Influenza virus vaccine

Abstract

The present invention provides a modified protein of influenza A matrix 1 (M1), the modified protein inducing a humoral immune reaction and a cellular immune reaction against influenza A virus. The modified protein has the following characteristics: (a) at least one region of the M1 protein is substituted by a membrane fusion peptide derived from influenza A virus hemagglutinin; and (b) at least one T cell epitope in the M1 protein is preserved.

Description

Influenza virus vaccine

[0001] The present invention relates to a modified protein capable of inducing a humoral immune response and a cellular immune response against influenza virus.

[0002] Influenza viruses that repeatedly undergo antigenic mutations through zoonotic infections have historically caused repeated pandemics. There are also highly pathogenic influenza viruses, and when they infect humans, the fatality rate can reach about 50%. Therefore, the development of a powerful vaccine against influenza virus is an urgent issue.

[0003] Various vaccines against influenza virus have been developed so far. In the production of influenza vaccines, the HA protein of influenza virus is used as one of the antigens. The influenza HA protein is present on the outer envelope of influenza virus particles and is a protein composed of an HA1 domain and an HA2 domain. The HA1 domain constitutes the global head, and the HA2 domain constitutes the stem structure. The global head contains a region that binds to the surface receptor of host cells, and this region can be a suitable antigen. However, this region is very prone to amino acid mutations. As a result, vaccines using this region as an antigen generally have low versatility. HA2 is called the stem and is less prone to amino acid mutations compared to the global head, so it is useful for the production of highly versatile vaccines, but the problem may be its low immunogenicity.

[0004] As a result of comprehensively considering such a background, the present inventors have succeeded in producing an influenza virus vaccine capable of inducing a humoral immune response and a cellular immune response against influenza virus by fusing HA lacking the head portion with matrix 1 (M1) protein (Patent Document 1).

[0005] U.S. Patent No. 11576962

[0006] The influenza virus vaccine developed by the present inventors as described in Patent Document 1 is a useful influenza virus vaccine that can induce humoral and cellular immune responses against a wide range of A subtype influenza virus strains. In this invention, in order to further enhance the convenience of the vaccine, we have attempted to miniaturize the vaccine antigen.

[0007] As a result of diligent research, the inventors have confirmed that a modified protein incorporating a membrane fusion peptide (hereinafter sometimes simply referred to as "fusion peptide" or "FP (Fusion Peptide)") present in the HA protein into the M1 protein can induce humoral immune responses (antibody production) against multiple subtypes of influenza virus strains. Furthermore, since the modified M1 protein maintains known T cell epitopes and oligomeric active sites, it is considered possible to induce cellular immune responses (cytotoxic T cell proliferation) and oligomerization as well. Based on these findings, the inventors conducted further research and completed the present invention. That is, the present invention is as follows:

[0008] [1] A modified protein of influenza A matrix 1 (M1) that induces humoral and cellular immune responses against influenza A virus, wherein the modified protein has the following characteristics: (a) at least one region of the M1 protein is replaced by a membrane fusion peptide derived from influenza A virus hemagglutinin; and (b) at least one T cell epitope in the M1 protein is maintained. [2] The modified protein according to [1], wherein two or more regions of the M1 protein are replaced by membrane fusion polypeptides. [3] The modified protein according to [1] or [2], wherein the membrane fusion peptide comprises the amino acid sequence represented by SEQ ID NO: 3 or 4. [4] The modified protein according to any one of [1] to [3], wherein the M1 protein comprises the amino acid sequence represented by SEQ ID NO: 1 or a fragment thereof having self-assembly activity. [5] The modified protein according to any one of [1] to [4], comprising the amino acid sequence represented by SEQ ID NO: 9. [6] A pharmaceutical composition comprising the modified protein according to any one of [1] to [5]. [7] The pharmaceutical composition according to [6] for inducing an immune response against influenza A virus. [8] The pharmaceutical composition according to [6] for the prevention or treatment of influenza A virus infection. [9] A nucleic acid encoding the modified protein according to any one of [1] to [5]. [10-A] A method for inducing an immune response against influenza A virus in a subject, comprising the step of administering the modified protein according to any one of [1] to [5] to the subject. [10-B] A method for preventing or treating influenza A virus infection, comprising the step of administering the modified protein according to any one of [1] to [5] to the subject. [10-C] The modified protein according to any one of [1] to [5] for use in inducing an immune response against influenza A virus in a subject. [10-D] The modified protein according to any one of [1] to [5] for use in the prevention or treatment of influenza A virus infection.[10-E] Use of a modified protein described in any of [1] to [5] in the manufacture of a pharmaceutical product for inducing an immune response to influenza A virus in a subject. [10-F] Use of a modified protein described in any of [1] to [5] in the manufacture of a pharmaceutical product for the prevention or treatment of influenza A virus infection.

