Recombinant hemagglutinin protein derived from influenza virus surface protein forming trimer and its use

Abstract

The present invention relates to a recombinant hemagglutinin (HA) protein derived from an influenza virus surface protein that forms a trimer and uses thereof. Specifically, the present invention relates to a recombinant vector for producing an HA protein derived from an influenza virus surface protein that forms a trimer, a transformant transformed with the recombinant vector, a method for producing an HA protein derived from an influenza virus surface protein that forms a trimer using the recombinant vector, and uses of the HA protein derived from an influenza virus surface protein that forms a trimer produced by the method and for preventing, ameliorating, or treating influenza virus infection.

Description

[Technical field]

[0001] The present invention relates to a recombinant hemagglutinin (HA) protein derived from an influenza virus surface protein that forms a trimer and uses thereof. Specifically, the present invention relates to a recombinant vector for producing an HA protein derived from an influenza virus surface protein that forms a trimer, a transformant transformed with the recombinant vector, a method for producing an HA protein derived from an influenza virus surface protein that forms a trimer using the recombinant vector, and the HA protein derived from an influenza virus surface protein that forms a trimer produced by the method and uses thereof for preventing, ameliorating or treating influenza virus infection. [Background technology]

[0002] Recently, the possibility of producing recombinant proteins from plants at low cost has been proposed, and various attempts are being made. In particular, research is being conducted to confirm the possibility of producing various medical proteins. There are various advantages to producing recombinant proteins from plants, one of which is that there are almost no toxins such as endotoxins present in microorganisms such as E. coli, and there are no pathogens that can infect the human body. It is also known that there are no harmful proteins such as prions, and recombinant proteins can be produced that are safer than animal cells or microorganisms. The production cost is also much cheaper than animal cells, and depending on the cultivation method of plants, it is more economical than microorganisms such as E. coli for large-scale production. In order to realize this possibility, several essential technologies need to be developed. The most important technology is the development of expression vectors to induce high gene expression in plants. Gene expression can be induced in plants through various methods. Various methods are possible, such as introducing recombinant genes into the genome of the plant body, introducing them into the genome of chloroplasts, and expressing a transient gene using Agrobacterium. The method of fusing recombinant genes into the nuclear genome or chloroplast genome basically involves the production of proteins from plants through the process of obtaining transformants. On the other hand, when producing proteins by infiltrating Agrobacterium into plant tissues and inducing transient expression of genes, the protein production period is short because the process of producing transformants is not involved, and the protein production level is generally significantly higher than that of protein production by transformants. In addition, the expression level of proteins can be further increased because the expression suppression mechanisms of different genes in plants can be suppressed by co-infiltrating a gene silencing suppressor. However, there is a disadvantage that each time transient expression is performed, a culture of Agrobacterium introduced with a binary vector containing the target gene and a culture of Agrobacterium introduced with a binary vector expressing the p38 gene silencing suppressor must be separately prepared, and the two must be mixed in an appropriate ratio and co-infiltrated. In particular, there are limitations in terms of time and cost when culturing two types of Agrobacterium.

[0003] Influenza virus is an RNA virus belonging to the Orthomyxoviridae family that induces inflammation in the respiratory tract and is highly contagious, being transmitted directly through the air via the cough or saliva of an infected person, or indirectly through contact with objects of an influenza patient. The incubation period is about 24 to 30 hours, and the virus is classified into serotypes A, B, and C. Of these, types B and C have only been confirmed to infect humans, while type A has been confirmed to infect humans, horses, pigs, other mammals, and various types of poultry and wild birds. Therefore, it is necessary to develop a vaccine to prevent infection with such highly contagious influenza viruses.

