Universal influenza mRNA vaccine and its use

Abstract

A universal influenza mRNA vaccine is provided, the protein encoded by the universal influenza mRNA vaccine consists of the extracellular domain (M2e), hemagglutinin (HA) stem LAH region, and nucleoprotein (NP) of the influenza A virus matrix protein 2. Furthermore, the application of the universal influenza mRNA vaccine in a mouse animal model is further provided, the universal influenza mRNA vaccine can induce a robust humoral and cellular immune response, protecting mice in the animal model from infection by multiple influenza A viruses. The universal influenza mRNA vaccine is safe, effective, and has a rapid production platform.

Description

[Technical Field]

[0001] This invention relates to the field of biotechnology, and more specifically to a universal influenza mRNA vaccine. Furthermore, this invention also relates to the application of said mRNA vaccine. [Background technology]

[0002] Influenza viruses are RNA viruses that cause influenza in humans or animals, causing acute upper respiratory tract infections and being easily transmitted through the air, thus regularly causing pandemics worldwide (generally occurring every winter, spring, and summer). An estimated 1 billion people worldwide contract influenza each year, with 3 to 5 million developing severe symptoms and 290,000 to 650,000 dying from influenza-related respiratory illnesses. Influenza cases are expected to increase globally and in China from 2022 onwards. The World Health Organization (WHO) recommends annual influenza vaccination, as it is the most effective way to prevent influenza. However, the hemagglutinin (HA) of influenza viruses often undergoes antigenic drift and / or antigenic shift, resulting in antigenic mismatch between vaccine virus strains and circulating virus strains. This means that current influenza vaccines are not effectively preventing both seasonal and pandemic influenza. The global effectiveness of seasonal influenza vaccines has been reported to be only 10% to 60%. Therefore, there is an urgent need in the market for the development of a universal vaccine targeting the antigenic epitopes of conserved influenza viruses.

[0003] Currently, commercially available influenza vaccines are mainly split influenza vaccines, attenuated influenza vaccines, and protein subunit vaccines. Whether it is a conventional chicken embryo-produced split influenza vaccine or an influenza vaccine produced by a cell-based matrix or recombinant protein method, the production time is long. The successful application of mRNA technology in COVID-19 vaccines has drawn significant attention to the high efficiency, safety, and potent immunogenicity characteristics of mRNA technology. The development of influenza-related mRNA vaccines can avoid complex biological activity processes such as cell fermentation and requires only in vitro mRNA synthesis, thus greatly accelerating development and simplifying the process. Whether it is a specific vaccine targeting a particular influenza virus strain or a universal vaccine, mRNA technology allows candidate influenza vaccines to rapidly enter the clinical trial stage. [Overview of the project] [Problems that the invention aims to solve]

[0004] The objective of this invention is to provide an mRNA vaccine that encodes universal influenza and its applications. Specifically, the first technical problem that the present invention aims to solve is to provide an mRNA vaccine that encodes universal influenza.

[0005] The second technical problem that the present invention aims to solve is to provide a method for preparing an mRNA vaccine encoding universal influenza. The third technical problem that this invention aims to solve is to provide an application for mRNA vaccines encoding universal influenza. [Means for solving the problem]

[0006] A first aspect of the present invention provides a recombinant protein having a structure represented by the following formula (I) or (II) from the N-terminus to the C-terminus, Equation (I): SMHN Equation (II): SHMN In the formula, Each "-" independently represents a bound or linked peptide. S is either absent or a signal peptide. M is either absent or consists of n tandem-bound extracellular domains (M2e) of two influenza virus matrix proteins or their immunogenic fragments. H is either absent or consists of m tandem-bound hemagglutinin (HA) stem (LAH) regions or their immunogenic fragments. N is a nucleoprotein (NP), or an immunogenic fragment thereof. Here, n is an integer between 1 and 6, and m is an integer between 1 and 6. Furthermore, M and H are not absent at the same time.

[0007] In another preferred example, n is 1, 2, 3, or 4. In another preferred example, m is 1, 2, or 3. In another preferred example, the signal peptide is selected from the group consisting of tissue-type plasminogen activator signal peptides, serum immunoglobulin E signal peptides, or combinations thereof.

[0008] In another preferred example, S has the amino acid sequence shown in SEQ ID NO:11. In another preferred example, the recombinant protein does not enter the cell nucleus when synthesized within the cell.

[0009] In another preferred example, the nucleoprotein (NP) is an NP protein derived from the PR8 virus strain, or a homologous sequence thereof. In another preferred example, the NP protein derived from the PR8 virus strain is generated by one or more amino acid mutations in the nuclear localization signaling region of the wild-type PR8 virus strain NP protein.

[0010] In another preferred example, the nuclear localization signal region is positions 2-12, 197-215, 339-344, or a combination thereof, of the sequence shown in SEQ ID NO:10. In another preferred example, the mutation is located at the amino acids at positions 6, 7, 212, 213, 214, 215, 341, and 342 of the sequence shown in SEQ ID NO:10, or in combination thereof.

[0011] In another preferred example, the mutation includes a mutation selected from the group consisting of K6P, R6P, R212P, K213P, R215P, R341P, V342L, or a combination thereof, wherein the amino acid position number corresponds to the position number of the mutated sequence SEQ ID NO:10.

[0012] In another preferred example, the amino acid sequence of the nuclear protein is: (N1) The amino acid sequence shown in SEQ ID NO:10, (N2) Based on the sequence shown in SEQ ID NO:10, one or more amino acid residues are substituted, deleted, modified, or inserted, or 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues are added to the N-terminus or C-terminus. The obtained amino acid sequence is selected from the group consisting of (N2) and (N1), and has sequence identity of ≥85% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98%, or ≥99%) with the sequence shown in SEQ ID NO:10, and has immunogenicity homologous or similar to the sequence in (N1), and does not nuclear localize.