[0009] According to the present invention, it is possible to provide a miniaturized influenza virus vaccine that cross-reacts to a wide range of influenza A subtype viruses, including seasonal influenza viruses and expected highly pathogenic influenza viruses, and that is extremely easy to prepare.

[0010] Figure 1 is a schematic diagram of the HA structure of influenza virus type A (Michigan strain (H1N1)). In A), positions 1 to 17 of HA are the signal peptide region. Positions 18 to 354 are the H1 region. Positions 76 to 308 are the head region. Positions 355 to 554 are the H2 region. FP indicates the membrane fusion region. A indicates the helix region, B indicates the loop region, and C indicates the helix region. Positions 539 to 554 are the transmembrane region (TM). Positions 555 to 566 are the cytoplasmic region. S-S indicates an intramolecular disulfide bond. The sequence excluding the signal peptide region corresponds to the amino acid sequence of the HA of a mature influenza A virus. (Those skilled in the art can determine the corresponding regions in the HA of other subtypes using methods known to them.) Figure 2 is a schematic diagram of the first-generation influenza vaccine and the second-generation influenza vaccine of the present invention. The following shows the SDS-PAGE (A) and Native-PAGE (B) results for influenza virus type A vaccine antigen fusion polypeptide (head-deficient HA-M1 fusion protein (+GS linker + 6×His Tag)) expressed in a wheat cell-free system. The figure shows the results of quantifying cytokines (IL-10 and IFN-γ) in the supernatant after cells were collected from the lymph nodes of mice inoculated with HA-M1 antigen, HA-M1 antigen was added (0 (control), 25, 50, or 100 μg / ml), and the cells were cultured for 92 hours. The figure shows the results of counting the number of cells after cells were collected from the lymph nodes of mice inoculated with HA-M1 antigen, HA-M1 antigen was added (0 (control), 25, 50, or 100 μg / ml), and the cells were cultured for 92 hours. This demonstrates that mice immunized with the antigen protein of the present invention acquire resistance to influenza virus and have a higher survival rate. Figure 7 shows the amino acid sequence (FLM1 protein, 321 amino acids, SEQ ID NO: 9) of the modified M1 protein of the present invention, which was prepared by substituting four sites of the M1 protein of influenza A3 virus with the HA membrane fusion peptide (underlined) of influenza A1 virus. Figure 8 is a schematic diagram showing the predicted three-dimensional structure of the FLM1 protein. The region where the membrane fusion peptide is thought to exist is indicated by a circle.Figure 9 shows the results when monomers and polymers of a modified M1 protein (FLM1 protein) in which membrane fusion peptides were substituted at four locations were subjected to SDS-PAGE or Native PAGE Blue gel and stained with CBB.

[0011] 1. Modified Protein The present invention provides a modified protein of influenza A matrix 1 (M1) that induces humoral and cellular immune responses against influenza A virus, wherein the modified protein has the following characteristics (hereinafter sometimes referred to as "the modified protein of the present invention"): (a) at least one region of the M1 protein is replaced by a membrane fusion peptide derived from influenza A virus hemagglutinin; and (b) at least one T cell epitope in the M1 protein is maintained.

[0012] In this specification, humoral immune response to influenza virus means induction of antibody production against the virus. The antibody may be a neutralizing antibody. Neutralizing antibodies bind to influenza virus antigens, inhibiting infection and proliferation of the virus, or promoting its elimination from the body. Cellular immune response to influenza virus means proliferation of cytotoxic T cells (CTLs) that kill cells infected with the virus. Cytotoxic T cells recognize viral antigen-derived peptides presented on major histocompatibility class I molecules using T cell receptors, and suppress the spread of viral infection by destroying virus-infected cells that have presented these peptides.

[0013] The modified protein of the present invention is characterized in that at least one region of the M1 protein of influenza A virus is replaced by a membrane fusion peptide derived from the hemagglutinin (HA) protein of influenza A virus. Theoretically, 198 subtypes (HA: 1 to 18, NA: 1 to 11) of influenza A virus are known, but the M1 protein and HA protein of influenza A virus may be derived from any subtype as long as the desired effects of the present invention are achieved.

