Vaccine composition for preventing influenza virus infection having enhanced neutralizing activity

Abstract

The present invention relates to a vaccine composition for preventing influenza virus infection, having enhanced neutralizing activity. A variant protein or fragment thereof according to the present invention has a stable pre-fusion structure, thereby enabling rapid preparation for influenza viruses with sequence variations that are difficult to predict each year. In addition, the variant protein or fragment thereof according to the present invention exhibits high neutralizing activity, thereby serving as an effective vaccine with superior protective ability against influenza virus. In particular, sequence optimization enables the efficient production of structurally stable proteins with high expression levels, while reducing production costs.

Description

Neutralizing ability improved vaccine composition for the prevention of influenza virus infection

[0001] The present invention relates to a vaccine composition with improved neutralizing ability for the prevention of influenza virus infection.

[0002] Influenza remains a persistent threat to global public health, causing severe morbidity and mortality through periodic outbreaks and pandemics. The key to the influenza virus's ability to infect host cells is the hemagglutinin (HA) protein, which is crucial for viral penetration and serves as a primary target for neutralizing antibodies. HA enables the virus to bind to sialic acid on the surface of host cells, facilitating the fusion of the viral membrane with the host cell membrane and allowing for viral penetration. This fusion process depends on the conformational change of HA, transitioning from a pre-fusion state to a post-fusion state. Therefore, since the pre-fusion form is the most effective target for inducing a protective immune response, stabilizing the pre-fusion form of HA is critical for vaccine development.

[0003] The HA protein is synthesized from the precursor polypeptide HA0, which is cleaved into two subunits, HA1 and HA2, connected by disulfide bonds. HA1 possesses a receptor-binding domain, while HA2 facilitates membrane fusion. Structurally, HA is expressed as a trimer on the viral surface, with each monomer containing both HA1 and HA2 subunits. The trimer configuration is essential for HA's function, enabling effective sialic acid receptor binding in host cells and subsequent intracellular invasion. Acidification within the endosome induces a conformational change in HA, exposing the fusion peptide of the HA2 subunit and inducing membrane fusion. Importantly, the pre-fusion form of HA is translocationally stable, requiring stabilization to preserve the intrinsic antigenic structure crucial for neutralizing antibody recognition.

[0004] Although the pre-fusion form of HA plays an important role in vaccine development, its stability outside the virus is a significant challenge, and various strategies have been used to stabilize the pre-fusion form of HA trimer to overcome this problem. One common approach is to promote trimer formation by incorporating external trimerization domains, such as T4 Foldon or ferritin cages.

[0005] For example, Weldon et al. demonstrated that by incorporating a trimerization sequence tag into the H3N2 HA protein, engineered HA forms a trimer similar to the natural one and exhibits increased vaccine activity in mice. In the case of subunit ferritin cages, monomeric ferritin subunits are assembled into a ternary array at the vertices. Attaching HA monomers to ferritin cages forms a stable ternary array, providing potent and broad-spectrum protection against various influenza strains. Ferritin-based HA vaccines have shown promising results in preclinical and early clinical trials, demonstrating potent immunogenicity and protective efficacy. Additionally, isolated HA stem proteins stabilized by N- and C-terminal trimerization domains and disulfide bridges are referred to as mini-HA. This variant has demonstrated broad-spectrum protection against various influenza viruses.

[0006] The inventors of the present invention confirmed that optimizing a few pre-selected locations in the HA stem region can effectively stabilize the protein in a trimer pre-fusion form, and completed the present invention by confirming that the strategy according to the present invention not only improves the thermal stability of HA but also preserves the pre-fusion structure, thereby enabling rapid vaccine development.

[0007] The inventors of the present invention intended to develop a vaccine with improved neutralizing ability based on a recombinant protein of the ectodomain HA2 subunit of viral hemagglutinin as an influenza virus vaccine.

[0008] As a result, the present invention was completed by preparing a vaccine composition with improved neutralizing ability for preventing influenza virus infection using a variant of the ectodomain HA2 subunit protein of influenza virus hemagglutinin as the antigen protein.

[0009] Accordingly, the object of the present invention is to provide a variant protein or a fragment thereof comprising amino acid substitutions at the 95th and 103rd residues of the ectodomain HA2 subunit protein of influenza virus hemagglutinin.

[0010] Another objective of the present invention is to provide a gene encoding the variant protein or a fragment thereof, a recombinant vector containing the gene, and a host cell transformed with the recombinant vector.

[0011] Another objective of the present invention is to provide a vaccine composition for preventing or treating influenza virus infection, comprising the above-mentioned variant protein or a fragment thereof.

[0012] Another objective of the present invention is to provide a method for preventing or treating influenza virus infection, comprising the step of administering a vaccine composition containing the variant protein or a fragment thereof to a subject in need thereof.

[0013] Another objective of the present invention is to provide a method for preventing or treating influenza virus infection, comprising the step of administering a vaccine composition containing the variant protein or a fragment thereof to a subject.

[0014] Another objective of the present invention is to provide a use of the variant protein or a fragment thereof in the manufacture of a drug for the prevention or treatment of influenza virus infection disease.

[0015] Another objective of the present invention is to provide a use of the variant protein or a fragment thereof in the manufacture of a drug for the prevention or treatment of influenza virus infection.

[0016] Another objective of the present invention is to provide a vaccine composition comprising the variant protein or a fragment thereof for the prevention or treatment of influenza virus infection.

[0017] Another objective of the present invention is to provide a vaccine composition comprising the variant protein or a fragment thereof for the prevention or treatment of influenza virus infection.

[0018] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0019] Furthermore, the terms used in this invention are for illustrative purposes only and should not be interpreted as intended to be limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this invention, terms such as "comprising" or "having" are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0020] Furthermore, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the embodiments pertain. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0021] Furthermore, to prevent clutter with overlapping content, redundant details have been omitted below. In other words, the content of the invention is not limited solely to the following description, and should be interpreted in accordance with the overall context of the invention.

[0022] Stabilizing the pre-fusion structure of antigen proteins can improve the efficacy and universality of antiviral vaccines. The pre-fusion form of hemagglutinin (HA) of the influenza virus generally adopts a stable triple structure. However, we unexpectedly discovered that the HA recombinant ectodomain of the A / California / 04 / 2009 (H1N1) influenza virus forms a monomer in solution rather than the expected triple.

[0023] In one embodiment of the present invention, the addition of a T4 poldon tag induced trimerization, but the resulting trimer did not exhibit a stable pre-fusion structure when analyzed by cryo-electron microscopy (cryo-EM). Accordingly, to promote the formation of a pre-fusion structure, five important amino acid residues involved in the triplication of the stem region of HA were redesigned using a computer sequence optimization program.

[0024] The engineered HA protein formed a stable trimer without a poldon sequence at both pH 8.0 and pH 5.6. In addition, the thermal stability of the modified protein was enhanced, with the melting temperature (Tm) increasing by approximately 10°C. High-resolution cryo-EM analysis at 2.2 Å resolution confirmed that the mutant hemagglutinin protein adopts a stable pre-fusion structure.

[0025] By optimizing the same five sites in the hemagglutinin of other influenza viruses, a stable pre-fusion structure of the hemagglutinin protein as described above was confirmed, and the therapeutic efficacy against influenza infection resulting from the production of neutralizing antibodies using this was confirmed in experimental animals.

[0026] Accordingly, the present invention aims to provide a therapeutic use of the 5-site mutant fragment optimized through the above method for influenza viruses.

[0027]

[0028] As a preferred embodiment for achieving the above-mentioned purpose, the present invention provides a variant protein or a fragment thereof comprising amino acid substitutions at the 95th and 103rd residues of the ectodomain HA2 subunit protein of influenza virus hemagglutinin.

[0029] More preferably, the influenza virus is a Group 1 influenza virus, which may be selected from influenza A viruses including, for example, H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18 subtypes.

[0030] Hemagglutinin is a glycoprotein on the surface of the influenza virus that helps the virus bind to cells and enter them. Hemagglutinin is divided into the HA1 subunit and the HA2 subunit; the HA1 subunit binds to sialic acid on the surface of host cells, while the HA2 subunit facilitates membrane fusion, enabling the virus to inject its nucleolus into the cell.

[0031] Hemagglutinin is the most abundant glycoprotein on the surface of the influenza virus, serves as the virus's major antigen, and is the most variable and rapidly evolving part. Because hemagglutinin plays a crucial role in the processes of viral binding, fusion, and entry, it is a promising target for developing anti-influenza drugs. In other words, it can be used as an epitope capable of producing neutralizing antibodies.

[0032] As a most preferred example, the naming of residues according to the present invention is based on the full-length sequence of the HA2 subunit protein of influenza hemaclutinin and the sequence number.

[0033] The HA2 subunit protein of hemagglutinin of the influenza virus according to the present invention may mean any one amino acid sequence selected from SEQ ID NOs 47 to 55 (Table 1).

[0034]

[0035] More preferably, the amino acid substitution may be substituted with leucine. Even more preferably, the 95th and 103rd residues of the ectodomain HA2 subunit protein of group 1 influenza virus hemagglutinin may be commonly substituted with leucine to impart stabilization to the pre-fusion structure of the isolated and purified protein fragment. Most preferably, the variant protein or its fragment further comprises an amino acid substitution at any one of the 47th, 102nd, and 109th residues. At this time, the amino acid substitution at the 47th residue may be substituted with any one amino acid selected from glycine, tyrosine, isoleucine, and alanine. The amino acid substitution at the 102nd residue may be substituted with phenylalanine or leucine, and the amino acid substitution at the 109th residue may be substituted with any one amino acid selected from glutamic acid, asparagine, alanine, and serine. Preferably, the mutant protein or its fragment may include a Foldon tag at the C-terminus, in which case trimerization may be promoted. However, the stable pre-fusion structure of the isolated and purified protein fragment is derived from 5-site mutations at positions 47, 95, 102, 103, and 109.