[0004] Recombinant protein antigens as vaccines are safe to produce and use, but they have the disadvantage of being less immunogenic than live virus-based vaccines and generally have a high production cost. Therefore, in order to increase the efficacy of recombinant proteins as vaccines, it is essential to develop a recombinant protein vaccine delivery technology that can induce various immune responses and induce a high immune response. In addition, if multiple types of antigens can be delivered simultaneously, rather than just one type of antigen, this will be an even more effective vaccine. In fact, the recent trend is to develop multiple types of antigens as a single injection. The most effective way to increase the immunogenicity of an antigen is to use a strong adjuvant. If the efficacy of the adjuvant is high, it can effectively induce an immune response even with a small amount of antigen, which can reduce the price of the vaccine, so the development of a strong adjuvant is very important in the development of a protein-based vaccine. In addition, different types of adjuvants can induce different immune responses, so it is very important to use the appropriate adjuvant depending on the type of antigen. Currently, injectable adjuvants such as aluminum hydroxide have been developed and are used in humans, and cholera toxin B subunit (CTB) is used for oral vaccines. Many kinds of adjuvants have been developed and used for animals such as livestock. For experimental purposes, Freund's complete adjuvant (Freund's complete adjuvant) is often used for mice. However, it is not yet clear how these enhance the immunogenicity of antigens in humans, livestock, and experimental animals.

[0005] Many different types of adjuvants have been developed, and there are many different ways to deliver vaccines, so different types of adjuvants are required for each delivery method. Recently, much research has been conducted into using bacteria as oral vaccine delivery vehicles and adjuvants. In particular, Lactococcus is a bacterium that has been granted "Generally Recognized as Safe (GRAS) status" by the U.S. Food and Drug Administration (FDA) and is considered safe for the human body, and is being developed as an oral adjuvant and antigen delivery agent. Bacteria themselves are highly antigenic, and it has been reported that they produce a very high immune response to the antigens they deliver.

[0006] The full-length hemagglutinin (HA) protein is difficult to produce at high levels from cells because it is in a membrane-bound form with a transmembrane domain. On the other hand, if the ectodomain alone is expressed, without the transmembrane domain of HA, it is produced in a soluble form in cells and can be produced with high efficiency. However, if only the ectodomain of HA is produced in a soluble form, the trimer form that the original full-length HA has when it is present on the surface of the influenza virus is not often produced. We attempted to develop a technique to induce trimer formation when expressing and producing recombinant HA ectodomain proteins from plants for use in vaccine purposes. We also devised an HA ectodomain recombinant protein that can be made capable of binding to peptidoglycan and can be combined with genes that can bind to surfaces such as lactococci or chitosan particles to deliver antigens in various ways by giving the protein produced in this way the ability to bind to peptidoglycan and by combining them with genes that can bind to surfaces such as lactococci or chitosan particles. We constructed a binary vector that can express the recombinant HA gene produced in this way in plants at a high level. The HA recombinant protein highly expressed from the plant was administered to mice infected with influenza virus, and the therapeutic effect was confirmed. Furthermore, the HA recombinant protein antigen and CTB, which is said to greatly enhance immune response, were heated and treated with trichloroacetic acid to remove the water-soluble proteins and nucleic acids of the bacteria from GRAS-state lactococci, and then coated on the killed lactococcus cells. The mice were then treated with an injectable vaccine to infect influenza virus, and the therapeutic effect was confirmed to be increased. Furthermore, the soluble H5N6 HA trimer or the H5N6 HA trimer coated on the surface of killed lactococcus cells was administered to chickens infected with influenza virus, and a high immune effect was confirmed. CTB, soluble H5N6 HA trimer, and soluble H9N2 HA trimer were each coated on killed lactococcus cells, and mixed to prepare an immune composition. Analysis of hemagglutination revealed that the group containing CTB showed increased hemagglutination, confirming that CTB increases immunogenicity. A vaccine composition was prepared by mixing lactococci coated with H5N6 HA trimer and lactococci coated with H9N2 HA trimer in a 1:1 ratio, and then used to immunize mice, confirming that immunogenicity was increased.The H5N6 HA trimer and H9N2 HA trimer were mixed in a 1:1 ratio and coated onto lactococci. This was then prepared into a vaccine composition and injected into mice, confirming that immunogenicity was increased. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been devised to solve the above-mentioned problems, and an object of the present invention is to provide a recombinant vector for producing a recombinant influenza virus-derived HA protein that forms a trimer, comprising: (i) a gene encoding a protein lacking the transmembrane protein portion in influenza virus-derived HA (hemagglutinin); and (ii) a gene encoding a protein in the trimerization motif region of coronin 1.