[0013] In another preferred example, M is n tandem-linked identical or different influenza virus matrix protein 2 extracellular domains (M2e). In another preferred example, the influenza virus is derived from human influenza, avian influenza, swine influenza, or a combination thereof. In another preferred example, the influenza virus is an influenza A virus.

[0014] In another preferred example, the amino acid sequence of the M2e is (M1) an amino acid sequence shown in any of SEQ ID NOs: 3 - 6, and (M2) an amino acid sequence obtained by substituting, deleting, modifying or inserting one or more amino acid residues based on the sequence shown in any of SEQ ID NOs: 3 - 6, or adding 1 - 3 amino acid residues, preferably 1 - 2 amino acid residues, more preferably 1 amino acid residue to its N-terminus or C-terminus, and the obtained amino acid sequence has a sequence identity of ≧85% (preferably ≧90%, more preferably ≧95%, such as ≧96%, ≧97%, ≧98% or ≧99%) with the sequence shown in any of SEQ ID NOs: 3 - 6, and the obtained amino acid sequence has an immunogenicity homologous or similar to the sequence in (M1).

[0015] In another preferred example, M has a structure represented by the following formula (III) from the N-terminus to the C-terminus, Formula (III): M1 - M2 - M3 - M4 In the formula, “-” is independently a bond or a linking peptide, M1, M2, M3, and M4 are each independently absent or an M2e or its immunogenic fragment, and M1, M2, M3, and M4 are not all absent at the same time.

[0016] In another preferred example, the amino acid sequences of M1, M2, M3, and M4 are each independently selected from the group consisting of SEQ ID NO:3, 4, 5, and 6. In another preferred example, the M1, M2, M3, and M4 sequences are different from each other.

[0017] In another preferred example, M1 has the amino acid sequence shown in SEQ ID NO:3, M2 has the amino acid sequence shown in SEQ ID NO:4, M3 has the amino acid sequence shown in SEQ ID NO:5, and M4 has the amino acid sequence shown in SEQ ID NO:6.

[0018] In another preferred example, the amino acid sequence of M is as shown in SEQ ID NO:12, or has a sequence identity of ≧85% (preferably ≧90%, more preferably ≧95%, for example ≧96%, ≧97%, ≧98% or ≧99%) with SEQ ID NO:12, and also has an immunogenicity homologous or similar to SEQ ID NO:12.

[0019] In another preferred example, the H is m tandem-linked identical or different hemagglutinin stem LAH regions. In another preferred example, the HA stem LAH region contains a long α-helix region derived from HA2 of H1, H3, or H5 subtype A influenza virus, or a homologous sequence thereof.

[0020] In another preferred example, the amino acid sequence of the HA stem LAH region is selected from the group consisting of: (L1) the amino acid sequence shown in any of SEQ ID NO:7 to 9, and (L2) Based on the sequence shown in any of SEQ ID NO: 7 to 9, one or more amino acid residues are substituted, deleted, modified, or inserted, or 1 to 10 amino acid residues, preferably 1 to 5 amino acid residues, more preferably 1 to 3 amino acid residues are added to the N-terminus or C-terminus. The obtained amino acid sequence is selected from the group consisting of (L2) and (L1), and has sequence identity of ≥85% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98%, or ≥99%) with the sequence shown in any of SEQ ID NO: 7 to 9, and has immunogenicity homologous or similar to the sequence in (L1).

[0021] In another preferred example, H has a structure represented by the following formula (IV) from the N-terminus to the C-terminus, Formula (IV): H1-H2-H3 In the formula, Each "-" is independent and acts as a conjunction; H1, H2, and H3 are each independently either absent or located in the hemagglutinin stem LAH region or its immunogenic fragment, and H1, H2, and H3 are not absent simultaneously.

[0022] In another preferred example, the amino acid sequences of H1, H2, and H3 are each independently selected from the group consisting of SEQ ID NO: 7, 8, and 9. In another preferred example, the H1, H2, and H3 sequences are different from each other. In another preferred example, H1 has the amino acid sequence shown in SEQ ID NO:7, H2 has the amino acid sequence shown in SEQ ID NO:8, and H3 has the amino acid sequence shown in SEQ ID NO:9.

[0023] In another preferred example, the amino acid sequence of H is as shown in SEQ ID NO:13, or has ≥85% (preferably ≥90%, more preferably ≥95%, e.g., ≥96%, ≥97%, ≥98%, or ≥99%) sequence identity with SEQ ID NO:13, and also has homologous or similar immunogenicity with SEQ ID NO:13. In another preferred example, the linked peptide is selected from the group consisting of GG, GGS, (G3S)4, or a combination thereof.

[0024] In another preferred example, the recombinant protein has the amino acid sequence shown in SEQ ID NO:1, or has sequence identity of ≥85% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98%, or ≥99%) with SEQ ID NO:1, and also has homologous or similar immunogenicity with SEQ ID NO:1. In another preferred example, the recombinant protein further comprises an optional tag sequence that aids in expression and / or purification.

[0025] In another preferred example, the tag array includes the 6His tag. In another preferred example, the recombinant protein is a monomer, a dimer, or a polymer.

[0026] A second aspect of the present invention provides a polynucleotide that encodes a recombinant protein according to the first aspect of the present invention. In another preferred example, the polynucleotide is mRNA.

[0027] In another preferred example, the mRNA has a structure represented by formula (V), Formula (V): Z1-Z2-Z3-Z4-Z5 In the formula, Each "-" is independently a linker or connector. Z1 is either absent or a 5' capping element. Z2 is a 5'-UTR element, Z3 is the coding sequence of the recombinant protein described in claim 1, Z4 is a 3'-UTR element, The Z5 is either absent or a polyA tail element.