[0036] More preferably, the variant protein or fragment thereof has amino acid substitutions at the 47th, 95th, 102nd, 103rd, and 109th positions of the HA2 subunit protein of the influenza virus hemagglutinin of SEQ ID NO. 47, most preferably having a variant of E47G / N95L / L102F / E103L / D109E based on SEQ ID NO. 47, and the polypeptide sequence of hemagglutinin (A / California / 04 / 2009 (H1N1) (H1 / Cal09) HA protein) containing the same is as shown in SEQ ID NO. 3.

[0037] More preferably, the variant protein or fragment thereof has amino acid substitutions at the 95, 102, 103, and 109 positions of the HA2 subunit protein of the influenza virus hemagglutinin of SEQ ID NO. 48, most preferably having a variant N95L / L102F / E103L / D109E based on SEQ ID NO. 48, and the polypeptide sequence of hemagglutinin (A / Malaysia / 1842338 / 2007 (H1N1) (H1 / Mal07) HA protein) containing the same is as shown in SEQ ID NOs. 9 and 10. SEQ ID NO. 10 includes a Foldon tag at the 3' end.

[0038] More preferably, the variant protein or fragment thereof has amino acid substitutions at the 95th and 103rd positions of the HA2 subunit protein of the influenza virus hemagglutinin of SEQ ID NO. 49, most preferably having a variant of N95L / E103L based on SEQ ID NO. 49, and the polypeptide sequence of hemagglutinin (HA protein of A / swine / Hong Kong / 2106 / 98 (H9 / N2) (H9 / HK98)) containing the same is as shown in SEQ ID NOs. 17 and 18. SEQ ID NO. 18 includes a Foldon tag at the 3' end.

[0039] More preferably, the variant protein or fragment thereof has amino acid substitutions at the 47th, 95th, 102nd, 103rd, and 109th positions of the HA2 subunit protein of the influenza virus hemagglutinin of SEQ ID NO. 50, most preferably having a variant of G47Y / N95L / M102L / E103L / D109N based on SEQ ID NO. 50, and the polypeptide sequence of hemagglutinin (HA protein of A / Vietnam / 1194 / 2004 (H5N1)) containing the same is as shown in SEQ ID NO. 24.

[0040] More preferably, the variant protein or fragment thereof has amino acid substitutions at the 47th, 95th, 103rd, and 109th positions of the HA2 subunit protein of the influenza virus hemagglutinin of SEQ ID NO. 51, most preferably having a variant of G47I / N95L / E103L / D109A based on SEQ ID NO. 51, and the polypeptide sequence of hemagglutinin (HA protein of A / Taiwan / 2 / 2013 (H6N1)) containing the same is as shown in SEQ ID NO. 26.

[0041] More preferably, the variant protein or fragment thereof has amino acid substitutions at the 95th, 103rd, and 109th positions of the HA2 subunit protein of the influenza virus hemagglutinin of SEQ ID NO. 52, most preferably having a variant N95L / E103L / D109S based on SEQ ID NO. 52, and the polypeptide sequence of hemagglutinin (HA protein of A / turkey / Ontario / 6118 / 1968 (H8N4)) containing the same is as shown in SEQ ID NO. 28.

[0042] More preferably, the variant protein or fragment thereof has amino acid substitutions at the 47th, 95th, and 103rd positions of the HA2 subunit protein of the influenza virus hemagglutinin of SEQ ID NO. 53, most preferably having a variant of N47A / N95L / E103L based on SEQ ID NO. 53, and the polypeptide sequence of hemagglutinin (HA protein of A / duck / Alberta / 60 / 1976 (H12N5)) containing the same is as shown in SEQ ID NO. 30.

[0043] More preferably, the variant protein or fragment thereof has amino acid substitutions at the 47th, 95th, 103rd, and 109th positions of the HA2 subunit protein of the influenza virus hemagglutinin of SEQ ID NO. 54, most preferably having a variant N47Y / N95L / E103L / D109A based on SEQ ID NO. 54, and the polypeptide sequence of hemagglutinin (HA protein of A / gull / Maryland / 704 / 1977 (H13N6)) containing the same is as shown in SEQ ID NO. 32.

[0044] More preferably, the variant protein or fragment thereof has amino acid substitutions at the 95th, 103rd, and 109th positions of the HA2 subunit protein of the influenza virus hemagglutinin of SEQ ID NO. 55, most preferably having a variant of N95L / E103L / D109S based on SEQ ID NO. 55, and the polypeptide sequence of hemagglutinin (HA protein of A / flat-faced bat / Peru / 033 / 2010 (H18N11)) containing the same is as shown in SEQ ID NO. 34.

[0045] In addition, it may include polypeptides in which amino acids of the above-described polypeptide sequence are substituted by conservative substitution, and polypeptides having 80 to 99%, 85 to 99%, preferably 90 to 99%, sequence homology therewith, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence homology therewith. The same applies to corresponding polynucleotides.

[0046] The variant protein or fragment thereof according to the present invention is structurally stable and has a small size compared to the full sequence of the influenza virus, making it very easy to produce and having the effect of producing a neutralizing antibody that is significantly superior to that of the wild type.

[0047] In other words, while protein antigens generally have the problem of low immunogenicity, the variant protein or its fragment according to the present invention has excellent stability and immunogenicity and can be usefully utilized as a vaccine because it has a high expression level within host cells.

[0048] In addition, according to one embodiment of the present invention, the variant protein or a fragment thereof exhibits an excellent protective effect against influenza virus infection by improving the survival rate and weight recovery rate after viral infection.

[0049] If necessary, the variant protein or a fragment thereof according to the present invention may be fused with an Fc fragment (Fc antibody fragment). The Fc fragment (Fc antibody fragment) refers to a portion capable of binding to an antigen-presenting cell (APC), comprising a CH2 domain and / or a CH3 domain derived from the antibody Fc fragment. The Fc fragment may be derived from any one selected from the group consisting of IgG, IgA, IgD, IgE, and IgM, and preferably may be an Fc fragment derived from IgG. The IgG may be further divided into IgG1, IgG2, IgG3, and IgG4, and the Fc fragment of the present invention may most preferably be an Fc fragment derived from IgG1. The 'Fc fragment' is a fragment obtained when an immunoglobulin (Ig) molecule is digested into papain, and is a region from which the variable region (VL) and constant region (CL) of the light chain and the variable region (VH) and constant region 1 (CH1) of the heavy chain have been removed. That is, the Fc fragment refers to a dimer of two CH2-CH3 domain chains, wherein the two chains form a dimer structure by disulfide bonds. Additionally, the Fc fragment may include all or part of a hinge region peptide in the constant region of the heavy chain. Furthermore, it may be an extended Fc fragment including part or all of the constant region 1 (CH1) and / or the constant region 1 (CL1) of the heavy chain, provided that it has an effect substantially equivalent to or enhanced to the natural form. Additionally, it may be a fragment from which a significantly long partial amino acid sequence corresponding to CH2 and / or CH3 has been removed.

[0050]

[0051] In another preferred embodiment for achieving the above-mentioned purpose, the present invention relates to a gene encoding the variant protein or a fragment thereof, a recombinant vector containing the same, and a host cell transformed with said recombinant vector.

[0052] In a specific embodiment of the present invention, the gene may be characterized as being represented by the nucleotide sequences of SEQ ID NOs. 6, 13 to 14, 21 to 22, 36, 38, 40, 42, 44, and 46:

[0053] Sequence No. 6 is encoded to have the variant E47G / N95L / L102F / E103L / D109E based on the A / California / 04 / 2009 (H1N1) (H1 / Cal09) HA2 subunit protein.

[0054] SEQ ID NOs 13 and 14 are encoded to have the N95L / L102F / E103L / D109E variant based on the A / Malaysia / 1842338 / 2007 (H1N1) (H1 / Mal07) HA2 subunit protein. Additionally, SEQ ID NO 14 contains a sequence encoding a Foldon tag at the 3' end.

[0055] Sequence Nos. 21 and 22 are encoded to have the N95L / E103L variant based on the HA2 subunit protein of A / swine / Hong Kong / 2106 / 98 (H9 / N2) (H9 / HK98). Additionally, Sequence No. 22 contains a sequence encoding a Foldon tag at the 3' end.

[0056] Sequence No. 36 is encoded to have the G47Y / N95L / M102L / E103L / D109N variant based on the HA2 subunit protein of A / Vietnam / 1194 / 2004 (H5N1).

[0057] Sequence No. 38 is encoded to have the G47I / N95L / E103L / D109A variant based on the HA2 subunit protein of A / Taiwan / 2 / 2013 (H6N1).

[0058] Sequence No. 40 is encoded to have the N95L / E103L / D109S variant based on the HA2 subunit protein of A / turkey / Ontario / 6118 / 1968 (H8N4).

[0059] Sequence No. 42 is encoded to have the N47A / N95L / E103L variant based on the HA2 subunit protein of A / duck / Alberta / 60 / 1976 (H12N5).

[0060] Sequence No. 44 is encoded to have the N47Y / N95L / E103L / D109A variant based on the HA2 subunit protein of A / gull / Maryland / 704 / 1977 (H13N6).

[0061] Sequence No. 46 is encoded to have the N95L / E103L / D109S variant based on the HA2 subunit protein of A / flat-faced bat / Peru / 033 / 2010 (H18N11).