[0014] The amino acid sequence of the M1 protein of influenza A virus is publicly known and can be appropriately determined by those skilled in the art. One example is the amino acid sequence of the M1 protein of influenza A virus Michigan strain (H1N1) (SEQ ID NO: 1), but it is not limited to this. The M1 protein derived from influenza A1 virus that constitutes the modified protein of the present invention may have sequences other than the region substituted by the membrane fusion peptide that are those of the wild-type M1 protein, or it may have multiple amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50) added, deleted, or substituted. In a preferred embodiment of the present invention, the modified protein of the present invention forms an oligomer by the self-assembly activity inherent in the wild-type M1 protein, and as a result, its antigenicity may be enhanced (the region exhibiting the self-assembly activity of M1 is shown in SEQ ID NO: 2). Therefore, when the sequence other than the region substituted by the membrane fusion peptide is modified, it is preferable that the self-assembly activity is maintained. Since M1 usually forms 2 to 20-mers, when the modified protein of the present invention oligomerizes, it may usually be 2 to 20-mers, preferably 8 to 20-mers, but is not limited to these.

[0015] Furthermore, the amino acid sequences of membrane fusion peptides derived from the HA protein of influenza A virus are also known to those skilled in the art, and the membrane fusion peptide may be derived from any subtype of the HA protein. In the HA gene of influenza A virus, genetic exchange occurs between subtypes, and antigenic mutations occur due to mutations in the base sequence, but these mutations are concentrated in the head of the three-dimensional structure of HA. On the other hand, the stem region undergoes few mutations, and the amino acid sequence is conserved among subtypes, and it is thought that the common B cell epitope for the A subtype HA stem region is a three-dimensional structure including a membrane fusion domain (PS Lee and IA Wilson, Structural characterization of viral epitopes recognized by broadly cross-reactive antibodies. Curr. Top. Microbiol. Immunol. 386, 323-341 (2015)). It has been reported that this three-dimensional structural epitope within the stem region is conserved among influenza A subtypes, and therefore, it is possible to induce cross-reactive antibodies between subtypes (PS Lee and IA Wilson, see above). Influenza A virus HA can be classified phylogenetically into Group 1 (H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18) and Group 2 (H3, H4, H7, H10, H14, and H15), and the three-dimensional structural epitopes within the stem region are particularly well conserved in these groups. As shown in the following examples, even when a membrane fusion peptide of H3-derived hemagglutinin was used as a B-cell epitope, neutralizing antibodies against the H1N1 strain could be induced. Therefore, it is considered that neutralizing antibodies that cross-reference with all influenza A viruses can be induced by using membrane fusion peptides derived from hemagglutinin of any subtype of Group 1 or Group 2. As an example, the amino acid sequence (SEQ ID NO: 3) of a membrane fusion peptide derived from the HA protein of influenza A virus Michigan strain (H1N1) can be used.In another embodiment, the amino acid sequence (SEQ ID NO: 4) of a membrane fusion peptide derived from the HA protein of influenza A virus, Hong Kong strain (H3N2) can be used. When multiple regions of the M1 protein are substituted with membrane fusion peptides, all substituted regions may be replaced by the same membrane fusion peptide, or they may be replaced by different membrane fusion peptides.

[0016] Furthermore, the amino acid sequence of the membrane fusion peptide used for substitution of the M1 protein may be the wild-type sequence of the membrane fusion peptide, or it may have multiple amino acids (e.g., 1, 2, 3, 4, or 5) added, deleted, or substituted, as long as it achieves the desired effect of the present invention. The length of the membrane fusion peptide used in the modified protein of the present invention is not particularly limited as long as it functions as a B cell epitope that induces the desired antibody, but a shorter length is preferable from the viewpoint of miniaturization. For example, the length of the membrane fusion peptide is usually 50 amino acids or less, preferably 40 amino acids or less, 30 amino acids or less, or 25 amino acids or less, but is not limited to these. Also, the lower limit is usually 15 amino acids or more, preferably 16 amino acids or more, 17 amino acids or more, or 18 amino acids or more, but is not limited to these. In one embodiment, the length of the membrane fusion peptide is usually 15 to 50 amino acids, preferably 16 to 40 amino acids, 17 to 30 amino acids, or 18 to 25 amino acids, but is not limited to these.

