of viral proteins by the proteasome of cells, thereby inhibiting the replication ability of viruses; when the proteasome system of cells is blocked Following inhibition, viral replication capacity was restored, consistent with the design rationale for proteolytically targeting influenza viruses.

Test case 3

Immunogenicity and protection of proteolytically targeted influenza viruses at the animal level:

The immunogenicity and protective properties of the proteolytically targeted influenza viruses were evaluated at the animal level. Taking inactivated influenza vaccine (IIV) as a control (the inactivated influenza virus vaccine was prepared by the inventor with homologous influenza virus particles according to the method provided by the Chinese Pharmacopoeia), and the cold-adapted attenuated vaccine used in clinical practice was used as a control at the same time. Six proteolytically targeted influenza virus strains (M1-PTD ^KLHDC2 , M1-PTD ^KLHDC3 , M1-PTD ^APPBP2 , M1-PTD ^KLHL20 , M1-PTD ^FBXO31 , M1-PTD ^β-TrCP ) were selected as proteolytically targeted influenza strains. Virus representatives were evaluated for immunogenicity and protection of proteolytically targeted influenza viruses.

The specific steps of immunogenicity and protective investigation are as follows:

(1) 180 7-week-old female BALB/c mice or C57BL/6J mice were divided into 9 groups, 20 in each group;

(2) Each mouse in the first group was intranasally inoculated with PBS (Vehicle), each mouse in the second group was inoculated with 1×10 ⁵ TCID ₅₀ cold-adapted influenza vaccine (CAIV), and each mouse in the third group was inoculated intranasally with PBS (Vehicle). Rats were inoculated intramuscularly with 1×10 ⁵ TCID ₅₀ inactivated influenza vaccine (IIV), and the fourth to ninth groups were inoculated with 10 ⁵ TCID ₅₀ of M1-PTD ^KLHDC2 , M1-PTD ^KLHDC3 , M1-PTD ^APPBP2 , M1-PTD ^KLHL20 , M1-PTD ^FBXO31 , or M1-PTD ^β-TrCP ;

(3) One week after inoculation, 5 mice were taken from each group, and their lung tissues and spleens were taken to detect the immune response of T cells therein;

(4) Three weeks after the inoculation, 5 mice were taken from each group, and blood was taken for hemagglutination inhibition (HI) test, neutralizing (NT) antibody detection and ELISA detection, respectively, to detect the antibody immune response therein;

(5) Three weeks after the inoculation, each group of mice was inoculated with 2×10 ⁵ PFU of homologous wild-type WSN influenza virus or heterologous wild-type influenza virus A/X-31(H3N2);

(6) Three days after inoculation with wild-type influenza virus, take 5 mice in each group, get their lung tissues, and detect the virus titer therein;

(7) Continue to observe and monitor the body weight and death of the remaining 5 mice in each group for 14 days.

The results shown in Figure 5 show that all proteolytically targeted influenza viruses can induce high levels of hemagglutination inhibition (HI) antibody titers, neutralizing (NT) antibody titers, anti-HA IgG, anti-NP IgG in animals , anti-NP IgA, T cell immune response, etc.; and the induced hemagglutination inhibitory antibody, neutralizing antibody, anti-NP IgG, anti-NP IgA, T cell immune response levels were significantly higher than those induced by inactivated influenza vaccine Antibody levels, the level of immune response induced by some strains was higher than that of cold-adapted attenuated vaccines. The results shown in Figure 6 show that the vaccination of proteolysis-targeted influenza virus vaccine can significantly reduce the titer of wild-type WSN influenza virus and X-31 (H3N2) virus in the lung tissue of animals, and can protect all animals from survival, so the protein Hydrolysis-targeting influenza virus vaccines can provide cross-immune protection; proteolysis-targeting influenza virus vaccines provide significantly better protection than inactivated influenza vaccines. Therefore, the proteolysis-targeted influenza virus vaccine has better immunogenicity and protective effect. In addition, the results shown in Figure 7 demonstrate that proteolytically targeted influenza virus vaccines in aged (15-month-old) mice And in the ferret model, it can also exert a level of immune protection effect.

Test case 4

To investigate the potential of proteolytically targeting influenza virus as an oncolytic virus:

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

The specific test steps are as follows:

(1) Inject melanoma subcutaneously on the back of the mouse, and raise it for 9 days. When the volume of the tumor reaches about 100 mm ³ , continue the experimental operation;

(2) Inject 50 μL of proteolytically targeted influenza virus M1-PTD ^β-TrCP (M1-PTD94) and PBS into the tumor, once every other day, for a total of 4 injections;

(3) Detect the volume of the tumor every day.