[0062] In the present invention, the term "vector" refers to a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA within a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Upon transformation into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, be incorporated into the genome itself. Since plasmids are the most commonly used form of vector currently, "plasmid" and "vector" are sometimes used interchangeably in the specification of the present invention. For the purposes of the present invention, it is preferable to use a plasmid vector.

[0063] A typical plasmid vector that can be used for this purpose has a structure comprising (a) a replication origin that enables efficient replication to include hundreds of plasmid vectors per host cell, (b) an antibiotic resistance gene that allows host cells transformed with the plasmid vector to be selected, and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Even if a suitable restriction enzyme cleavage site is not present, the vector and foreign DNA can be easily ligated using a synthetic oligonucleotide adapter or linker according to conventional methods.

[0064] In the present invention, the term "recombinant vector" typically refers to a recombinant carrier into which a fragment of heterogeneous DNA is inserted, generally meaning a fragment of double-stranded DNA. Here, heterogeneous DNA refers to atypical DNA that is not naturally found in host cells. Once inside a host cell, the recombinant vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (heterogeneous) DNA can be produced.

[0065] In one embodiment of the present invention, a fragment obtained by PCR amplifying the nucleotide sequence of a fragment of a variant protein according to the present invention was inserted into a pEG BacMam baculovirus delivery vector, and an expression vector loaded with a polynucleotide encoding a variant protein fragment of the ectodomain HA2 subunit of influenza virus hemagglutinin was constructed.

[0066] More specifically, a restriction enzyme site and a signal sequence may be attached to any one of the aforementioned sequence numbers 6, 13 to 14, 21 to 22, 36, 38, 40, 42, 44 and 46, and the prepared sequence may be loaded into an expression vector.

[0067] After ligation, the gene or the recombinant vector is transformed or transfected into a host cell. Various techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into a prokaryotic or eukaryotic host cell to perform "transformation" or "transfection," such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection, may be used.

[0068] As is well known in the art, in order to increase the expression level of a transfected gene in a host cell, the gene must be activatorily linked to transcription and translation expression regulatory sequences that are functional within the selected expression host.

[0069] In the present invention, the term "transformation" refers to the introduction of DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or through the completion of chromosomal integration. Of course, it must be understood that not all vectors function equally in expressing the gene sequence of the present invention. Likewise, not all hosts function equally for the same expression system. However, a person skilled in the art can make an appropriate selection among various vectors, expression regulatory sequences, and hosts without excessive experimental burden and without departing from the scope of the present invention. For example, when selecting a vector, the host must be considered, as the vector must be replicated within it. The copy number of the vector, the ability to control the copy number, and the expression of other proteins encoded by said vector must also be considered.

[0070] In the present invention, the transformed host cell is preferably selected from the group consisting of animal cells, plant cells, yeast, E. coli, and insect cells, but is not limited thereto.

[0071] Specifically, in the present invention, the microorganism used as the transformed host cell may be any Gram-negative bacteria such as Escherichia coli, Salmonella typhi, Salmonella typhimurium, Vibrio cholerae, Mycobacterium bovis, Shigella, etc., provided that it is non-toxic or attenuated when applied to the body, and any Gram-positive bacteria such as Bacillus, Lactobacillus, Lactococcus, Staphylococcus, Listeria monocytogenes, Streptococcus, etc. may be used, and preferably, edible microorganisms such as lactic acid bacteria may be used, but are not limited thereto.

[0072] The above lactic acid bacteria may include genera Lactobacillus sp., Streptococcus, and Bifidobacterium, and representatively, the genus Lactobacillus includes Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus delbrueckii, Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus bulgaricus, and Lactobacillus casei; For the genus Streptococcus, Streptococcus thermophiles; for the genus Bifidobacterium, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, and Bifidobacterium adolescentis can be used.

[0073] It may be a eukaryotic cell such as fungi like Aspergillus sp., yeasts like Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp., and Neurospora crassa, other lower eukaryotic cells, and cells of higher eukaryotes such as insects.

[0074] In another aspect, the present invention relates to a method for producing an influenza virus antigen by culturing the host cells.

[0075] When a recombinant vector capable of expressing the antigen of the above-mentioned influenza virus is introduced into a host cell, the antibody can be produced by culturing the host cell for a period sufficient to cause the antigen to be expressed in the host cell, or more preferably, for a period sufficient to cause the antigen to be secreted into the culture medium in which the host cell is cultured.

[0076] In some cases, the expressed antigen can be isolated from the host cell and purified to be homogeneous. The isolation or purification of the antigen can be performed by separation and purification methods used for conventional proteins, for example, by chromatography. The chromatography may include, for example, affinity chromatography including a protein A column or a protein G column, ion exchange chromatography, or hydrophobic chromatography. In addition to the chromatography, antibodies can be isolated and purified by combining filtration, ultrafiltration, salting out, dialysis, etc.

[0077]

[0078] In another preferred embodiment for achieving the above-mentioned purpose, the present invention provides a vaccine composition for preventing or treating influenza virus infection, comprising a variant protein or a fragment thereof comprising amino acid substitutions at the 95th and 103rd residues of the ectodomain HA2 subunit protein of influenza virus hemagglutinin.

[0079] Preferably, the influenza virus is a type A influenza virus, and more preferably, a Group 1 type A influenza virus.

[0080] The vaccine composition of the present invention induces neutralizing antibodies of high titers. Furthermore, by selecting and using a structurally stable protein as the antigen protein, the vaccine can stably exhibit an active effect. In other words, it has the advantage of being able to induce a strong host antibody response or protective immunity.

[0081] At this time, the composition may further include an immune enhancer, an excipient, or a carrier. The immune enhancer may be one or more selected from the group consisting of, as an example, complete and incomplete Freund immune enhancers; vitamin E; non-ionic blocking polymers; muramil dipeptide; Quil A; mineral oils and non-mineral oils; and Carbopol.

[0082] That is, the vaccine composition of the present invention may include, in addition to the variant protein or fragment thereof which is the antigen, one or more adjuvants, excipients, or carriers suitable for constituting the vaccine composition.

[0083] An adjuvant that may be included in the composition of the present invention refers to a substance that enhances the immune response of an injected animal, and numerous different adjuvants are known to those skilled in the art. The adjuvant includes, but is not limited to, Freund complete and incomplete adjuvants, vitamin E, non-ionic blocking polymers, muramil dipeptide, Quil A, mineral oils and non-mineral oils and Carbopol, water-in-oil emulsion adjuvants, etc.

[0084] Examples of excipients that may be included in the vaccine composition of the present invention include aluminum phosphate, aluminum hydroxide, aluminum potassium sulfate, etc.

[0085] A carrier that may be included in the vaccine composition of the present invention may be a pharmaceutically acceptable carrier, and examples of said pharmaceutically acceptable carriers include saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc.

[0086] The vaccine composition of the present invention may further include preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, inert gases, etc. The preservatives include formalin, thimerosal, neomycin, polymyxin B, and amphotericin B, etc. The vaccine composition of the present invention may include one or more suitable emulsifiers, such as Span or Tween. Additionally, the vaccine composition of the present invention may include a protective agent, and protective agents known in the art may be used without limitation, which may include, but are not limited to, lactose (LPGG) or trehalose (TPGG).

[0087] In a specific embodiment of the present invention, it is preferable to administer the vaccine composition 1 to 3 times at intervals of 1 to 3 weeks, and more preferable to administer it 2 times at intervals of 2 weeks.

[0088] In a specific embodiment of the present invention, the vaccine composition may be formulated using a method known in the art to enable rapid release, or sustained or delayed release, of the active ingredient when administered to a mammal. The formulations include powder, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.

[0089] In a specific embodiment of the present invention, the vaccine composition may be administered by one or more routes selected from the group consisting of intramuscular, intravenous, subcutaneous, intradermal, and intranasal.

[0090] In a specific embodiment of the present invention, the vaccine composition may be administered by a method of applying physical stimulation to increase delivery efficiency upon administration, examples of which include electroporation, gene gun, and jet injection methods.

[0091] The vaccine composition according to the present invention can be formulated into a suitable form with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oil, saline solution, aqueous glucose and glycol, and may additionally include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Additionally, the vaccine composition according to the present invention may appropriately include, if necessary depending on the method of administration or formulation, a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, an analgesic agent, a buffer, an antioxidant, etc.

[0092] The dosage of the above vaccine composition to a patient varies depending on many factors, including the patient's height, body surface area, age, specific compound administered, gender, time and route of administration, general health, and other drugs administered concurrently.

[0093] In addition, the vaccine composition of the present invention is administered in a therapeutically effective amount.

[0094] In the present invention, a therapeutically effective amount means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined according to factors including individual type and severity, age, sex, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The dosage of the vaccine composition of the present invention is in the range of 0.0001 to 1,000 mg / kg (body weight) per day, but may vary depending on the individual type and is not limited thereto.

[0095] Meanwhile, the above dosage can be appropriately adjusted according to the patient's age, gender, and condition.

[0096] As another preferred embodiment for achieving the above-mentioned purpose, the present invention provides a method for preventing or treating an influenza virus infection, comprising the step of administering to a subject in need of the vaccine composition comprising a variant protein or a fragment thereof, wherein the variant protein comprises amino acid substitutions at the 95th and 103rd residues of the ectodomain HA2 subunit protein of the influenza virus hemagglutinin.

[0097] As another preferred embodiment for achieving the above-mentioned purpose, the present invention provides a method for preventing or treating an influenza virus infection, comprising the step of administering a vaccine composition comprising a variant protein or a fragment thereof, comprising amino acid substitutions at the 95th and 103rd residues of the ectodomain HA2 subunit protein of influenza virus hemagglutinin, to a subject other than a human who requires such a vaccine composition.