[0017] In the modified protein of the present invention, the region in the M1 protein that is substituted by the membrane fusion peptide is at least one region, and preferably two or more regions (for example, two, three, or four regions) can be substituted by the membrane fusion peptide. The region of the M1 protein that is substituted by the membrane fusion peptide may be any region, as long as at least one function of the T cell epitope in the M1 protein is maintained and the self-assembly activity of the M1 protein is maintained.

[0018] In this specification, a T cell epitope refers to a specific structural unit of a viral antigen that is recognized and bound by a T cell receptor present on the surface of cytotoxic T cells induced in a host mammal infected with a virus. Cytotoxic T cells recognize a peptide containing a T cell epitope (or a peptide consisting of a T cell epitope) presented on a major histocompatibility class 1 antigen molecule using their T cell receptor, and destroy the virus-infected cell on which the peptide was presented. In this invention, the M1 protein, which is conserved among influenza virus subtypes, is used as the T cell epitope.

[0019] The M1 protein of influenza A virus is known to contain at least four T cell epitopes. In the case of the Michigan strain, these are located at positions 47-57 (KTRPILSPLTK, SEQ ID NO: 5), 52-60 (LSPLTKGIL, SEQ ID NO: 6), 123-132 (ALASCMGLIY, SEQ ID NO: 7), and 178-187 (RMVLASTTAK, SEQ ID NO: 8) in SEQ ID NO: 1 (Michigan strain M1 protein). The modified protein of the present invention is characterized in that at least one of these four T cell epitopes is modified to maintain its function as a T cell epitope.

[0020] Substitution regions that maintain the function of these four T cell epitopes include, for example, the following regions (1) to (4) in SEQ ID NO: 1 (Michigan strain M1 protein) in the Michigan strain: (1) positions 69 to 76 (2) positions 133 to 138 (3) positions 196 to 200 (4) positions 221 to 228.

[0021] Furthermore, the substitution of the (1) to (4) regions of M1 with membrane fusion peptides may involve substituting the entire region with a membrane fusion peptide, or substituting some of the amino acids in (1) to (4) with membrane fusion peptides while maintaining them. Specifically, for example, in the case of (1), the entire region from positions 69 to 76 may be substituted with a membrane fusion peptide, or the region from positions 70 to 75 may be substituted with a membrane fusion peptide (i.e., the amino acid residue at position 69 (P) and the amino acid residue at position 75 (Q) are maintained in the modified M1 protein, and the amino acid region between P and Q is substituted with a membrane fusion peptide).

[0022] In one preferred embodiment of the modified protein of the present invention, the number of regions in the M1 protein substituted by the membrane fusion peptide is 2, more preferably 3, and particularly preferably 4. The position of the substituted region is selected from any of (1) to (4) above.

[0023] In a preferred embodiment of the modified protein of the present invention, the entire region of the modified protein is replaced with a membrane fusion peptide at four locations (1) to (4). An example of the amino acid sequence of the modified protein of the present invention in this embodiment is shown in SEQ ID NO: 9.

[0024] In another preferred embodiment of the modified protein of the present invention, the modified protein may be a fragment of the M1 protein of influenza A virus, insofar as it has self-assembly activity. The self-assembly activity of the M1 protein of influenza A virus is known to depend on the N-terminal domain of the M1 protein (the region from position 3 to position 157 in SEQ ID NO: 1). Therefore, the modified protein of the present invention may be a peptide fragment containing this domain region.

[0025] In this embodiment, the M1 protein of influenza A virus is modified so that at least one of three T cell epitopes, located at positions 47-57 (KTRPILSPLTK, SEQ ID NO: 5), 52-60 (LSPLTKGIL, SEQ ID NO: 6), and 123-132 (ALASCMGLIY, SEQ ID NO: 7), maintains its function as a T cell epitope.

[0026] Substitution regions that maintain the function of these three T cell epitopes include, for example, the following regions in SEQ ID NO: 1 (Michigan strain M1 protein): (1) positions 69–76 (2) positions 133–138.

[0027] Furthermore, the mode of substitution by membrane fusion peptides in the relevant region is the same as described above.

[0028] The modified M1 protein of the present invention can be produced using a well-known recombinant protein production method.

[0029] 2. Pharmaceutical Compositions The present invention also provides pharmaceutical compositions comprising the modified protein of the present invention as described above. The pharmaceutical compositions of the present invention can be obtained by formulating the modified protein of the present invention according to conventional methods. The pharmaceutical compositions of the present invention comprise the modified protein of the present invention and a pharmaceutically acceptable carrier.