The results shown in Figure 8 show that the proteolysis-targeting influenza virus can effectively inhibit the increase in tumor volume, which proves that the proteolysis-targeting influenza virus has the potential to become an oncolytic virus.

In summary, the proteolysis-targeted influenza virus provided by this application can be recognized and hydrolyzed by the ubiquitin-proteasome system, has weak replication ability, and has high safety. It can be used as a live vaccine or an attenuated vaccine for the prevention of influenza; The proteolytically targeted influenza virus can be used as an oncolytic virus in tumor therapy. The subtypes of the proteolysis-targeted influenza virus provided by the application are rich in types, and play an important role in the research and development of influenza virus vaccines and tumor treatment.

The applicant declares that the above description is only a specific embodiment of the application, but the scope of protection of the application is not limited thereto, and those skilled in the art should understand that any person skilled in the art disclosed in this application Within the technical scope, easily conceivable changes or substitutions all fall within the scope of protection and disclosure of the present application.

Claims

a proteolytically targeted influenza virus comprising a hydrolytically targeted M1 protein;

Wherein, the C-terminus of the hydrolysis-targeted M1 protein is sequentially inserted into the TEVp recognition site and the proteolysis targeting molecule recognized by the ubiquitin-proteasome system; the proteolysis target recognized by the ubiquitin-proteasome system Molecules include the amino acid sequences shown in SEQ ID Nos. 1-353.
The proteolytically targeted influenza virus according to claim 1, wherein the TEVp recognition site comprises the amino acid sequence shown in SEQ ID No.354.
The proteolytically targeted influenza virus according to claim 1 or 2, wherein the C-terminus of the M1 protein and the TEVp recognition site are connected by a flexible linker 1;

Preferably, the flexible linker 1 includes the amino acid sequence shown in SEQ ID No.355;

Preferably, the TEVp recognition site and the proteolysis targeting molecule are connected by a flexible linker 2;

Preferably, the flexible linker 2 comprises the amino acid sequence shown in SEQ ID No.356.
The proteolysis-targeted influenza virus according to any one of claims 1-3, wherein the proteolysis-targeted influenza virus is a type A, type B, or type C virus;

Preferably, the subtypes of the proteolytically targeted influenza virus include H1N1, H1N2, H1N3, H1N8, H1N9, H2N2, H2N3, H2N8, H3N1, H3N2, H3N8, H4N2, H4N4, H4N6, H4N8, H5N1, H5N2, H5N3 , H5N6, H5N8, H5N9, H6N1, H6N2, H6N4, H6N5, H6N6, H6N8, H7N1, H7N2, H7N3, H7N7, H7N8, H7N9, H8N4, H9N1, H9N2, H9N5, H9N8, H10N3, H10N4, H10N7, H1 0N8, H10N9 , H11N2, H11N6, H11N9, H12N1, H12N3, H12N5, H13N6, H13N8, H14N5, H15N2, H15N8, H16N3, H17N10 or H18N11 any one or a combination of at least two.
A nucleic acid molecule encoding the hydrolysis-targeted M1 protein of any one of claims 1-4.
A recombinant vector containing at least one copy of the nucleic acid molecule of claim 5.
A method for preparing the proteolytic targeting influenza virus according to any one of claims 1-4, comprising the steps of:

(1) Constructing an expression vector for preparing proteolytic targeting influenza virus;

(2) Using the expression vector obtained in step (1) to replace the plasmid expressing the influenza virus M gene in the influenza virus rescue system, and co-transfecting the cell line to obtain the proteolytically targeted influenza virus.
[Corrected under Rule 26 02.03.2023]
The preparation method of proteolysis-targeted influenza virus according to claim 7, wherein, in step (1), the expression vector includes the coding sequence of the hydrolysis-targeted M1 protein;

Preferably, in step (2), the cell line is an artificial cell line overexpressing TEVp;

Preferably, in step (2), the influenza virus rescue system includes a 12-plasmid rescue system for WSN influenza virus.
The preparation method of the proteolysis-targeted influenza virus according to claim 7 or 8, wherein the preparation method further comprises replicating the proteolysis-targeted influenza virus in an artificially modified cell line overexpressing TEVp, steps in mass production.
An influenza virus vaccine comprising the proteolytic targeting influenza virus according to any one of claims 1-4;

Preferably, the influenza virus vaccine is any one of an attenuated vaccine, a replication-deficient live virus vaccine or a replication-controllable live virus vaccine.
The proteolytic targeting influenza virus according to any one of claims 1-4, the nucleic acid molecule according to claim 5, the recombinant vector according to claim 6, the protein according to any one of claims 7-9 Application of any one or a combination of at least two of the preparation method of the hydrolysis-targeted influenza virus or the influenza virus vaccine described in claim 10 in the preparation of a drug for treating influenza and/or an oncolytic drug.