[0028] In another preferred example, Z1 is selected from an m7GpppG cap, a 3'-o-methyl-m7GpppG cap, or an anti-reverse cap analogue. In another preferred example, the mRNA has the nucleotide sequence shown in SEQ ID NO:2.

[0029] In another preferred example, the mRNA sequence is natural or modified mRNA. In another preferred example, the modified mRNA is modified by partially or completely substituting native uridine with modified uridine. In another preferred example, the modified mRNA is modified by completely substituting the native uridine with 1-methylpsoidouridine.

[0030] A third aspect of the present invention provides an mRNA vaccine composition, the vaccine composition is (i) mRNA according to a second aspect of the present invention, (ii) comprising a vaccine-acceptable carrier.

[0031] In another preferred example, the carrier is a lipid nanoparticle. In another preferred example, the mRNA or recombinant protein is encapsulated in the lipid nanoparticles.

[0032] In another preferred example, the lipid nanoparticles include cationic lipids, neutral phospholipids, steroidal lipids, and polyethylene glycol-lipids. In another preferred example, the vaccine composition is in the form of an injectable or inhaled preparation.

[0033] A fourth aspect of the present invention provides an expression vector comprising a polynucleotide as described in the second aspect of the present invention. In another preferred example, the vector includes a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus (e.g., adenovirus, retrovirus), or other vectors.

[0034] A fifth aspect of the present invention provides a host cell comprising an expression vector as described in the fourth aspect of the present invention. In another preferred example, the host cell is a mammalian cell.

[0035] A sixth aspect of the present invention is: (S1) A step of providing mRNA according to a second aspect of the present invention, (S2) A method for preparing a vaccine composition according to a third aspect of the present invention is provided, comprising the step of mixing the mRNA with lipid nanoparticles to form liposome nanoparticles in which mRNA is encapsulated.

[0036] A seventh aspect of the present invention provides the use of a recombinant protein according to the first aspect of the present invention, a polynucleotide according to the second aspect of the present invention, a vaccine composition according to the third aspect of the present invention, an expression vector according to the fourth aspect of the present invention, and / or a host cell according to the fifth aspect of the present invention, for use in the preparation of a drug, the drug is (a) Prevention of influenza, and / or (b) Used to induce and produce humoral and / or cellular immunity against influenza viruses in mammals.

[0037] In another preferred example, the influenza is influenza A. In another preferred example, the influenza is selected from the group consisting of human influenza, avian influenza, swine influenza, or a combination thereof. In another preferred example, the influenza is caused by a virus selected from the group consisting of H1N1, H3N2, H9N2, or a combination thereof.

[0038] An eighth aspect of the present invention provides a method for preventing influenza, the method comprising administering to a target subject of interest a recombinant protein according to the first aspect of the present invention, a polynucleotide according to the second aspect of the present invention, a vaccine composition according to the third aspect of the present invention, an expression vector according to the fourth aspect of the present invention, and / or a host cell according to the fifth aspect of the present invention.

[0039] In another preferred example, the subject is a human or a non-human mammal. In another preferred example, the non-human mammals include mice, rats, poultry, pigs, dogs, cats, and rabbits.

[0040] Another aspect of the present invention provides the use of the universal influenza mRNA for inducing and producing humoral and cellular immune responses in a mouse model, and / or for protecting mice from infection by multiple influenza A viruses in a protected animal model. [Effects of the Invention]

[0041] It should be understood that, within the scope of the present invention, new or preferred technical solutions can be constructed by combining the above-described technical features of the present invention with the technical features specifically described below (e.g., in the examples). Due to space limitations, this will not be repeated here. [Brief explanation of the drawing]

[0042] [Figure 1]Figure 1A shows the structural schematic and intracellular expression of the universal influenza mRNA vaccine in Example 1 of the present invention. Here, Figure 1B shows the Western blotting identification diagram after introducing 293T cells with universal influenza mRNA, and Figure 1C shows the immunofluorescence identification diagram after introducing 293T cells with universal influenza mRNA. Here, a mutation in the nuclear localization signal at the NP portion of the mRNA sequence allows the M2e-LAH-NP protein encoded by the mRNA to exist in the cytoplasm, but the original sequence of the NP that has not been mutated allows the M2e-LAH-NP protein to enter the cell nucleus, which is unfavorable for the expression of the protein.

[0043] [Figure 2] The antibody titers of mice after two intramuscular immunizations with different doses of the universal influenza mRNA vaccine in Example 2 of the present invention are shown. [Figure 3] This shows the number of splenic lymphocyte spots secreting different doses of the universal influenza mRNA vaccine IFN-γ in Example 3 of the present invention.

[0044] [Figure 4] This document describes the preventive and protective effects of different doses of the universal influenza mRNA vaccine in Example 4 of the present invention on mice infected with lethal doses of H1N1, H3N2, and H9N2 viruses. Here, Figure 4A shows a schematic diagram of weight loss and survival curve for mice infected with the H1N1 virus, Figure 4B shows a schematic diagram of weight loss and survival curve for mice infected with the H3N2 virus, and Figure 4C shows a schematic diagram of weight loss and survival curve for mice infected with the H9N2 virus.

[0045] [Figure 5]A representative sequence of the recombinant protein of the present invention is shown, where positions 1-23 are signal peptides, the underlined portion represents the M2e protein, the bold portion represents the LAH protein, the italicized portion represents the NP protein, the gray background represents the nuclear localization region of the NP protein, and the double underlined portion represents the site of mutation. [Modes for carrying out the invention]

[0046] As a result of extensive and meticulous research, the inventors selected the conserved region M2e, HA stem, and NP protein of influenza A virus as target genes for a universal influenza vaccine. M2 is a transmembrane protein on the surface of the influenza virus, and its amino acid sequence is highly conserved. In particular, the extracellular region M2e, consisting of only 23 amino acid residues at the N-terminus, is a candidate target point for a universal influenza vaccine. The M2e sequences of human, porcine, and avian influenza viruses differ slightly, and the present invention intends to cover a wider range of influenza viruses by combining the human, porcine, and avian influenza M2e regions in a tandem-linked configuration.