[0030] Examples of pharmaceutically acceptable carriers include, but are not limited to, excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium glycol starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; fragrances such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium bisulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersants such as surfactants; diluents such as water and physiological saline; and base waxes such as cocoa butter, polyethylene glycol, and kerosene.

[0031] The pharmaceutical composition of the present invention may further contain an adjuvant to enhance the immune response-inducing effect of the modified protein of the present invention. Examples of adjuvants include, but are not limited to, aluminum hydroxide, complete Freund's adjuvant, incomplete Freund's adjuvant, Bordetella pertussis adjuvant, poly(I:C), CpG-DNA, etc.

[0032] Such pharmaceutical compositions are provided in dosage forms suitable for oral or parenteral administration (preferably parenteral administration).

[0033] For parenteral administration, for example, injectable preparations, suppositories, etc., can be used, and injectable preparations may include dosage forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip infusion injection. Such injectable preparations can be prepared according to known methods. For example, an injectable preparation can be prepared by dissolving or suspending the modified protein of the present invention in a sterile aqueous solvent commonly used for injectable preparations. As aqueous solvents for injection, for example, distilled water; physiological saline; phosphate buffer, carbonate buffer, Tris buffer, acetate buffer, etc., can be used. The pH of such aqueous solvents is typically 5 to 10, preferably 6 to 8. The prepared injectable solution is preferably filled into a suitable ampoule.

[0034] Furthermore, a powder formulation of the modified protein of the present invention can be prepared by subjecting the modified protein to processes such as vacuum drying or freeze-drying. The modified protein of the present invention can be stored in powder form and used by dispersing the powder in an aqueous solvent for injection when needed.

[0035] The content of the modified protein of the present invention in the pharmaceutical composition is usually about 0.1 to 100% by weight of the total pharmaceutical composition, preferably about 1 to 99% by weight, and more preferably about 10 to 90% by weight.

[0036] 3. Pharmaceutical Uses The modified proteins and pharmaceutical compositions of the present invention can be used to induce an immune response (humoral and cellular immune response) against influenza A virus and to prevent or treat infections caused by the virus. By administering the modified proteins or pharmaceutical compositions of the present invention to mammals (primates such as humans, rodents such as mice, etc.), an immune response (humoral and cellular immune response) against influenza A virus can be induced in the mammals, and infections caused by the virus can be prevented or treated. The modified proteins of the present invention contain B cell epitopes (i.e., membrane fusion peptides) and T cell epitopes (i.e., M1 proteins) that are conserved among subtypes of influenza A virus, and thus exhibit cross-reactivity to a wide range of influenza A subtypes and induce humoral and cellular immune responses against the virus. Therefore, by using the modified proteins and pharmaceutical compositions of the present invention as a vaccine, infections caused by influenza A virus can be prevented or treated.

[0037] 4. Nucleic acids encoding the modified protein of the present invention The present invention also provides nucleic acids encoding the modified protein of the present invention (hereinafter sometimes referred to as "the nucleic acids of the present invention").

[0038] The nucleic acid of the present invention may be DNA, RNA, or a chimera thereof. The nucleic acid of the present invention can be produced using well-known genetic engineering techniques. The nucleic acid of the present invention is not particularly limited as long as it encodes the modified protein of the present invention, and may be produced by applying well-known genetic engineering techniques such as codon optimization. Furthermore, the nucleic acid of the present invention may be incorporated into a cloning vector or an expression vector.

[0039] The present invention will be described in more detail in the following examples, but the present invention is not limited in any way by these examples.

[0040] [Reference Example]Headless HA-M1 Fusion Protein (First Generation) [Gene Synthesis] From the sequence of the influenza virus Michigan strain, nucleotides encoding a headless hemagglutinin and nucleotides encoding a matrix protein were ligated with a nucleotide encoding a GS linker sandwiched therebetween, and a gene was designed by adding nucleotides encoding a 6×His tag to the C-terminus (SEQ ID NO: 10). From this sequence, a sequence with an EcoRV sequence and a start codon (ATG) added to the 5'-terminus and a stop codon (TGA) and a NotI sequence added to the 3'-terminus was artificially synthesized. The synthesized sequence was inserted between EcoRV and NotI within the MCS of pEU-EU1-MCS (Cell-Free Sciences Co., Ltd.), a protein expression vector dedicated to the wheat cell-free system, using the added restriction enzyme sites. The inserted sequence was confirmed to have no mutations by sequence analysis. The obtained plasmid was prepared in large quantities using Escherichia coli DH5.