[0008] Another object of the present invention is to provide a recombinant vector for producing a recombinant influenza virus-derived HA protein that forms a trimer, which is obtained by additionally inserting a gene encoding a LysM domain protein into the aforementioned recombinant vector.

[0009] Another object of the present invention is to provide a method for producing a recombinant HA protein from an influenza virus that forms a trimer in a plant, the method comprising the steps of: (a) producing the recombinant vector; (b) introducing the recombinant vector into a cell to produce a transformant; (c) culturing the transformant; and (d) infiltrating a culture of the transformant into a plant; and (e) crushing the plant to obtain a recombinant influenza virus HA protein that forms a trimer.

[0010] Another object of the present invention is to provide a trimer-forming recombinant influenza virus HA protein produced by the above-mentioned method.

[0011] Another object of the present invention is to provide a vaccine composition for preventing or treating influenza virus infections with increased immunogenicity, which comprises a recombinant HA protein derived from an influenza virus that forms a trimer as described above.

[0012] Another object of the present invention is to provide a vaccine composition for preventing or treating influenza virus infection with increased immunogenicity comprising a bacterium or chitosan encoding the recombinant HA protein derived from the aforementioned trimer-forming influenza virus.

[0013] Another object of the present invention is to provide a vaccine composition for preventing or treating influenza virus infection, which further comprises a cholera toxin B subunit in the above-mentioned vaccine composition, and has enhanced immunogenicity.

[0014] Another object of the present invention is to provide a vaccine composition for preventing or treating infections with influenza viruses of different genotypes, which contains recombinant HA proteins derived from two or more different influenza viruses that form the above-mentioned trimers and has increased immunogenicity.

[0015] Another object of the present invention is to provide a method for preventing or treating influenza virus infection, comprising administering the above-mentioned vaccine composition in various forms to an individual in need thereof. [Means for solving the problem]

[0016] In order to solve the above-mentioned problems, the present invention provides a recombinant vector for producing a recombinant influenza virus-derived HA (hemagglutinin) protein that forms a trimer, comprising: (i) a gene encoding an influenza virus-derived HA protein lacking a transmembrane protein portion; and (ii) a gene encoding a protein in the trimerization motif region of coronin 1. According to a preferred embodiment of the present invention, the influenza virus may be any one or more selected from the group consisting of influenza A viruses H5N6, H7N9 and H9N2. According to another preferred embodiment of the present invention, in the influenza virus-derived HA, the protein lacking the transmembrane protein portion may include the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:18.

[0017] According to another preferred embodiment of the present invention, in the HA derived from the influenza virus, the gene encoding a protein lacking a transmembrane protein portion may include the base sequence of SEQ ID NO: 1 or the base sequence of SEQ ID NO: 17. According to another preferred embodiment of the present invention, the protein of the trimerization motif region of coronin 1 may include the amino acid sequence of SEQ ID NO:4. According to another preferred embodiment of the present invention, the gene encoding the protein of the trimerization motif region of coronin 1 may include the base sequence of SEQ ID NO:3. According to another preferred embodiment of the present invention, a gene encoding a protein having a LysM domain may be additionally inserted into the recombinant vector. According to another preferred embodiment of the present invention, the LysM domain protein may comprise the amino acid sequence of SEQ ID NO:14. According to another preferred embodiment of the present invention, the gene encoding the LysM domain protein may include the base sequence of SEQ ID NO:13.

[0018] According to another preferred embodiment of the present invention, the above-mentioned recombinant vector may further include any promoter selected from the group consisting of a 35S promoter derived from cauliflower mosaic virus, a 19S RNA promoter derived from cauliflower mosaic virus, a Mac promoter, a plant actin protein promoter, and a ubiquitin protein promoter.