[0047] The HA stem is a key component of influenza virus-mediated membrane fusion. Compared to the HA head region, the sequence and structure of the HA stem are more conserved across different influenza subtypes, and antibodies that broadly neutralize this domain are considered a potential approach against various influenza virus strains. The LAH region is one of the most conserved regions of the HA stem. The NP protein is a conserved structural protein with a very low mutation rate during viral evolution and exhibits stronger cross-immunity, making it a widely studied target point for universal influenza vaccines.

[0048] This invention involves tandem ligation of the immunogenic and conserved regions of these three influenza viruses to prepare them as an mRNA-LNP vaccine. This universal influenza mRNA vaccine exhibits robust humoral and cellular immune responses in immunogenicity evaluations of mouse models and effectively prevents and protects mice from infectious lethal doses of H1N1, H3N2, and H9N2 viruses. This vaccine is expected to have good applications as a universal influenza vaccine.

[0049] The inventors have found that by mutating the nuclear localization signaling region of the NP protein, the M2e-LAH-NP protein encoded by the mRNA of the present invention can reside in the cytoplasm rather than entering the cell nucleus, thereby promoting protein expression.

[0050] term To facilitate understanding of this disclosure, we first define certain terms. Unless otherwise specified herein, each of the following terms should have the meanings set forth below, as used in this invention. Other definitions are explained throughout the invention.

[0051] The term "approximately" can refer to a specific value or configuration within a set acceptable margin of error determined by those skilled in the art, which depends in part on how the value or configuration is measured or measured.

[0052] As used herein, the terms “contain” or “include” may be open, semi-closed, or closed. In other words, the terms also include “essentially consisting of” or “consisting of.”

[0053] As used herein, the terms “host,” “subject,” and “required subject” refer to any mammal or non-mammalian. Mammals include, but are not limited to, cats, other vertebrates such as rodents, humans, and non-human primates. For example, cattle, horses, dogs, pigs, sheep, goats, giraffes, deer, camels, antelopes, rats, mice, hares, and rabbits.

[0054] Recombinant protein The present invention provides a recombinant protein comprising a structure in which immunogenic and conserved regions of three influenza viruses are tandem-linked, wherein the recombinant protein possesses immunogenicity of the influenza viruses and can induce humoral and cellular immune responses against the influenza viruses.

[0055] As used herein, the terms “recombinant protein” or “M2e-LAH-NP protein” are defined as follows: (1) The extracellular domain (M2e) of influenza virus matrix protein 2, or an immunogenic fragment thereof, (2) The hemagglutinin (HA) stem LAH region, or an immunogenic fragment thereof, (3) This refers to a recombinant protein of an antigen peptide or structural protein selected from the group consisting of a nucleoprotein (NP) or an immunogenic fragment thereof.

[0056] In a preferred embodiment, the recombinant protein of the present invention is encoded by the mRNA vaccine of the present invention. M2 is a transmembrane protein on the surface of the influenza virus, and its amino acid sequence is highly conserved. In particular, the N-terminus is the extracellular region M2e, consisting of only 23 amino acid residues, making it a candidate target point for a universal influenza vaccine. The M2e sequences of human, porcine, and avian influenza viruses differ slightly. This invention covers a wider range of influenza viruses by combining the human, porcine, and avian influenza M2e regions in a tandem configuration.

[0057] In a preferred embodiment, the recombinant protein of the present invention is SEQ ID NO:3:SLLTEVETPIRNEWGSRSN, SEQ ID NO:4:SLLTEVETPTRSEWESRSS, SEQ ID NO:5:SLLTEVETPTRNEWESRSS, and SEQ ID NO:6: Contains one or more M2e sequences selected from the group consisting of SLLTEVETLTRNGWGCRCS.

[0058] Preferably, the recombinant protein of the present invention comprises a sequence formed by tandem linking of SEQ ID NO:3~6. The HA stem is a major component of influenza virus-mediated membrane fusion. Compared to the HA head region, the sequence and structure of the HA stem are highly conserved across different influenza subtypes, and antibodies that broadly neutralize this domain are considered a potential method against various influenza virus strains. The LAH region is one of the most conserved regions within the HA stem.

[0059] In a preferred embodiment, the recombinant protein of the present invention is SEQ ID NO:7:RMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNA, SEQ ID NO:8:RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENA, and SEQ ID NO:9: Contains one or more LAH sequences selected from the group consisting of RIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNA.

[0060] Preferably, the recombinant protein of the present invention comprises a sequence formed by tandem linking of SEQ ID NO: 7-9.

[0061] NP proteins are conserved structural proteins with a very low mutation rate during viral evolution, possessing stronger cross-immunity and being widely studied as target points for universal influenza vaccines. Wild-type NP proteins have a nuclear localization signal and readily enter the cell nucleus after expression, which is detrimental to vaccine secretion. Through screening, this invention unexpectedly discovered that partially mutating the NP nuclear localization signal allows the mRNA-encoded M2e-LAH-NP protein to be present in the cytoplasm, promoting its secretion into the extracellular space.