[0098] The above prevention may also refer to the efficacy of the vaccine.

[0099] The above-mentioned subjects may be mammals or birds, excluding humans. As a preferred example, mammals include, but are not limited to, rats, mice, hamsters, pigs, rabbits, horses, donkeys, goats, sheep, guinea pigs, llamas, and bats. Additionally, birds include, but are not limited to, seagulls, chickens, ducks, etc.

[0100] Diseases caused by the influenza virus may be respiratory diseases. Symptoms may appear after an incubation period of 2 to 14 days following infection with the influenza virus. These symptoms include any symptoms caused by the virus infection, such as, for example, high fever, cough, shortness of breath, pneumonia, gastrointestinal symptoms like diarrhea, organ failure (renal failure, renal dysfunction, etc.), septic shock, and death in severe cases.

[0101] In the present invention, the term "prevention" means suppressing the occurrence of a disease or illness in a subject who has not been diagnosed with having such a disease or illness but is at risk of developing such a disease or illness.

[0102] In the present invention, the term "treatment" means (a) inhibition of the progression of a disease, illness, or symptom; (b) alleviation of a disease, illness, or symptom; or (c) elimination of a disease, illness, or symptom. The composition of the present invention serves to inhibit the progression of symptoms, eliminate them, or alleviate them by activating an immune response against the virus in an individual suffering from a disease caused by an influenza virus infection. Accordingly, the composition of the present invention may serve as a therapeutic composition in itself, or it may be applied as an adjuvant for the treatment of the said disease by being administered together with other pharmacological components.

[0103] Accordingly, in this specification, the terms "treatment" or "therapeutic agent" include the meaning of "therapeutic aid" or "therapeutic adjuvant."

[0104] The term "immune response" in the present invention refers to a cell-mediated (T-cell) immune response and / or an antibody (B-cell) response. Preferably, it is an antibody (B-cell) response following antigen treatment.

[0105] In the present invention, the vaccine composition may be characterized by further comprising a pharmaceutically acceptable carrier, excipient, or diluent. Examples of excipients that may be included in the vaccine composition of the present invention include aluminum phosphate, aluminum hydroxide, aluminum potassium sulfate, etc.

[0106] A carrier that may be included in the vaccine composition of the present invention may be a pharmaceutically acceptable carrier, and examples of said pharmaceutically acceptable carriers include saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc.

[0107] The vaccine composition of the present invention may further include preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, inert gases, etc. The preservatives include formalin, thimerosal, neomycin, polymyxin B, and amphotericin B, etc. The vaccine composition of the present invention may include one or more suitable emulsifiers, such as Span or Tween. Additionally, the vaccine composition of the present invention may include a protective agent, and protective agents known in the art may be used without limitation, which may include, but are not limited to, lactose (LPGG) or trehalose (TPGG).

[0108] In a specific embodiment of the present invention, it is preferable to administer the vaccine composition 1 to 3 times at intervals of 1 to 3 weeks, and more preferable to administer it 2 times at intervals of 2 weeks.

[0109] In a specific embodiment of the present invention, the vaccine composition may be formulated using a method known in the art to enable rapid release, or sustained or delayed release, of the active ingredient when administered to a mammal. The formulations include powder, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.

[0110] In a specific embodiment of the present invention, the vaccine composition may be administered by one or more routes selected from the group consisting of intramuscular, intravenous, subcutaneous, intradermal, and intranasal.

[0111] In a specific embodiment of the present invention, the vaccine composition may be administered by a method of applying physical stimulation to increase delivery efficiency upon administration, examples of which include electroporation, gene gun, and jet injection methods.

[0112] The vaccine composition according to the present invention can be formulated into a suitable form with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oil, saline solution, aqueous glucose and glycol, and may additionally include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Additionally, the vaccine composition according to the present invention may appropriately include, if necessary depending on the method of administration or formulation, a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, an analgesic agent, a buffer, an antioxidant, etc.

[0113] The dosage of the above vaccine composition to a patient varies depending on many factors, including the patient's height, body surface area, age, specific compound administered, gender, time and route of administration, general health, and other drugs administered concurrently.

[0114] In addition, the vaccine composition of the present invention is administered in a therapeutically effective amount.

[0115] In the present invention, a therapeutically effective amount means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined according to factors including individual type and severity, age, sex, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The dosage of the vaccine composition of the present invention is in the range of 0.0001 to 1,000 mg / kg (body weight) per day, but may vary depending on the individual type and is not limited thereto.

[0116] Meanwhile, the above dosage can be appropriately adjusted according to the patient's age, gender, and condition.

[0117] The optimal dosage of the vaccine composition of the present invention can be determined by standard studies including the observation of an appropriate immune response in subjects. After initial vaccination, subjects may be treated with one or several booster immunizations at appropriate intervals.

[0118] The suitable dosage of the vaccine composition of the present invention varies depending on factors such as the formulation method, method of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response responsiveness, and can be determined by a person skilled in the art by taking the above into consideration.

[0119] The present invention provides the use of the variant protein or a fragment thereof in the manufacture of a drug for the prevention or treatment of influenza virus infection disease.

[0120] The present invention provides the use of the variant protein or a fragment thereof in the manufacture of a drug for the prevention or treatment of influenza virus infection.

[0121] The present invention provides a vaccine composition comprising the variant protein or a fragment thereof for the prevention or treatment of influenza virus infection.

[0122] The present invention provides a vaccine composition comprising the variant protein or a fragment thereof for the prevention or treatment of influenza virus infection.

[0123] The present invention provides a vaccine composition comprising the variant protein or a fragment thereof for the prevention or treatment of influenza virus infection.

[0124] As another preferred embodiment for achieving the above-mentioned purpose, the present invention provides a method for producing a monoclonal antibody that specifically binds to an influenza virus, comprising the steps of: (a) injecting a variant protein or a fragment thereof, comprising amino acid substitutions at the 95th and 103rd residues of the ectodomain HA2 subunit protein of the influenza virus hemagglutinin into an animal; and (b) recovering an antibody from the animal.

[0125] As another preferred embodiment for achieving the above-mentioned purpose, the present invention provides a method for producing a monoclonal antibody that specifically binds to an influenza virus, comprising the steps of: (a) injecting a variant protein or a fragment thereof, comprising amino acid substitutions at the 95th and 103rd residues of the ectodomain HA2 subunit protein of the influenza virus hemagglutinin, into an animal other than a human; and (b) recovering the antibody from the animal.

[0126] The introduction of the above protein or a fragment thereof can be performed by injecting it into the skin, subcutaneous tissue, or muscle tissue.

[0127] The term "antibody" refers to a component produced within the immune system in response to antigenic stimulation; it is a protein that specifically binds to a particular antigen, circulates in the lymph and blood, and triggers an antigen-antibody reaction. Antigen-antibody reactions exhibit high specificity for each antigen. This means that when antibodies are produced by B cells of lymphocytes, antibodies generated by a specific antigen do not, in principle, react with other antigens. This high specificity is utilized in tests for immunity, allergies, and determining the type and form of various diseases and infections.

[0128] In this case, the animal may be a mammal. Examples include, but are not limited to, humans, rats, mice, hamsters, pigs, rabbits, horses, donkeys, goats, sheep, guinea pigs, llamas, etc. For example, mice are preferred to be used to produce monoclonal antibodies.

[0129] In one embodiment, a neutralizing antibody produced in a non-human mammal can be converted into a humanizing antibody. In this case, the method of the present invention may further include the step of humanizing the neutralizing antibody produced in a non-human mammal.

[0130] The neutralizing antibody of the present invention can be used to prevent or treat diseases caused by viral infection in mammals. The neutralizing antibody is used in a dose sufficient to neutralize the virus.

[0131] In addition, the present invention provides an antibody or a fragment thereof that specifically binds to a protein or a fragment thereof of the ectodomain HA2 subunit of influenza virus hemagglutinin.

[0132] Meanwhile, it is important to possess multiple sets of stabilized mutations to prepare for pandemics against influenza and other viruses with unpredictable sequence variations.

[0133] To this end, the present invention provides a high-resolution cryo-EM method for identifying additional key residues for optimization to impart a stable prepolymerized structure as a protein fragment of the influenza virus.

[0134] Previous studies on the pre-fusion structure of HA rely primarily on X-ray crystallography, which requires crystallizing protein samples at high protein concentrations under non-physiological conditions containing high concentrations of precipitating reagents such as ammonium sulfate or polyethylene glycol. These conditions can lead to erroneous conclusions because they induce the ternation of monomeric proteins under physiologically more suitable protein concentrations and buffering conditions. Furthermore, as shown in Figure 2C, low-resolution techniques such as negatively stained EM have limitations in confirming the stabilization of the pre-fusion structure because they cannot detect small but significant structural deviations of the protein.

[0135] On the other hand, the present invention has confirmed that high-resolution cryo-EM is useful for definitive structural confirmation. In particular, the potential threat of an influenza pandemic caused by the H5N1 avian influenza virus highlights the urgency of preparedness efforts, and the method according to the present invention can rapidly redesign new virus strains with significant sequence variations using high-resolution cryo-EM and computational tools such as MPNN programs.

[0136] The variant protein or its fragment according to the present invention has a stable pre-fusion structure, which offers the advantage of enabling rapid preparation against influenza viruses with unpredictable sequence variations each year. Furthermore, the variant protein or its fragment according to the present invention possesses high neutralizing ability, thereby exhibiting efficacy as a vaccine through excellent defense capabilities against influenza viruses. In particular, through sequence optimization, it is possible to efficiently produce a protein with a stable structure and excellent protein expression levels, along with reduced production costs.