[0019] The present invention also provides a transformant transformed with the aforementioned recombinant vector. According to a preferred embodiment of the present invention, the transformant may be a prokaryote or a eukaryote. Furthermore, the present invention provides a method for producing a recombinant HA protein derived from a trimer-forming influenza virus in a plant, the method comprising the steps of: Furthermore, the present invention provides a vaccine composition for preventing or treating influenza virus infections, which contains the above-mentioned trimer-forming influenza virus-derived recombinant HA protein and has increased immunogenicity.

[0020] According to a preferred embodiment of the present invention, the trimer-forming recombinant HA protein derived from influenza virus may be coated on the surface of bacteria or chitosan that contain peptidoglycan in the cell wall. According to another preferred embodiment of the present invention, the bacterium containing peptidoglycan in its cell wall may be a bacterium with Generally Recognized As Safe (GRAS) status. According to another preferred embodiment of the present invention, the vaccine composition may further comprise a cholera toxin B subunit.

[0021] According to another preferred embodiment of the present invention, the vaccine composition may be in the form of an injection. Furthermore, the present invention provides a vaccine composition for preventing or treating influenza virus infections of different genotypes with increased immunogenicity, which comprises recombinant HA proteins derived from two or more different influenza viruses that form the above-mentioned trimer.

[0022] According to a preferred embodiment of the present invention, the recombinant HA proteins derived from two or more different influenza viruses that form the trimer may be HA proteins derived from any influenza virus selected from the group consisting of H5N6, H7N9 and H9N2. According to another preferred embodiment of the present invention, the trimer-forming recombinant HA proteins derived from two or more different influenza viruses may be coated onto the surface of bacteria or chitosan that contain peptidoglycan in their cell walls. According to another preferred embodiment of the present invention, the bacterium containing peptidoglycan in its cell wall may be a bacterium with Generally Recognized As Safe (GRAS) status.

[0023] According to another preferred embodiment of the present invention, the trimer-forming recombinant HA proteins derived from two or more different influenza viruses may be coated onto the surface of bacteria or chitosan containing peptidoglycan in the cell wall by any of the following methods (i) to (iii): (i) Mixing recombinant HA proteins from two or more different influenza viruses that form the trimer, and then coating the mixture on the surface of bacteria or chitosan that contain peptidoglycan in the cell wall, or (ii) mixing recombinant HA proteins from two or more different influenza viruses that form the trimers described above by coating each of them on the surface of bacteria or chitosan that contain peptidoglycan in their cell walls; or (iii) Using the two methods described above in (i) and (ii), recombinant HA proteins derived from two or more different influenza viruses are coated onto the surface of bacteria or chitosan that contain peptidoglycan in their cell walls.

[0024] According to another preferred embodiment of the present invention, the vaccine composition may further comprise a cholera toxin B subunit. Furthermore, the present invention provides a method for preventing or treating influenza virus infection, which comprises administering the above-mentioned vaccine composition in various forms to an individual in need thereof. Effect of the Invention