[0062] The fusion protein of the present invention may contain a signal peptide. The signal peptide sequence is as shown in SEQ ID NO:11. MDAMKRGLCCVLLLCGAVFVSPS(SEQ ID NO:11)

[0063] In a preferred embodiment, the recombinant protein of the present invention comprises the NP sequence shown in SEQ ID NO:10. ASQGTPPSYEQMETDGERQNATEIRASVGKMIGGIGRFYIQMCTELKLSDYEGRLIQNSLTIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVNGKWMRELILYDKEEIRRIWRQANNG DDATAGLTHMMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELVRMIKRGINDRNFWRGENGPPTPIAYERMCNILKGKFQTAAQKAMMDQVRESRNPGNA EFEDLTFLARSALILRGSVAHKSCLPACVYGPAVASGYDFEREGYSLVGIDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLPLLSFIKGTKVVPRGKLSTRGVQIASNENMETMES STLELRSRYWAIRTRSGGNTNQQRASAGQISIQPTFSVQRNLPFDRTTIMAAFTGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKAASPIVPSFDMSNEGSYFFGDNAEEYDN(SEQ ID NO:10)

[0064] In the recombinant protein and the mRNA encoding it according to the present invention, the M2e protein, LAH protein, and NP protein sequences are tandem-linked in any order, as long as they are expressed and secreted by cells. In a preferred embodiment, because the NP protein has a large molecular weight and possesses a nuclear localization signal, the NP protein is ligated to the C-terminus of the recombinant protein of the present invention.

[0065] The present invention provides a scheme for designing a universal influenza vaccine sequence, the universal influenza vaccine comprising at least one antigenic peptide or structural protein, comprising the M2e of influenza A virus or its immunogenic fragment, the HA stem LAH region or its immunogenic fragment, NP or its immunogenic fragment. In a preferred embodiment, the recombinant protein of the present invention comprises the M2e shown in amino acid sequences SEQ ID NO:3-6, the LAH shown in SEQ ID NO:7-9, and / or the NP shown in SEQ ID NO:10.

[0066] In a preferred embodiment, the recombinant protein of the present invention has a structure represented by the following formula (I) or (II) from the N-terminus to the C-terminus, Equation (I): SMHN Equation (II): SHMN In the formula, Each "-" independently represents a bound or linked peptide. S is either absent or a signal peptide. M is either absent or tandem-bound n M2e or its immunogenic fragment, where n is an integer from 1 to 6, preferably 1, 2, 3, or 4. H is either absent or tandem-bound m hemagglutinin stem LAH regions or immunogenic fragments thereof, where m is an integer from 1 to 6, preferably 1, 2, or 3. N is a nuclear protein NP, or an immunogenic fragment thereof, and also, M and H are not absent at the same time.

[0067] Preferably, the amino acid sequence of M is as shown in SEQ ID NO:12, or has ≥85% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98%, or ≥99%) sequence identity with SEQ ID NO:12, and also has homologous or similar immunogenicity with SEQ ID NO:12. SLLTEVETPIRNEWGSRSNGGSLTEVETPTRSEWESRSSGGSLLTEVETPTRNEWESRSSGGSLLTEVETLTRNGWGCRCS(SEQ ID NO:12)

[0068] Preferably, the amino acid sequence of H is as shown in SEQ ID NO:13, or has sequence identity of ≥85% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98%, or ≥99%) with SEQ ID NO:13, and also has homologous or similar immunogenicity with SEQ ID NO:13. RMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAGGSRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAGGSRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNA(SEQ ID NO:13)

[0069] The fusion protein of the present invention may optionally include a peptide linker. The size and complexity of the peptide linker may affect the activity of the protein. Typically, the peptide linker should have sufficient length and flexibility to ensure sufficient spatial freedom for the two linked proteins to function. In a preferred embodiment of the present invention, the length of the peptide linker is generally 0 to 15 amino acids, preferably 0 to 10 amino acids, more preferably 0 to 5 amino acids, for example GG, GGS, GGGSGGGSGGGSGGGS (SEQ ID NO: 14).

[0070] Preferably, the amino acid sequence of the recombinant protein of the present invention is as shown in SEQ ID NO:1. (SEQ ID NO:1)

[0071] PhR vaccine As used herein, the term "mRNA" refers to a type of single-stranded RNA that is transcribed using a single strand of DNA as a template, carrying genetic information and guiding protein synthesis.

[0072] As used herein, the term "vaccine" refers to a composition suitable for application to animals (including humans) that induces an immune response after administration and whose strength is sufficient to minimally assist in the prevention, improvement, or cure of a clinical disease caused by a microbial infection.

[0073] As used herein, the terms “mRNA of the present invention,” “mRNA vaccine,” “universal influenza mRNA vaccine,” and “mRNA sequence of the present invention” are interchangeable and refer to the mRNA described in the second aspect of this application.

[0074] In a preferred embodiment, the mRNA has a structure represented by formula (V), Formula (V): Z1-Z2-Z3-Z4-Z5 In the formula, Each "-" is independently a linker or connector. Z1 is either absent or a 5' capping element. Z2 is a 5'-UTR element, Z3 is the coding sequence of the recombinant protein described in claim 1, Z4 is a 3'-UTR element, The Z5 is either absent or a polyA tail element.

[0075] Preferably, the mRNA vaccine of the present invention has the nucleotide sequence shown in SEQ ID NO:2.

[0076] The present invention further provides vaccine compositions comprising the mRNA vaccine of the present invention. In a preferred embodiment, the vaccine composition of the present invention is provided in the form of an mRNA-LNP vaccine. As used herein, the term “lipid nanoparticles” (LNP) refers to at least one nanometer-scale particle containing at least one lipid. In preferred embodiments, the lipid includes, but is not limited to, neutral phospholipids and polyethylene glycol-lipids. As used herein, the term “neutral phospholipid” refers to an uncharged, non-phosphoglycerol ester phospholipid molecule. As used herein, the term “polyethylene glycol-lipid” refers to a molecule containing a lipid moiety and a polyethylene glycol moiety.

[0077] The present invention provides polynucleotides that encode the mRNA nucleic acid sequence, the M2e, LAH, and NP protein sequences of the present invention, or the recombinant protein of the present invention. The present invention further provides a vector, the carrier comprising the polynucleotide of the present invention.