[0137] Figure 1 is a diagram showing the cryo-EM data processing process of H1 / Cal09-mut (A: Representative microscopic image, B: Selected 2D class mean, C: Cryo-EM data processing summary, D: Orientation distribution of particles used for 3D electron density reconstruction, E: FSC curve map).

[0138] Figure 2 illustrates the cryo-EM data processing process of H1 / Mal07-mut (A: Representative microscopic image, B: Selected 2D class mean, C: Cryo-EM data processing summary, D: Orientation distribution of particles used for 3D electron density reconstruction, E: FSC curve map).

[0139] Figure 3 illustrates the cryo-EM data processing process of H9 / HK98-mut (A: Representative microscopic image, B: Selected 2D class mean, C: Cryo-EM data processing summary, D: Orientation distribution of particles used for 3D electron density reconstruction, E: FSC curve map).

[0140] Figure 4 illustrates the trimerization status of the HA ectodomain of the A / California / 04 / 2009 (H1N1) virus (A: Domain structure of the HA ectodomain, B: Chromatographic elution profile of the A / California / 04 / 2009 (H1N1) (H1 / Cal09) HA ectodomain; predicted positions of peaks for trimer and monomeric proteins are indicated by arrows and labeled T and M, respectively. C: 2D class mean cryo-EM map of the H1 / Cal09 HA ectodomain).

[0141] Figure 5 illustrates the trimerization state of the Foldon-tagged ectodomain of the HA protein of the H1 / Cal09 virus (A: Domain structure of the HA ectodomain, B: Chromatographic elution profile of the Foldon-tagged H1 / Cal09 HA ectodomain; predicted positions of peaks for trimer and monomeric proteins are indicated by arrows and labeled T and M, respectively. C: 2D class-averaged cryo-EM map of the H1 / Cal09 HA ectodomain tagged with the Foldon sequence).

[0142] Figure 6 is a diagram showing the locations of computationally optimized amino acid sites (A: Structure of one of the monomers of the HA trimer; mutated amino acids are indicated by red dots, B: Structure of the HA trimer; the view is rotated approximately 30 degrees along the vertical axis).

[0143] Figure 7 is a close-up of the computationally optimized site (A: Close-up of E47 site, B: Close-up of N95 site, C: Close-up of L102 and E103 sites; D: Close-up of D109 site; a top view looking down at the central stem helix. Mutated residues are entered in red text, side chains of the wild-type structure are drawn in dark gray, and side chains of the mutated protein structure are drawn in red. Amino acid residues interacting with the mutated residues are drawn in green, and one of the three HA subunits of the HA triplicate is indicated).

[0144] Figure 8 shows the trimerization state and thermal stability of the H1 / Cal09 HA exoskeleton domain wild-type and mutant (A: Gel permeation chromatography elution profiles of wild-type (WT), wild-type mutant with Foldon tag (WT(tri)), and mutant protein with Foldon tag (mut) at three different pH values; B: Differential scanning fluorescence profiles of wild-type and mutant HA proteins).

[0145] Figure 9 shows the cryo-EM structure of the mutated H1 / Cal09 HA ectodomain (A: 2D class mean cryo-EM image of the mutated HA ectodomain, B: Reconstructed cryo-EM map of the mutated HA ectodomain, C: Close-up view of the mutation site; side chains of the wild-type structure with PDB id 7MEM are shown in blue, and side chains of the mutated protein determined by cryo-EM are shown in red, D: Another close-up of the mutated amino acid site; side chains of the mutated protein predicted by AlphaFold 2 are shown in gray, and side chains of the mutated protein determined by cryo-EM are shown in red).

[0146] Figure 10 shows the sequence alignment of the HA ectododomains of H1 / Mal07 and H1 / Cal09. The boundaries of the HA1 and HA2 chains are indicated by arrows, and the computationally optimized amino acid sites are highlighted in red boxes.

[0147] Figure 11 shows the trimerization state and temperature stability of the A / Malaysia / 1842338 / 07 (H1N1) (H1 / Mal07) HA exoskeleton domain wild-type and mutant (A: Gel permeation chromatography elution profiles of wild-type, Foldon-tagged wild-type, mutant, and Foldon-tagged mutant proteins at pH 8.0 and 5.5, B: Differential scanning fluorescence profiles of these proteins).

[0148] Figure 12 shows the cryo-EM structure of the mutated H1 / Mal07 HA ectodomain (A: 2D class mean cryo-EM image of the mutated HA ectodomain, B: 3D reconstructed cryo-EM map of the mutated HA ectodomain (compared to the X-ray crystallographic structure of the wild-type protein with PDB ID 7UMM and the mutant structure predicted by AlphaFold 3), C: Zoom in on the mutant site; side chains of the wild-type protein determined by X-ray crystallography are shown in gray, and side chains of the mutant residue are shown in red, D: Zoom in on another mutant site; side chains of the mutant protein predicted by AlphaFold 2 are shown in gray, and side chains of the mutant residue are shown in red).

[0149] Figure 13 shows the sequence alignment of the HA ectododomains of H9 / HK98 and H1 / Cal09. The boundaries of the HA1 and HA2 chains are indicated by arrows, and the computationally optimized amino acid sites are highlighted in red boxes.

[0150] Figure 14 illustrates the trimerization state, thermal stability, and cryo-EM structure of the mutated A / swine / Hong Kong / 2106 / 98 (H9N2) (H9 / HK98) HA ectodomain (A: Summary of the stabilizing mutation, B: Gel permeation chromatography elution profiles of wild-type and mutant proteins at three different pH levels, C: Differential scanning fluorescence profiles of wild-type and mutant ectodomains at three different pH levels; Tm values ​​of the proteins were recorded, D: 2D class mean cryo-EM map of the mutated HA ectodomain, E: Reconstructed cryo-EM map of the mutated HA ectodomain (structural comparison of the cryo-EM structure of the mutant protein with the X-ray crystallography of the wild-type protein (PDB ID: 1JSD) and the mutant structure predicted by AlphaFold 3), F: Zoom in on the mutation site; the side chain structure of the wild-type protein determined by X-ray crystallography is shown in gray, The side chains of the mutant protein determined by Cryo-EM are shown in red).

[0151] Figure 15 is a figure showing the sequence alignment of the HA ectododomains of H5 and H1 / Cal09. The boundaries of the HA1 and HA2 chains are indicated by arrows, and the computationally optimized amino acid sites are highlighted in red boxes.

[0152] Figure 16 is a figure showing the sequence alignment of the HA ectododomains of H6 and H1 / Cal09. The boundaries of the HA1 and HA2 chains are indicated by arrows, and the computationally optimized amino acid sites are highlighted in red boxes.

[0153] Figure 17 is a figure showing the sequence alignment of the HA ectododomains of H8 and H1 / Cal09. The boundaries of the HA1 and HA2 chains are indicated by arrows, and the computationally optimized amino acid sites are highlighted in red boxes.

[0154] Figure 18 is a figure showing the sequence alignment of the HA ectododomains of H12 and H1 / Cal09. The boundaries of the HA1 and HA2 chains are indicated by arrows, and the computationally optimized amino acid sites are highlighted in red boxes.

[0155] Figure 19 shows the sequence alignment of the HA ectododomains of H13 and H1 / Cal09. The boundaries of the HA1 and HA2 chains are indicated by arrows, and the computationally optimized amino acid sites are highlighted in red boxes.

[0156] Figure 20 is a figure showing the sequence alignment of the HA ectododomains of H18 and H1 / Cal09. The boundaries of the HA1 and HA2 chains are indicated by arrows, and the computationally optimized amino acid sites are highlighted in red boxes.

[0157] Figure 21 is a figure showing the trimerization status of the soft-mutated group 1 HA ectodomain (A: Summary of mutations introduced into the ectodomain of group 1 HA protein; no change in nc, B: Gel permeation elution profile of the ectodomain of group 1 influenza HA protein; predicted positions of the trimer and monomeric proteins are indicated as 'T' for the trimer and 'M' for the monomer, respectively).

[0158] Figure 22 shows the immune response in mice immunized with H1 / Mal07 HA antigen (a: schematic diagram of the viral infection experiment, b: results of antibody titer measurement via ELISA).

[0159] Figure 23 shows the results of viral infection in mice immunized with H1 / Mal07 HA antigen (a: results of survival rate measurement after viral infection, b: results of body weight recovery rate measurement after viral infection).

[0160] The present invention will be described in more detail below through examples. These examples are solely for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0161] Experimental method

[0162] 1. Expression and purification of H1 / Cal09 and H9

[0163] Genes encoding the ectodomains of H1 / Cal09 HA and H9 / HK98 HA were synthesized (Twist Bioscience) and replicated into a pEG BacMam baculovirus delivery vector.

[0164] Subsequently, a PreScission protease site, mCherry, a thrombin cleavage site (mCherry), and an ALFA tag were added to the C-terminus of HA. The plasmid was transformed into DH10Bac E. coli (ThermoFisher) and transposed into bacmid.

[0165] Recombinant baculovirus was produced by infecting Sf9 insect cells with bacmid DNA. Proteins were expressed in Expi293F cells cultured in Expi293 media (ThermoFisher) in an 8% CO2 stirred incubator. 10% (v / v) baculovirus was administered to the cells at a dose of 3 x 10⁶ 6 Infection was induced by treatment at a density of cells / mL. After 18–22 hours of infection, protein expression was increased by treatment with 10 mM sodium butyrate (Sigma-Aldrich), and the cells were cultured for an additional 5–7 days. The supernatant was collected by centrifugation at 4,500 rpm for 20 minutes and loaded onto a column packed with agarose resin conjugated with anti-ALFA nanobody.