[0041] [Synthesis of Antigen Protein] Using the plasmid as a template, an mRNA was synthesized by performing a transcription reaction at 37°C for 6 hours using the SP6 promoter on the plasmid. Using the synthesized mRNA, a translation reaction was performed at 15°C for 20 hours by the overlay method with a reaction scale of 6 mL scale × 12 reactions. For the wheat germ extract, WEPRO7240H, which was optimized for the synthesis / purification of His-tag fusion proteins, was used. The synthesized protein (crude) solution was centrifuged (21,600 x g, 4°C, 10 minutes), and the precipitate fraction was washed twice with the translation buffer. This precipitate fraction was solubilized with a solubilizing agent to obtain an antigen protein (headless HA-M1 fusion protein (+GS linker + 6×His tag), SEQ ID NO: 11) (Figure 3).

[0042] [Analysis of T cell antigen-specific response behavior to influenza virus antigen (HA-M1 antigen)] Test method Five BALB / cAJcl mice (female) were prepared and subcutaneously immunized with HA-M1 / Copete Friend Ajuvant (250 μg / ml) at 25 μL each in the foot-pad and 50 μL in the base of tail. Dissection was performed on day 7 and inguinal and popliteal lymph nodes were collected. The collected lymph nodes were ground through a mesh to prepare cell dispersions. Antigen dilutions for co-culture were prepared and the concentrations in the well were set to 100, 50, 25, and 0 μg / ml. Single-cell dispersion (5 × 10) ５ After stimulating cells (cells / well) with HA-M1 antigen solution (0, 25, 50, 100 μg / ml) for 92 hours, the degree of lymph node cell proliferation was evaluated. In addition, the concentrations of IL-10 and IFN-γ in the culture supernatant were measured by ELISA. Specifically, after collecting the supernatant, the culture medium was replenished, and 20 μl / well of CCK-8 (Cell Counting Kit-8: Dojin Chemical Co., Ltd.), diluted 2-fold with culture medium, was added to each well. The number of cells in each well was calculated by measuring the absorbance value (450 nm) 3 hours after addition. Meanwhile, the culture supernatant was diluted 2-fold (IL-10) and 20-fold (IFN-γ), and measurements were performed using the Mouse IL-10 Duoset ELISA (R&D) and Mouse IFN-γ Duoset ELISA (R&D) kits.

[0043] Results Figure 4 shows the results regarding the activation of HA-M1 antigen-specific T cells. Since Th1 T cells produce IFN-γ and Th2 T cells produce IL-10, the concentrations of these cytokines were measured, and it was found that the production of these cytokines was dramatically enhanced in an antigen-specific manner. Therefore, the HA-M1 antigen is considered an excellent candidate as a vaccine antigen that can induce humoral and cellular immunity. Figure 5 shows the results regarding HA-M1 antigen-specific lymph node cell proliferation. When lymph node cells from mice immunized with the HA-M1 antigen were cultured with the HA-M1 antigen added, significant cell proliferation was observed at antigen concentrations of 25, 50, and 100 μg / ml. There was no significant difference in cell proliferation activation depending on the antigen concentration, suggesting that the antigen concentration is saturated. However, it was shown that antigen-presenting cells present in the cultured cells took up the HA-M1 antigen, digested it to form a peptide antigen, and presented it using histocompatibility antigen (MHC class I), resulting in T cell activation.

[0044] [Influenza Virus Neutralizing Activity of HA-M1 Antigen-Administered Mouse Serum] Method Three BALB / cAJcl mice (female) were prepared for each test group. HA-M1 antigen solution was administered intraperitoneally at a dose of 50 or 100 μg / head + Alum 2 mg / head (Imject alum, Thermo: #77161, Lot TE267860B) to induce sensitization. Sensitization was performed twice, on day 0 and day 11. The test groups consisted of three groups: a group whose blood was collected 14 days after the first sensitization (#1-#3), a group whose blood was collected 21 days after the first sensitization (#4-#6), and a control group (#7-#9). Serum was collected by whole blood collection under anesthesia. The obtained serum was incubated at 56°C for 45 minutes to inactivate it. Each serum was serially diluted 2 times from a 10-fold dilution, and 50 μL of serum diluted solution was mixed with 200 TCID. ５０ 50 μL each of influenza virus A1 (A / Michigan / 45 / 2015 (H1N1pdm09)) and influenza virus A3 (A / Hong Kong / 4801 / 2014 (H3N2)) were mixed and reacted at 37°C for 30 minutes. After infecting MDCK cells cultured in a 48-well plate, 100 μL of trypsin-supplemented medium was added and the cells were cultured for 4 days. Neutralizing antibody titers were measured based on the presence or absence of cytopathic changes.