[0078] The present invention provides the use of a universal influenza mRNA vaccine for inducing humoral and cellular immune responses in a mouse model, and / or for protecting mice from infection by multiple influenza A viruses in a protected animal model.

[0079] Vaccine composition The present invention further provides a vaccine composition comprising (ii) a pharmaceutically acceptable carrier and (ii) mRNA as described in a second aspect of the present invention.

[0080] The drug composition may be in any suitable form depending on the patient's desired method of administration. It may be provided in the form of unit dosage forms in sealed containers, or as part of a kit. Such a kit may typically (but not necessarily) include instructions for use. It may contain multiple unit dosage forms.

[0081] The drug composition is suitable for any appropriate route of administration, including injection (including subcutaneous, intradermal, intramuscular, intraperitoneal, microneedle, or intravenous injection), inhalation, or oral, or nasal or rectal administration. The composition can be prepared by any method known in the pharmaceutical field, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions.

[0082] The main advantages of this invention are as follows: (a) The recombinant protein of the present invention comprises three conserved immunogenic regions and enhances its immunogenicity by tandem binding in multiple copies. (b) The vaccine of the present invention uses a highly efficient and safe mRNA vaccine form in order to accelerate research and development efficiency. (c) In the recombinant protein of the present invention, the nuclear localization region of the NP protein is mutated to reduce its nuclear localization, thereby promoting the extracellular secretion of the recombinant protein. (d) The universal influenza mRNA vaccine of the present invention exhibits a robust humoral and cellular immune response in immunogenicity evaluations of mouse models, and can effectively prevent and protect mice from attacks by infectious lethal doses of H1N1, H3N2, and H9N2 viruses.

[0083] The present invention will be further described below in conjunction with specific examples. These examples are used solely to illustrate the present invention and should not be used to limit its scope. In the following examples, experimental methods that do not specify conditions typically follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: An Experimental Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions suggested by the manufacturer. Unless otherwise specified, percentages and quantities are calculated by weight.

[0084] Example 1. Preparation of universal influenza mRNA vaccine and evaluation of cell expression. The structure of universal influenza mRNA is shown in Figure 1A, where the NP sequence mutates three nuclear localization signals, causing the encoded protein to be secreted extracellularly without entering the cell nucleus. A plasmid expressing universal influenza mRNA is artificially synthesized. After plasmid extraction, linearization enzyme digestion, and linearization plasmid purification, a high-purity linearization plasmid template is obtained and, after UV quantification, stored at -20°C for storage. Using the above linearization plasmid as a template, universal influenza mRNA is obtained using the T7 enzyme in vitro transcription kit, capping kit, and purification kit according to the kit instructions. The mRNA concentration is measured by UV, the mRNA mass is identified by capillary electrophoresis, and the plasmid is stored at -80°C for storage.

[0085] Prepared universal influenza mRNA was transfected into 293T cells with lipofectamine 3000, and the cells were lysed 48 hours after transfection. The cell lysates were separated by 12% SDS-PAGE, treated with semi-dry vent, and then blocked with 5% skim milk-PBS for 1 hour. The blocking solution was discarded, anti-M2 mouse monoclonal antibody was added as the primary antibody, and the cells were incubated at room temperature for 1 hour. After thoroughly washing the cells, donkey anti-mouse IgG-HRP was added as the secondary antibody and the cells were incubated at room temperature for 1 hour. After thoroughly washing the cells, ECL was added to induce staining, and the cells were imaged using an ECL imaging system. The exposed membrane was detached and the membrane was regenerated, then the cells were blocked again, β-actin antibody was added as the primary antibody, and donkey anti-rabbit-HRP was added as the secondary antibody, and the cells were stained with ECL and then imaged.

[0086] As shown in Figure 1B, cells transfected with universal influenza mRNA show a distinct band around 100kD, the size of which coincides with the location of the target protein, while untransfected cells do not show this band, indicating that the prepared mRNA is well expressed in 293T cells. As shown in Figure 1C, the M2e-LAH-NP protein encoded by this mRNA can be secreted extracellularly by being located in the cytoplasm, but the original sequence of the unmutated NP causes the M2e-LAH-NP protein to enter the cell nucleus, which is unfavorable for the expression of this protein.

[0087] The prepared universal influenza mRNA is encapsulated in a formulation containing cationic liposomes ALC-0315, and cationic lipids:neutral phospholipids:steroidal lipids:polyethylene glycol-lipids are dissolved and mixed in ethanol in a molar ratio of 46:9:43:2. The nanodrug is prepared at a total flow rate of 12 mL / min, and the mRNA solution and lipid mixture solution are encapsulated at a flow rate ratio of 3:1. After encapsulation is complete, the solution is replaced by ultrafiltration to obtain the universal influenza mRNA vaccine (mRNA-LNP preparation).

[0088] Example 2. Evaluation of humoral immunity of universal influenza mRNA vaccine in a mouse model. Female BALB / C mice aged 6-8 weeks were intramuscularly injected with 5 μg, 10 μg, and 20 μg of the universal influenza mRNA-LNP preparation prepared in Example 1 on days 0 and 21, respectively. Five mice were administered to each group, with PBS preparation serving as the negative control group. Blood was collected on day 21 (day 42) after secondary immunization, and the level of specific IgG antibodies in the serum was detected by enzyme-linked immunosorbent assay (ELISA). M2e synthetic peptide segment, LAH synthetic peptide segment, or NP protein were used as coating antigens, and 100 μl / well was used to coat 96-well microtiter plates, which were left overnight at 2-8°C. After blocking with blocking solution, serum was divided into two sections. 5 Starting with 2 24 Dilute 2-fold and add 100 μl / well to a 96-well plate, incubate at 37°C for 2 hours, and wash. Add diluted enzyme-labeled secondary antibody (donkey anti-mouse IgG-HRP), incubate at 37°C for 1 hour, and wash thoroughly. Add 100 μl of freshly prepared TMB substrate solution, let stand at room temperature for 5-10 minutes, and stop the reaction by adding 50 μl of 2 M H2SO4 to each well. Read the OD value at 450 nm using a microplate reader. Calculate the mean and standard difference of all readings in the PBS control group, set the threshold = mean + 2 × standard difference, and the largest dilution factor at which the number of readings in other groups is greater than the threshold is the antibody titer of the serum sample in that group.