[0166] The anti-ALFA nanobody was conjugated to NHS-activated Sepharose 4 resin (Cytiva) according to the manufacturer's protocol. A total of 20 μmol of anti-ALFA nanobody was conjugated to 1 mL of NHS-activated Sepharose 4 resin, and the unreacted NHS resin was blocked using 20 mM Tris-HCl pH 8.0. The anti-ALFA nanobody was washed with 20 column volumes of wash buffer containing 20 mM Tris-HCl pH 8.0 and 200 mM NaCl.

[0167] Subsequently, the protein was eluted by treating with 3% (w / w) thrombin (Lee Biosolutions) overnight. The eluted protein solution was concentrated using an ultracentrifugal filter (Merck Milipore) equipped with a 30 kDa barrier. The protein was further purified using a Superdex 200 increase 10 / 300 GL gel filtration column (Cytiva) equilibrated with a buffer containing 20 mM Tris-HCl and 200 mM NaCl at pH 8.0.

[0168] 2. Expression and Purification of H1 / Mal07 and Other Group 1 HAs

[0169] The gene encoding the ectodomain of HA for H5 and H9 was synthesized at Gene Universal, and the other subtype was synthesized at Twist Bioscience, and all were replicated into the pAcGP67a baculovirus delivery vector (ThermoFisher).

[0170] Subsequently, recombinant baculovirus was generated by co-infecting Sf9 insect cells with the linearized baculovirus genome, BestBac2.0 (Expression Systems). Proteins were expressed in Hi-Five insect cells cultured in ESF 921 medium (Expression Systems) with the addition of 4% (v / v) baculovirus for 72 hours at 21°C. The secreted proteins were bound to a cOmplete His-Tag Purification column (Roche) and eluted using an elution buffer containing 100 mM to 500 mM imidazole at pH 8.0, 20 mM Tris-HCl, and 200 mM NaCl. The proteins were further purified using a Superdex 200 increase 10 / 300 GL gel filtration column (Cytiva) equilibrated with a buffer containing 20 mM Tris-HCl and 200 mM NaCl at pH 8.0.

[0171] 3. Preparation of cryo-EM grids

[0172] Samples of purified HA were prepared by quenching at a concentration of 0.8–1.0 mg / mL. 300-mesh UltraAuFoil R 1.2 / 1.3 EM grids (Structure Probe) were discharged at 15 mA for 60 seconds using a glow-discharger (PELCO). All samples were prepared by applying 3.2 μl of sample to the EM grid, blotting for 4 seconds using a Vitrobot Mark IV (ThermoFisher) at 4°C and 100% humidity, waiting for 30 seconds, and then quenching in liquid ethane.

[0173] 4. Cryo-EM 2D Classification Analysis

[0174] Cryo-EM image files were preprocessed using patch motion correction and patch contrast transfer function (CTF) methods. The preprocessed data was imported into the CryoSPARC program with an upsampling factor of 1, after which patch motion correction and patch CTF estimation were performed. Micrographs of poor quality were manually filtered based on CTF estimates, ice thickness, and overall frame motion. Initial particles were selected using the Topaz model and refined using the 2D class mean method. High-quality 2D class means were selected to generate the Topaz picking model. The particle picking method was further optimized by repeating 2–3 rounds of Topaz analysis and multiple rounds of 2D classification. All cryo-EM data processing was performed using the cryoSPARC v4.6.0 program.

[0175] 5. Cryo-EM Data Processing

[0176] As shown in Figure 1, for the data processing of the trimeric H1 / Cal09-mut, particles were extracted with a box size of 320 pixels and binned to 100 pixels. The initial model was generated using Ab-initio reconstruction with C1 symmetry, followed by particle recentering and debinding. The electron density map was enhanced through non-uniform segmentation using C3 symmetry. Noise in the final map was reduced using the DeepEMhancer program, a deep learning-based automatic sharpening method.

[0177] As shown in Figure 2, for the data processing of trimeric H1 / Mal07-mut, particles were extracted with a box size of 420 pixels and binned to 100 pixels. The initial model was generated using Ab-initio reconstruction with C1 symmetry, followed by particle recentering and debinding. To obtain a more refined electron density map, additional particle extraction was performed using Topaz training and Topaz extraction, followed by map refinement through non-uniform refinement using C3 symmetry. Noise in the final map was reduced using the DeepEMhancer program.

[0178] As shown in Figure 3, for the data processing of the trimeric H9 / HK98 HA mutation, particles were extracted with a box size of 420 pixels and binned to 100-pixel units. The initial model was generated using Ab-initio reconstruction with C1 symmetry, followed by particle recentering and debinding. To obtain a more refined electron density map, additional particle extraction was performed using Topaz training and Topaz extraction, after which the map was refined through non-uniform refinement using C3 symmetry. Noise in the final map was reduced using the DeepEMhancer program. All cryo-EM data processing was performed using the cryoSPARC v4.6.0 program.

[0179] 6. Model Construction

[0180] The initial model for the H1 / Cal09-mut mutation was generated using AlphaFold2, and models for the H1 / Mal07-mut and H9 / HK98-mut mutations were generated using AlphaFold3. These models were fitted to a cryo-EM density map using the ChimeraX program. The resulting structure was improved through multiple manual model builds using the Coot program and real-space segmentation using the Phenix program.

[0181] 7. Differential Scanning Fluorometry and Gel Permeation Chromatography

[0182] The melting temperature of the purified HA protein was measured using DSF with a Prometheus NT.48 (NanoTemper). Samples were placed in DSF capillaries and exposed to thermal stress from 25°C to 95°C at a beam ramping rate of 1°C / min. Fluorescence emission of tryptophan after UV excitation at 280 nm was collected at 330 nm and 350 nm using a dual UV detector. Thermal stability parameters, including toneset and Tm, were calculated using PR.ThermControl software (NanoTemper).

[0183] For gel permeation chromatography, purified HA protein was diluted to 0.2 mg / mL in pH 4.7 and pH 5.5 sodium acetate buffer (20 mM Na Acetate, 75 mM NaCl) and 20 mM Tris, 200 mM NaCl at pH 8.0. After incubating the protein at 4°C for 30 minutes, it was loaded onto Superdex 200 increase 10 / 300 or Superdex 200 increase 5 / 150.

[0184] 8. Preparation of Immunized Mouse Model and Virus Challenge Experiment

[0185] BALB / c mice (6 weeks old, female) were purchased from Koatech (Korea). To prepare an immunized mouse model, HA protein was subcutaneously injected into the right flank of mouse groups of 6 mice each, at 3-week intervals for a total of 2 times. The HA protein was dissolved in 50 μl of PBS at a dose of 3 μg per injection, mixed with 50 μl of Sigma Adjuvant System, and administered.

[0186] Blood samples were collected via retro-orbital plexus puncture before the first immunization and 2 weeks after the first and second immunizations. The collected blood was centrifuged at 3000 rpm for 10 minutes to separate the serum, and then stored at -80℃ for ELISA analysis.

[0187] In the viral infection (challenge) experiment, 3 days after the second blood draw, each mouse group was given 5 times the 50% lethal dose (5xLD) of the mouse-adapted pandemic H1N1 virus (A / Korea / 2785 / 2009; National Pathogen Resource Bank, Korea). 50 The experiment was conducted by infecting the nasal cavity with ). The body weight and survival rate of the mouse group were observed daily, and mice that lost more than 20% of their initial body weight were euthanized humanely. All animal experiments were conducted in accordance with relevant guidelines under the approval of the Institutional Animal Care and Use Committee (IACUC) of the Vaccine Commercialization Technology Support Center (ICV) under the Gyeongbuk Bioindustry Research Institute (GIB).

[0188] 9. Enzyme Immunoassay (ELISA)

[0189] Serum concentrations of HA-specific antibodies were measured via ELISA analysis. Immunogenicity was evaluated using serum collected from the retro-orbital venous plexus. 96-well microtiter plates (Komabiotech, Korea) were coated with 90 μl of recombinant HA protein at a concentration of 5 μg / ml dissolved in coating buffer (pH 9.6; Komabiotech, Korea) and incubated overnight at 4°C. Subsequently, the plates were washed three times with wash buffer (1 x PBS containing 0.05% Tween-20) and then blocked with PBS solution containing 2% BSA at room temperature for 2 hours. Serum was initially diluted 1:100 in PBS solution containing 0.1% BSA, followed by serial dilutions in tenfold increments. Diluted serum was added as a duplicate to the BSA-blocked wells, and the mixture was incubated at room temperature for 2 hours using a shaker. Subsequently, the plates were washed three times with wash buffer, and HRP (horseradish peroxidase)-conjugated goat anti-mouse IgG (GeneTex, USA) was added after being diluted 1:5000 in PBS solution containing 0.1% BSA. The plates were incubated at room temperature for 1 hour and washed three times, after which color development was initiated with 100 μl of TMB substrate solution (Komabiotech, Korea). After 5 minutes, the reaction was stopped by adding 100 μl of 0.5 M sulfuric acid, and the absorbance was measured at 450 nm using a microplate reader (Molecular Devices, USA). The endpoint titer was defined as the highest serum dilution factor that produced at least twice the ELISA signal compared to pre-immune sera diluted 1:100.

[0190] Example 1. Determination of the structure of influenza hemagglutinin

[0191] The structure of the pre-fusion form of hemagglutinin (HA) from human group 1 influenza was used as the structural input for Protein MPNN, and five amino acid positions selected at the trimerization interface were redesigned. Outputs were generated for Protein MPNN computation using a temperature of 0.1 and random seeds. 10,000 sequences were generated by applying ternary symmetry. The three sequences with the lowest scores were selected for structure prediction and manual verification. The structures were predicted by AlphaFold2 after undergoing five recycling steps. The best model predicted by AlphaFold2 was manually evaluated and selected for experimental testing.