[0045] Results: In the antigen-administered group where blood was collected on the 14th day, there was no difference in the neutralizing activity against influenza A virus subtypes H1N1 and H3N2 compared to the control group. However, in the antigen-administered group where blood was collected on the 21st day, dominant neutralizing activity was obtained (Table 1). Since cross-neutralizing activity against the H3N2 type was obtained by immunization with the fusion protein antigen of the head-deleted HA of H1N1 and M1 of influenza A virus, it was confirmed that the antigen constructed in the present invention has a neutralizing epitope common to the subtypes of influenza A virus.

[0046]

[0047] [Pharmacological efficacy test using influenza virus-infected mice] Using mice (BALB / c, female, 5 weeks old), the following four groups of tests were conducted.

[0048] Test group 1: Antigen protein administration × H1N1 type influenza virus inoculation (6 mice) Test group 2: Phosphate-buffered saline administration × H1N1 type influenza virus inoculation (5 mice) Test group 3: Antigen protein administration × H3N2 type influenza virus inoculation (6 mice) Test group 4: Phosphate-buffered saline administration × H3N2 type influenza virus inoculation (5 mice)

[0049] The mice were housed at 3 - 5 mice per cage in an environment with a room temperature of 24 ± 3°C, humidity of 50 ± 20%, ventilation of 10 - 25 times / hour, and lighting of 12 hours. The feed was MF (Oriental Yeast Co., Ltd.) and was fed by allowing free intake. As the method of administering the antigen protein, an antigen protein (protein concentration 250 μg / mL) mixed with an equal amount of Imject Alum Adjuvant (Thermo Fisher Scientific) was prepared, and 0.2 mL per mouse was administered subcutaneously twice at intervals of 7 days for a total of 0.4 mL. Test groups 2 and 4 were administered with phosphate-buffered saline added with the same adjuvant as test groups 1 and 3 in the same manner. The influenza virus inoculation was performed on the 7th day after the second administration of the antigen protein under isoflurane anesthesia by nasal inoculation of 50 μL of the inoculation virus. The influenza virus was H1N1 (strain name: A / PR / 8 / 34, ATCC No.: VR-1469, BSL: 2, virus titer: 1.6x10 ８ TCID５０ ( / mL) and H3N2 (strain name: A / Port Chalmers / 1 / 73, ATCC No.: VR-810, BSL: 2, viral titer: 1.3 x 10) ７ TCID ５０ Two subtypes ( / mL) were used. To assess the condition of the mice, their weight was measured on the day of arrival, 14 days before (Day-14), 7 days before (Day-7) virus inoculation, on the day of virus inoculation (Day 0), 3 days after (Day 3), 7 days after (Day 7), 10 days after (Day 10), and 14 days after (Day 14) virus inoculation. In addition, the general condition of the mice (decreased activity and rough coat) was evaluated from 14 days before to 14 days after virus inoculation.

[0050] The results are shown in Tables 2-1 and 2-2 (general condition), Table 3 (weight), and Table 4 (survival rate) below. A graph of the survival rate is shown in Figure 6.

[0051]

[0052]

[0053]

[0054]

[0055] As shown in Tables 2-1, 2-2, 3, and 4, and Figure 6, mice inoculated with the antigen protein of the present invention (groups 1 and 3) acquired resistance to influenza viruses (H1N1 and H3N2) compared to groups not inoculated with the antigen protein (groups 2 and 4). Therefore, the antigen protein of the present invention is useful as a vaccine that can confer cross-immunity between subtypes of influenza A virus.

[0056] [Example] Modified M1 protein substituted with membrane fusion peptide (second generation) The following experiment was conducted with the aim of miniaturizing the first generation influenza virus vaccine.