[0089] The results are shown in Figure 2, where serum specific IgG antibody titers against M2e, LAH, or NP increase with increasing dose after secondary immunization. The antibody titer against M2e increases with increasing dose. 19 ~2 21 The titer of the antibody against LAH was 2. 15 ~2 17reached, and the titer of the 20 μg group was higher than that of the 5 μg group (p < 0.01) and the 10 μg group (p < 0.05). There was no significant difference between the 10 μg group and the 5 μg group. The antibody titer against NP was 2 18 ~2 21 reached, and the titer of the 20 μg group was higher than that of the 5 μg group (p < 0.01) and the 10 μg group (p < 0.05). There was no significant difference between the 10 μg group and the 5 μg group.

[0090] Example 3. Immunological evaluation of universal influenza mRNA vaccine in mouse model cells On the 21st day (42nd day) after the secondary immunization, spleen tissues were collected from the mice in each group in Example 2, lymphocytes were separated, and the proportion of spleen lymphocytes secreting IFN-γ cytokine was detected by an enzyme-linked immunosorbent spot (ELISpot) test. The mice were sacrificed by cervical dislocation after enucleation of the eyeballs and exsanguination, immersed in 75% ethanol, placed in a biosafety cabinet, and the spleen was removed using sterile surgical instruments. The spleen was ground with lymphocyte separation medium until no tissue pieces remained, filtered through a 40 μm nylon cell strainer into a 15 mL centrifuge tube, and filled with lymphocyte separation medium until it reached 5 mL. Approximately 7 mL of PRMI 1640 medium was slowly added along the tube wall to make the interface between the upper layer medium and the lower layer lymphocyte separation medium have a clear stratification. Centrifuged at 800 g for 30 minutes at room temperature to aggregate the lymphocytes at the interface. The lymphocytes were aspirated, placed in a 15 mL centrifuge tube containing 1640 medium, the cells were washed, and centrifuged at room temperature at 250 g for 10 minutes. After centrifugation, the supernatant was discarded, the lymphocytes were resuspended in 1 mL of medium, the cells were collected, the cells were counted, and the viable cell density and survival rate were recorded. Based on the counting results, the lymphocytes were diluted to 5×10 6 cells / mL and stored.

[0091] The enzyme-linked immunosorbent spot experiment was carried out using the Mouse IFN-γ ELISpotPlus (HRP) kit from Mabtech and performed according to the instructions. The prepared 5×10 6Add 100 μL of lymphocytes (cells / mL) to an ELISpot plate and prepare three duplicate wells for each sample. Use M2e synthetic peptide segment, LAH synthetic peptide segment, or NP protein as the stimulant. Set up positive and negative control wells simultaneously, add PHA-positive stimulant to the positive wells, and add culture medium to the negative wells. Place the plate in a humidifying chamber and incubate at 37°C in a 5% CO2 constant temperature incubator for 12-48 hours. Remove the ELISpot plate, discard the culture medium, and wash the plate five times repeatedly with sterile PBS.

[0092] Dilute the primary antibody from the kit to 1 μg / mL with 0.5% FBS-containing sterile PBS, mix uniformly, add 100 μL to each well of the plate, and incubate at room temperature for 2 hours. Discard the primary antibody and wash the plate 5 times with sterile PBS. Dilute the enzyme-labeled secondary antibody from the kit with 0.5% FBS-containing sterile PBS at a volume ratio of 1:1000, mix uniformly, add 100 μL to each well of the plate, and incubate at room temperature for 1 hour. Discard the secondary antibody and wash the plate 5 times with sterile PBS. After draining the plate, add 100 μL of the TMB chromogenic solution from the kit to each well until clear spots appear. Rinse the plate with sterile pure water to stop the color development, air dry the plate in the dark, and read it with an ELISPOT plate reader.

[0093] The results, as shown in Figure 3, show that after two immunizations, M2e and NP as stimulants stimulated splenic lymphocytes capable of secreting IFN-γ cytokines at high levels, and the number of stimulated spots increased with increasing dose. LAH sequences, lacking T cell epitopes, are virtually incapable of stimulating IFN-γ cytokine-secreting splenic lymphocytes.

[0094] Example 4. Evaluation of the preventive effect of universal influenza mRNA vaccine in mice. Female BALB / C mice aged 6-8 weeks were intramuscularly injected with 5 μg, 10 μg, and 20 μg of the universal influenza mRNA-LNP preparation prepared in Example 1 on day 0 and day 21, respectively, with PBS serving as the negative control group. On day 21 (day 42) after secondary immunization, 20 μl of 5×LD was administered. 50 The Jiaxing influenza virus subtypes H1N1 (A / Puerto Rico / 8 / 1934), H3N2 (A / Guizhou / 54 / 1989), and H9N2 (A / Chicken / Jiangsu / 11 / 2002) were administered intranasally, with 10 mice given to each group. The survival and weight loss of the mice were monitored daily until day 14 after infection.