[0192] Specifically, to determine the structure of HA, the wild-type ectodomain of influenza strain A / California / 04 / 2009 (H1N1) (H1 / Cal09) was produced using Genbank ID ACP41105 as shown in Fig. 4 (A). Unexpectedly, the purified protein eluted as a monomer during gel permeation chromatography, indicating that a stable trimer pre-fusion structure may not be formed (B). 2D classification analysis using cryo-EM confirmed that the ectodomain of H1 / Cal09 HA exists mainly as a monomer (C).

[0193] As shown in Fig. 5, a Foldon triplication sequence was attached to the C-terminus of the HA ectodomain to enhance the formation of the pre-fusion triplicate structure (A). This is a short sequence consisting of 12 amino acids derived from the triplication domain of T4 fibrin. Due to its highly stable triplicate structure and rapid folding kinetics, the Foldon can induce triplication of the host protein. As expected, this modification to the HA ectodomain shifted the protein's gel permeation chromatography elution profile from monomer to trimer (B).

[0194] However, after collecting the trimer fraction and examining its structure using cryo-EM, it was discovered that the protein is actually a trimer but does not have a stable structure and high-resolution 3D reconstruction of the structure is impossible (C).

[0195] These results indicate that trimerization induced by the poldon sequence tag does not guarantee the formation of a pre-fusion structure of the H1 / Cal09 HA protein.

[0196] Example 2. Selection of Mutation Sites and Sequence Optimization via Computational Design

[0197] 1. Selection of mutation sites through computational design

[0198] To stabilize the pre-trimeric fusion structure of H1 / Cal09 HA with minimal mutation, the trimerization interface formed by the central stem alpha helices of the previously reported structure as shown in Fig. 6 was investigated using PDB id 7MEM. As shown in Fig. 7, the following five amino acid residues that do not appear optimal for the stable ternation of the HA monomer were identified.

[0199] The negatively charged E47 of the HA2 chain is located near the hydrophobic L30 of the adjacent subunit. The hydrophilic N95 is located in the center of the hydrophobic core of the trimerization interface and is surrounded by I91, W92, Y94, L98, and L99. Additionally, the hydrophobic L102 is flanked by the charged residues E103 and R106, with the charged E103 in contact with the hydrophobic L102. Finally, D109 is located at the periphery of the trimerization interface. At this position, the longer amino acid appears capable of enhancing interactions with the other two subunits of the trimerization interface. Residues N95, L102, E103, and D109 are located in the central stem alpha helices and are directly involved in trimerization. Although E47 is not in the trimerization interface, it is part of the dimerization interface as it mediates interactions with the adjacent HA monomer.

[0200] These five amino acids underwent computational optimization using the protein design program Protein MPNN 16. The previously reported structure of H1 / Cal09 HA was used as a structural template for the computation. This deep learning-based program calculates the amino acid sequence that best fits the backbone structure provided as a template. After a rapid optimization run, replacing E47 with Gly, N95 with Leu, L102 with Phe, E103 with Leu, and D109 with Glu significantly lowered the computational score. In the context of Protein MPNN, a lower score indicates a more stable mutation associated with lower free energy.

[0201] 2. Stabilization of the pre-fusion structure of H1 / Cal09 HA protein

[0202] To test the stabilization effect of sequence optimization, wild-type ectodomain (H1 / Cal09-WT), wild-type (H1 / Cal09-WT(tri)) with a poldon triplet tag, and mutant (H1 / Cal09-mut) proteins with five mutations were produced, and their specific sequences are shown in Tables 2 and 3 below.

[0203]

[0204] Underline: signal peptide

[0205] Bold: Foldon tag

[0206] Bold and Underline: mutation

[0207]

[0208]

[0209]

[0210] These proteins were analyzed using gel permeation chromatography to evaluate their trimerization status at three different pH levels. As shown in Figure 8A, at both pH 8.0 and 5.5, wild-type HA eluted as a monomer, whereas H1 / Cal09-WT(tri) and five mutant variants, H1 / Cal09-mut, eluted as trimers. At pH 4.7, all three proteins exhibited instability, with less than half of the protein injected into the column eluted, suggesting that a significant portion aggregated in the column and remained uneluted. Meanwhile, to evaluate the thermal stability of the wild-type and mutant proteins, the protein unfolding temperature (Tm) was measured using differential scanning fluorescence (DSF). This method monitored protein folding through intrinsic tryptophan fluorescence. As shown in Figure 8B, the poldon sequence tag (WT(tri)) stabilized at only 3–4°C in terms of thermal stability at all three pH levels. However, the mutant improved the stability of the HA protein by about 10°C. These results indicate that mutations at the triplet interface stabilize the protein structure and promote triplet. The structures of the five mutant HA proteins were analyzed using high-resolution cryo-EM and compared with the pre-fusion structure of H1 / Cal09, previously determined by the PDB ID 7MEM. As a result, it was determined that the antibody bound to the 7MEM structure stabilizes the pre-fusion structure of the HA protein.

[0211] As shown in Fig. 9, the cryo-EM structure of the stabilized mutant at 2.2 Å resolution was nearly identical to the pre-fusion structure of the wild-type stabilized by the bound antibody (A and B). A close examination of the mutation site reveals that the mutation did not disrupt the overall structure of the protein, and the side chains of the mutant residues fit perfectly into the structure, requiring only minor adjustments (C). Additionally, the mutant structure predicted by AlphaFold3 closely matched the cryo-EM structure (D). In conclusion, the above 5-site mutation strategy effectively stabilized the pre-fusion structure of the H1 / Cal09 HA protein.

[0212] 3. A / Malaysia / 1842338 / 2007 (H1N1) Pre-fusion Structure Stabilization of HA Protein

[0213] To determine whether the 5-site mutation strategy is applicable to other H1 HA proteins, the mutation was applied to the A / Malaysia / 1842338 / 2007 (H1N1) (H1 / Mal07) HA protein.

[0214] The H1 / Cal09-WT, H1 / Cal09-WT(tri), H1 / Mal07-WT, and H1 / Mal07-WT(tri) datasets were collected using a 300 kV Titan Krios microscope equipped with an energy filter and a Falcon4i direct electron detector (ThermoFisher) operating in a counting mode of 130,000x magnification with a pixel size of 0.938 Å.

[0215] The H1 / Cal09-mut, H1 / Mal07-5mut, and H9 / HK98-mut HA datasets were collected using a 300 kV Titan Krios microscope (ThermoFisher) equipped with a K3 direct electron detector (Gatan) operating in energy filter and counting mode. The H1 / cal09-mut data was collected at 105,000x magnification operating with a pixel size of 0.85 Å, whereas the other datasets were collected at 135,000x magnification with a pixel size of 0.651 Å.

[0216] Each cryo-EM image has a total dose of 70 e / Å. 2 It was recorded in the raw tag image file format (TIFF). All datasets were automatically collected using EPU software (ThermoFisher) for single-particle analysis.

[0217] Data collection statistics are summarized in Tables 4 and 5.

[0218]

[0219]

[0220] Meanwhile, as shown in Figure 10, the HA protein sequences of H1 / Cal09 and H1 / Mal07 exhibited 79% sequence identity. Sequence variations were mainly concentrated in the head region, whereas the stem region was highly conserved. Of the five regions selected for mutation, four—N95, L102, E103, and D109—were conserved in the H1 / Mal07 HA protein. Interestingly, the remaining site, E47, was changed to glycine in the H1 / Mal07 HA variant. Mutations occurred in four of these five conserved regions, similar to those in H1 / Cal09 HA. The E47 site was not altered because it is occupied by glycine in both the wild-type sequence and the sequence predicted by the protein MPNN. Wild-type HA protein (H1 / Mal07-WT), wild-type protein with a poldon tag (H1 / Mal07-WT(tri)), mutant protein (H1 / Mal07-mut), and mutant protein with a poldon tag (H1 / Mal07-mut(tri)) were constructed, and their specific sequences are shown in Tables 6 and 7 below. Their ternary states were evaluated using gel permeation chromatography.

[0221]

[0222]

[0223] Underline: signal peptide

[0224] Bold: Foldon tag

[0225] Bold and Underline: mutation

[0226]

[0227]

[0228]

[0229]

[0230] As shown in Figure 11, the wild-type ectodomain of the H1 / Mal07 HA protein eluted as a mixture of trimers and monomers. The addition of a poldon tag promoted the trimerization of the wild-type protein. Meanwhile, the mutant protein without a poldon tag eluted as a trimer, confirming that the 5-site mutation successfully stabilized the trimer structure. At low pH levels, the peaks of the wild-type and wild-type poldon-tagged proteins decreased due to aggregation, whereas the mutant protein mostly maintained a trimer state (A). Additionally, the thermal stability of the mutant protein was evaluated using the DSF method. At pH 8.0 and 5.5, the mutant protein exhibited higher Tm values, confirming that the protein structure was successfully stabilized through the 5-site mutation (B). Meanwhile, the structure of the mutant H1 / Mal07 HA protein without a poldon tag was analyzed at a resolution of 2.3 Å using Crio-EM.

[0231] As shown in Fig. 12, the 2D class mean map clearly revealed the trimer pre-fusion structure (A), and the purified structure could be superimposed with the structure of PDB id 7UMM at rmsd 1.06 Å (B). The sequence of 7UMM HA is 100% identical to the sequence of H1 / Mal07 HA, and a close-up of the area near the mutation site revealed that the overall structure of the protein was not disrupted by the mutation (C and D).