[0025] The trimer-forming recombinant HA protein derived from influenza virus of the present invention contains a protein of the ectodomain portion of the HA derived from highly pathogenic influenza A virus H5N6 lacking a transmembrane protein, a trimer motif of mouse coronin A, and a LysM domain, which is a cell wall binding domain of a protein containing the LysM peptidoglycan-binding domain of Lactococcus lactis, and can be produced in large quantities from plants, forms a trimer to increase immunity, and can be bound to or coated on bacteria such as Lactococcus or chitosan particles to deliver antigens in a high-speed manner. The HA recombinant protein highly expressed from the plant was administered to mice infected with influenza virus, and a therapeutic effect was observed. In addition, the HA recombinant protein antigen and the cholera toxin B subunit, which is said to greatly enhance immune responses, were coated on killed bacteria produced by heating and treating GRAS-state Lactococcus with trichloroacetic acid to remove the bacterial water-soluble proteins and nucleic acids, and the vaccine was administered to mice infected with influenza virus, and showed excellent therapeutic effects. Furthermore, the soluble H5N6 HA trimer, soluble H9N2 HA trimer or these were coated on the surface of killed lactococcus cells according to the present invention, and the resulting injectable vaccine was used to treat chickens infected with influenza virus, and showed high immune effects. CTB (cholera toxin B subunit), water-soluble H5N6 HA trimer and water-soluble H9N2 HA trimer were coated on killed lactococcus cells, respectively, and the immune composition was prepared by mixing the cells to analyze hemagglutination. As a result, it was confirmed that hemagglutination of the group including CTB was increased, and that CTB has an effect of enhancing immunogenicity. The vaccine composition prepared by mixing killed lactococcus cells coated with H5N6 HA trimer and killed lactococcus cells coated with H9N2 HA trimer in a 1:1 ratio was injected into mice for immunization, and showed an effect of increasing immunogenicity. A vaccine composition was also prepared by mixing H5N6 HA trimer and H9N2 HA trimer in a 1:1 ratio and coating this onto killed Lactococcus cells. When this was injected into mice, it similarly showed the effect of increasing immunogenicity.Thus, by coating different types of antigens onto killed Lactococcus cells respectively and then mixing them, or by simultaneously mixing different types of antigens and then coating them onto killed Lactococcus cells, or by using the above two methods together, multiple antigens can be delivered simultaneously to effectively enhance immunogenicity. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1a and FIG. 1b are schematic diagrams showing the construction of the recombinant vector used to produce the recombinant influenza virus-derived HA protein that forms a trimer according to the present invention. [Diagram 2] Figure 2a shows the results of Western blot analysis using anti-His antibody after separation of proteins expressed in Nicotiana benthamiana by SDS / PAGE, and Figure 2b shows the results of Coomassie Brilliant Blue staining of proteins expressed in Nicotiana benthamiana after separation by SDS / PAGE. [Diagram 3] Figures 3a and 3b show the results of gel filtration column chromatography confirming that mH5N6 and tH5N6 form monomers and trimers, respectively. Specifically, Figure 3a shows the results of fractionation (sections) by gel filtration column chromatography, and Figure 3b shows the results of Western blot analysis using anti-His antibody after SDS / PAGE of the fractions corresponding to each peak. Figures 3c and 3d show the results of gel filtration column chromatography confirming that mH9N2 and tH9N2 form monomers and trimers, respectively. Specifically, Figure 3c shows the results of fractionation (sections) by gel filtration column chromatography, and Figure 3d shows the results of Western blot analysis using anti-His antibody after SDS / PAGE of the fractions corresponding to each peak. [Figure 4]Figures 4a and 4b confirm the effect of trimers formed by mCor1 on the binding of LysM to Lactococcus lactis. Specifically, Figure 4a shows the results of binding of GFP-LysM and GFP-mCor-LysM to lactococci, which were then separated by SDS / PAGE, subjected to Western blot analysis, and stained with Coomassie brilliant blue. Figure 4b shows the results of observing the degree to which GFP-LysM and GFP-mCor-LysM bound to TCA-treated lactococci under a fluorescence microscope. [Diagram 5] Figure 5 shows the results of binding of H5N6 HA monomer (mH5N6) and trimer (tH5N6) produced in Nicotiana benthamiana leaf cells to TCA-treated lactococci, then running the resulting gel on SDS / PAGE and staining with Coomassie brilliant blue. [Figure 6] Figure 6a shows the administration schedule for confirming the immunogenic response of mice to soluble tHA and tHA coated on the surface of lactococci, and Figure 6b shows the results of ELISA measurement of the immunogenic response of mice to soluble tHA and tHA coated on the surface of lactococci. [Figure 7] Figures 7a and 7b show the results of an analysis of the extent to which soluble tHA and tHA coated on the surface of lactococci inhibit hemagglutination. [Figure 8] Figure 8a shows the results of an antibody induction experiment in chickens using PBS, lactococcal cells, soluble H5N6 HA trimer, and H5N6 HA trimer coated on the surface of lactococcus as antigens. Figure 8b shows the results of an antibody induction experiment in chickens using PBS, lactococcal cells, soluble H9N2 HA trimer, and H5N6 HA trimer coated on the surface of lactococcus as antigens. [Figure 9]Figure 9 shows the results of confirming immunogenicity by mixing lactococci coated with CTB (cholera toxin B subunit), soluble H5N6 HA trimer, and soluble H9N2 HA trimer. [Figure 10] Figure 10 shows the results of immunizing mice with a vaccine composition prepared by mixing lactococci coated with H5N6 HA trimer and lactococci coated with H9N2 HA trimer in a 1:1 ratio, and confirming strong immunogenicity against the two antigens. [Figure 11] Figure 11 shows the results of immunizing mice with a vaccine composition prepared by mixing H5N6 HA trimer and H9N2 HA trimer in a 1:1 ratio and coating the mixture onto lactococci, confirming strong immunogenicity against the two antigens. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present invention will be described in detail below. As mentioned above, the full-length protein of HA (hemagglutinin) derived from influenza virus is in a membrane-bound form with a transmembrane domain, and is therefore difficult to produce at a high level from cells. For this reason, in order to increase the production level, the transmembrane domain of HA is removed and only the ectodomain is expressed and made into a soluble form in cells, which has the advantage of enabling highly efficient production. However, when only the ectodomain of HA is produced in a soluble form, there is a limitation that the trimer form that the original full-length HA has when present on the surface of the influenza virus is not often produced. For this reason, the present inventors have attempted to develop a technology for inducing trimer formation when expressing and producing a recombinant protein of HA ectodomain from plants for use as a vaccine with increased immunogenicity. In addition, they have attempted to develop an HA ectodomain recombinant protein that can deliver antigens in various ways by binding to a gene that can bind to the surface of lactococcus or chitosan particles, so that the protein produced in this way has the ability to bind to peptidoglycan. As a result, we designed HA that expresses only the ectodomain excluding the transmembrane region of influenza virus-derived HA, which can be mass-produced, and a protein containing the trimer motif of mouse coronin A that forms a trimer structure and the LysM peptidoglycan-binding domain of Lactococcus lactis that binds to the surface of lactococci or chitosan particles, etc., to effectively deliver antigens. By expressing these in a single vector, we constructed a binary vector that can mass-express the trimer-forming influenza virus-derived recombinant HA protein and the above proteins in plants.