[0095] The results, as shown in Figure 4, show that mice in the PBS control group exhibited clear influenza-like symptoms 3-4 days after H1N1, H3N2, or H9N2 virus infection and all died on day 9 or 10 post-infection. In contrast, mice immunized with the universal influenza mRNA vaccine obtained partial or complete protection. As shown in Figure 4A, the protection rates for mice with 5, 10, and 20 μg of the universal influenza mRNA vaccine against H1N1 virus infection were 50%, 80%, and 100%, respectively. Mice in the 20 μg group began to recover weight on day 5 post-infection. Mice in the 10 μg and 5 μg groups began to recover weight on day 8 post-infection.

[0096] As shown in Figure 4B, the protection rates for mice immunized with 5, 10, and 20 μg of universal influenza mRNA vaccine against H3N2 virus infection were 30%, 70%, and 100%, respectively. Mice in the three immunization groups began to regain weight on day 7–8. As shown in Figure 4C, the protection rates for mice immunized with 5, 10, and 20 μg of universal influenza mRNA vaccine against H9N2 virus infection were 50%, 80%, and 100%, respectively. Mice in the three immunization groups began to regain weight on day 7–8. In summary, two doses of 20 μg of universal influenza mRNA-LNP vaccine completely protect mice from lethal attacks by H1N1, H3N2, and H9N2 subtypes of influenza A virus.

[0097] All documents referenced in this invention are cited as references in this application, as if each document were cited individually. Furthermore, after reading the above teachings of this invention, persons skilled in the art can make various changes or modifications to the invention, and these equivalent forms are also included within the scope defined by the claims appended to this application.

Claims

1. Recombinant protein, From the N-terminus to the C-terminus, it has a structure represented by the following formula (I) or (II): Formula (I): S-M-H-N Formula (II): SHM In the formula, Each "-" independently represents a bound or linked peptide. S is either absent or a signal peptide. M is either absent or consists of n tandem-bound extracellular domains (M2e) of two influenza virus matrix proteins or their immunogenic fragments. H is either absent or consists of m tandem-linked hemagglutinin (HA) stem (LAH) regions or their immunogenic fragments. N is a nucleoprotein (NP), or an immunogenic fragment thereof. Here, n is an integer from 1 to 6, m is an integer from 1 to 6, and also, The recombinant protein characterized in that M and H are not simultaneously absent.

2. The nucleoprotein (NP) is generated by one or more amino acid mutations in the nuclear localization signal region of the NP protein of the wild-type PR8 virus strain, wherein the nuclear localization signal region includes positions 2-12, 197-215, 339-344, or a combination thereof, of the sequence shown in SEQ ID NO:

10. The recombinant protein according to claim 1.

3. The mutation is characterized by being located at the amino acids at positions 6, 7, 212, 213, 214, 215, 341, and 342 of the sequence shown in SEQ ID NO: 10, or a combination thereof. The recombinant protein according to claim 2.

4. The amino acid sequence of the nuclear protein is (N1) The amino acid sequence shown in SEQ ID NO: 10, (N2) An amino acid sequence obtained by substituting, deleting, modifying, or inserting one or more amino acid residues based on the sequence shown in SEQ ID NO: 10, or by adding 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, to its N-terminus or C-terminus, is selected from the group, wherein the obtained amino acid sequence has ≥85% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98%, or ≥99%) sequence identity with the sequence shown in SEQ ID NO: 10, and the obtained amino acid sequence has homologous or similar immunogenicity to the sequence in (N1), and does not nuclear localize. The recombinant protein according to claim 1.

5. M has a structure from the N-terminus to the C-terminus that is represented by the following formula (III): Formula (III): M1-M2-M3-M4 In the formula, Each "-" independently represents a bound or linked peptide. M1, M2, M3, and M4 are each independently either absent or M2e or its immunogenic fragment, and M1, M2, M3, and M4 are not absent simultaneously. The recombinant protein according to claim 1.

6. The amino acid sequences of M1, M2, M3, and M4 are each independently selected from the group consisting of SEQ ID NO: 3, 4, 5, and 6. The recombinant fusion protein according to claim 5.

7. The amino acid sequence of M is as shown in SEQ ID NO: 12, or has sequence identity of ≥85% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98%, or ≥99%) with SEQ ID NO: 12, and is characterized by having homologous or similar immunogenicity to SEQ ID NO:

12. The recombinant fusion protein according to claim 5.

8. H has a structure represented by the following formula (IV) from the N-terminus to the C-terminus, Formula (IV): H1-H2-H3 In the formula, Each "-" independently represents a bound or linked peptide. H1, H2, and H3 are each independently either absent or are hemagglutinin stem LAH region or immunogenic fragment thereof, and H1, H2, and H3 are not absent simultaneously. The recombinant protein according to claim 1.

9. The amino acid sequences of H1, H2, and H3 are each independently selected from the group consisting of SEQ ID NO: 7, 8, and 9. The recombinant fusion protein according to claim 8.

10. The amino acid sequence of H is as shown in SEQ ID NO: 13, or has sequence identity of ≥85% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98%, or ≥99%) with SEQ ID NO: 13, and is characterized by having homologous or similar immunogenicity to SEQ ID NO:

13. The recombinant fusion protein according to claim 8.

11. The recombinant protein is characterized by having the amino acid sequence shown in SEQ ID NO:1, or having sequence identity of ≥85% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98%, or ≥99%) with SEQ ID NO:1, and having immunogenicity homologous or similar to SEQ ID NO:

1. The recombinant protein according to claim 1.

12. It is a polynucleotide, The polynucleotide characterized by encoding the recombinant protein described in claim 1.

13. The polynucleotide is mRNA and is characterized by having the nucleotide sequence shown in SEQ ID NO:

2. The polynucleotide according to claim 12.

14. mRNA vaccine composition, (i) The polynucleotide according to claim 12, which is mRNA, (ii) The vaccine composition comprising a vaccinologically acceptable carrier.

15. A characteristic feature is that the vaccinologically acceptable carrier is lipid nanoparticles. The vaccine composition according to claim 14.

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