[0232] 4. Pre-fusion structure stabilization of A / swine / Hong Kong / 2106 / 98 (H9N2) HA protein

[0233] To evaluate whether the 5-site optimization strategy is applicable to proteins other than the H1 HA protein, it was applied to the HA protein of the H9 influenza virus A / swine / Hong Kong / 2106 / 98 (H9 / N2) (H9 / HK98).

[0234] As shown in Figure 13, alignment of the HA protein sequences of H1 / Cal09 and H9 / HK98 confirmed 49% sequence identity. Sequence variations were observed in both the head and stem regions, but fewer variations were found in the stem region. Four of the five regions we selected—N95, L102, E103, and D109—are conserved in H9 / HK98. The remaining E47 residue was changed to lysine in H9 HA.

[0235] Computer optimization methods were applied to these five regions using the protein MPNN program. The structure template used was the structure of the previously reported PDB ID 1JSD.

[0236] As shown in Figure 14A, only two of the five regions were optimized by the program. Wild type (WT), wild type with a poldon tag (WT-tri), mutant protein (mut), and mutant protein with a poldon tag (mut-tri) were produced, and their specific sequences are shown in Tables 8 and 9 below. Their triplication status was analyzed using gel permeation chromatography.

[0237]

[0238]

[0239] Underline: signal peptide

[0240] Bold: Foldon tag

[0241] Bold and Underline: mutation

[0242]

[0243]

[0244]

[0245]

[0246] As shown in Figure 14, the wild-type ectodomain of the H9 HA protein eluted as a mixture of trimers and monomers. The addition of a poldon tag promoted the triplication of the wild-type protein. The mutant protein without the poldon tag eluted as a trimer, confirming that the 5-site mutation successfully stabilized the trimer structure. Furthermore, at low pH levels, the peaks of both the wild-type and wild-type poldon proteins decreased, which is presumed to be due to aggregation. On the other hand, the mutant protein mostly maintained the trimer state, suggesting that the 5-site mutation enhanced resistance to pH shock (B). Additionally, the thermal stability of the mutant protein was measured using the DSF method. At pH 8.0, the addition of the poldon triplication tag increased the unfolding temperature to approximately 20°C. In addition, the mutation increased the Tm value by 5.9°C, confirming that the protein structure was successfully stabilized by the 5-site mutation (C). The structure of the mutated H9 / HK98 HA protein was evaluated using cryo-EM, and the structure was purified to a resolution of 1.9 Å. The 2D class mean map clearly showed the trimer pre-fusion structure (D). The purified structure overlapped with the previously reported pre-fusion structure of PDB ID 1JSD (E). A close examination near the mutation site reveals that the overall structure of the protein was not disrupted by the mutation (F).

[0247] 5. Other group 1: Stabilization of the trimer state of influenza HA protein

[0248] To evaluate whether the 5-site optimization strategy could stabilize the HA triplicate of other group 1 influenza viruses, particularly viruses other than H1 and H9, six HA proteins of H5, H6, H8, H12, H13, and H18 were redesigned as shown in Figures 15 to 20. The ectodomains of the HA proteins of A / Vietnam / 1194 / 2004 (H5N1), A / Taiwan / 2 / 2013 (H6N1), A / turkey / Ontario / 6118 / 1968 (H8N4), A / duck / Alberta / 60 / 1976 (H12N5), A / gull / Maryland / 704 / 1977 (H13N6), and A / flat-faced bat / Peru / 033 / 2010 (H18N11) viruses were used in the experiments. Five pre-selected amino acid residues in each HA protein were analyzed using the protein MPNN program, and the optimal mutation for this region was selected.

[0249] The specific sequence of this is shown in Tables 10 and 11 below.

[0250]

[0251]

[0252]

[0253]

[0254]

[0255] Underline: signal peptide

[0256] Bold and Underline: mutation

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269] As shown in Figure 21, the previously reported structures of PDB id, 4BGW, 5BR0, 6V46, 7A9D, 4KPQ, and 4K3X were used as structural templates for protein MPNN calculations (A). Then, both wild-type and mutant HA ectodomains were produced, and the trimerization state was analyzed using gel permeation chromatography at pH 8.0. The wild-type ectodomains of H5 and H18 HA proteins formed mainly monomers in solution. In contrast, the mutant proteins eluted as trimers, demonstrating that 5-site mutagenesis effectively stabilized the trimer structure. Meanwhile, for H8 and H13, the wild-type ectodomains appeared as a mixture of monomers and trimers in solution. The proteins after mutation were almost entirely trimers, and no monomer peaks were detected. For H6 and H12 HA proteins, the wild-type ectodomain is not produced in HEK293 cells, so only mutant proteins that elute mainly as trimers were analyzed (B).

[0270] These results indicate that the 5-site mutation not only stabilized the triplicate state but also enhanced protein expression in HEK293 cells.

[0271] In conclusion, it was confirmed that the 5-site optimization strategy effectively stabilizes the trimer pre-fusion structure of most group 1 HA proteins.

[0272] Example 3. Protective effect of mutated HA protein against viral infection

[0273] To confirm the protective effect of the mutated H1 / Mal07 HA protein against H1N1 virus infection, a virus infection experiment was performed.

[0274] Specifically, four mouse groups immunized with each antigen (H1 / Mal07-WT, H1 / Mal07-WT(tri), H1 / Mal07-mut, H1 / Mal07-mut(tri)) prepared in Examples 2-3 were intranasally infected with a lethal dose of H1N1 virus (A / Korea / 2785 / 2009) two weeks after the second immunization (Fig. 22a).

[0275] As a result of analyzing the antibody titers in the above mouse group by ELISA, it was confirmed that, with the exception of the antibody titer for the H1 / Mal07-WT(tri) antigen, the antibody titers for each antigen were generally induced at similar levels (Fig. 22b).

[0276] Next, the survival rate and body weight recovery rate after viral infection were analyzed for the non-infected control group (PBS+no virus), the infected control group (PBS+virus), and the experimental group of mice immunized with each antigen.

[0277] As a result, it was confirmed that only 2 out of 6 mice in the group immunized with wild-type HA antigen (HA WT+virus) survived, showing a survival rate of about 33%, whereas 5 out of 6 mice in the group immunized with mutant HA antigen (HA mut+virus) survived, showing a survival rate of about 83% (Fig. 23a).

[0278] Meanwhile, regarding body weight recovery rate, the mouse group immunized with the mutant HA antigen (HA mut+virus) rapidly recovered its body weight after decreasing by about 15% or less, reaching about 95% of its initial body weight within 8 days of infection. In contrast, the mouse group immunized with the wild-type HA antigen (HA WT+virus) showed a decrease in body weight of about 25%, and the recovery speed was also found to be relatively slower (Fig. 23b).

[0279] In summary, antibody titers against the mutated HA antigen were induced to a level similar to that of the wild type, but the survival rate and body weight recovery rate after viral infection were superior to those of the wild type antigen.

[0280] Therefore, through the above experiments, it was confirmed that the HA protein mutated according to the 5-site optimization strategy exhibited an enhanced protective effect against viral infection.

Claims

1. A variant protein or fragment thereof comprising amino acid substitutions at the 95th and 103rd residues of the ectodomain HA2 subunit protein of influenza virus hemagglutinin.

2. In paragraph 1, the influenza virus is a variant protein or a fragment thereof which is a Group 1 influenza virus.

3. In claim 1, the HA2 subunit protein of the hemagglutinin of the influenza virus is a variant protein or a fragment thereof, wherein the amino acid sequence is selected from any one of SEQ ID NOs 47 to 55.

4. A variant protein or a fragment thereof, wherein the amino acid substitution is substituted with leucine in claim 1.

5. The variant protein or fragment thereof according to claim 1, wherein the variant protein or fragment thereof further comprises an amino acid substitution at any one of the 47th, 102nd, and 109th residues.

6. A variant protein or a fragment thereof, wherein the amino acid substitution at the 47th residue is substituted with any one amino acid selected from glycine, tyrosine, isoleucine, and alanine.

7. A variant protein or a fragment thereof, wherein, in paragraph 5, the amino acid substitution at the 102nd residue is substituted with phenylalanine or leucine.

8. A variant protein or a fragment thereof, wherein the amino acid substitution at the 109th residue is substituted with any one amino acid selected from glutamic acid, asparagine, alanine, and serine.

9. A gene encoding a variant protein or a fragment thereof according to any one of paragraphs 1 through 8.

10. In claim 9, the gene is any one selected from the group consisting of SEQ ID NOs 6, 13 to 14, 21 to 22, 36, 38, 40, 42, 44 and 46.

11. A recombinant vector containing the gene of claim 9.

12. Host cells transformed with the recombinant vector of paragraph 11.

13. A vaccine composition for preventing or treating influenza virus infection disease comprising a variant protein or a fragment thereof according to any one of claims 1 to 8.

14. A vaccine composition according to claim 13, wherein the influenza virus is a type A influenza virus.

15. A vaccine composition according to claim 13, wherein the composition further comprises an immunoadjuvant, an excipient, or a carrier.

16. A vaccine composition according to claim 15, wherein the immune adjuvant is one or more selected from the group consisting of Proint complete and incomplete immune adjuvants; vitamin E; non-ionic blocking polymers; muramil dipeptide; Quil A; mineral oil and non-mineral oil; and Carbopol.

17. A method for preventing or treating an influenza virus infection, comprising the step of administering a vaccine composition containing a variant protein or a fragment thereof according to any one of claims 1 to 8 to a subject who requires it.

18. Use of a variant protein or a fragment thereof according to any one of claims 1 to 8 in the manufacture of a drug for the prevention or treatment of influenza virus infection.

19. A vaccine composition comprising a variant protein or a fragment thereof according to any one of claims 1 to 8 for the prevention or treatment of influenza virus infection disease.

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