[0057] First, using a known genetic engineering technique, we created a nucleic acid encoding a modified M1 protein by substituting four regions (positions 69-76, 133-138, 196-200, and 221-228) of the M1 protein (SEQ ID NO: 1) derived from influenza A virus (H1) with a membrane fusion peptide (SEQ ID NO: 4) derived from influenza A virus (H3) and a polypeptide with an S added to its C-terminus (hereinafter, this polypeptide may be referred to as "FLM1 protein"). The primary amino acid sequence of the FLM1 protein is shown in Figure 7. Furthermore, the results of predicting the three-dimensional structure of the FLM1 protein using Alphafold2 are shown in Figure 8.

[0058] The nucleic acid encoding the FLM1 protein was synthesized by a malt embryo cell-free protein synthesis method, similar to the reference example described above, and recovered as a precipitate. The recovered precipitate was dissolved in a 5 mM PBS solution of the nonionic surfactant 1-O-n-Octyl-β-D-glucopyranoside. When the sample dissolved in PBS was subjected to SDS-PAGE (monomer), a single band was observed around 30 kDa. Furthermore, when subjected to Native-PAGE (multimer), in addition to the 30 kDa band indicating the monomer, multiple bands indicating larger molecular weights were observed (Figure 9). From these results, it was confirmed that the intended FLM1 protein was appropriately prepared and that the FLM1 protein maintained its self-assembly activity.

[0059] Next, an adjuvant was added to a PBS solution containing FLM1 protein under the conditions shown in Table 5 below. The resulting mixture was sonicated and then immunized in rabbits. The increase in antigen-specific antibody titers was measured by ELISA. When the antibody titer reached a plateau, blood was collected to obtain serum. After inactivation, neutralizing antibody titers against infection and proliferation of influenza A H1N1 and H3N2 strains were measured by TCID50. The results are shown in Table 5 below.

[0060]

[0061] *100TCID ５０An immunoassay was performed using serum dilution ratios below ** to neutralize the virus. Antigen dose: 100 μg / Rabbit / 1 dose [Administration method] 1st: Intramuscular administration, 2nd, 3rd & 4th: Subcutaneous administration [Adjuvant] 1st: FCA (Adjuvant Complete Friend) 2nd: Non 3rd & 4th: FIA (Adjuvant Incomplete Friend)

[0062] As shown in Table 5, the FLM1 protein has been shown to induce antibodies against H1N1 strains of influenza A virus group 1 and H3N2 strains of influenza A virus group 2. Furthermore, as shown in the reference example above, the M1 protein possesses multiple T cell epitopes, and as a result can induce cellular immunity against influenza viruses. Since the FLM1 protein also maintains all of the T cell epitopes possessed by the M1 protein, it is thought that it can induce cellular immunity in the same way as the M1 protein. Therefore, the FLM1 protein can induce both humoral and cellular immunity against influenza A viruses in general and can be used as a small, easy-to-handle influenza virus vaccine.

[0063] According to the present invention, it is possible to provide a miniaturized influenza virus vaccine that cross-reacts to a wide range of influenza A subtype viruses, including seasonal influenza viruses and expected highly pathogenic influenza viruses, and that is extremely easy to prepare.

[0064] This application is based on Japanese Patent Application No. 2024-169021 (filing date: September 27, 2024), the contents of which are fully incorporated herein.

Claims

1. A modified protein of influenza A matrix 1 (M1) that induces humoral and cellular immune responses against influenza A virus, wherein the modified protein has the following characteristics: (a) at least one region of the M1 protein is replaced by a membrane fusion peptide derived from influenza A virus hemagglutinin; and (b) at least one T cell epitope in the M1 protein is maintained.

2. The modified protein according to claim 1, wherein two or more regions of the M1 protein are replaced by membrane fusion polypeptides.

3. The modified protein according to claim 1 or 2, wherein the membrane fusion peptide comprises the amino acid sequence represented by SEQ ID NO: 3 or 4.

4. The modified protein according to claim 1 or 2, wherein the M1 protein comprises the amino acid sequence represented by Sequence ID No. 1 or a fragment thereof having self-assembly activity.

5. The modified protein according to claim 1 or 2, comprising the amino acid sequence represented by Sequence ID No.

9.

6. A pharmaceutical composition comprising the modified protein described in claim 1 or 2.

7. The pharmaceutical composition according to claim 6, which is for inducing an immune response against influenza A virus.

8. The pharmaceutical composition according to claim 6, for the prevention or treatment of influenza A virus infection.

9. A nucleic acid encoding the modified protein according to claim 1 or 2.

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