[0028] Therefore, a first aspect of the present invention relates to a recombinant vector for producing a trimer-forming recombinant influenza virus-derived HA (hemagglutinin) protein, comprising: (i) a gene encoding a protein lacking a transmembrane protein portion in influenza virus-derived HA (hemagglutinin); and (ii) a gene encoding a protein in the trimerization motif region of coronin 1.

[0029] In the recombinant vector of the present invention, the influenza virus includes, without limitation, various types of influenza viruses that infect humans, dogs, pigs, horses, poultry, wild birds, fur seals, etc., and may include the conventionally known influenza viruses type A, type B, type C, Isavirus, or Thogotovirus.

[0030] Influenza A viruses are responsible for seasonal and pandemic influenza epidemics. Wild aquatic birds are the natural host for a large variety of influenza A viruses. Occasionally, the virus can infect other species and cause devastating outbreaks in poultry or cause human influenza pandemics. Type A viruses are the most lethal human pathogens of the three influenza types, inducing the most severe disease. Influenza A viruses are subdivided into different serotypes based on the antibody response to the virus. The serotypes identified in humans (in order of number of notified human pandemic deaths) are: H1N1 (caused by the 1918 Spanish influenza), H2N2 (caused by the 1957 Asian influenza), H3N2 (caused by the 1968 Hong Kong influenza), H5N1 (pandemic threat for the 2007-2008 influenza season), H7N7 (potential pandemic threat), H1N2 (endemic in humans and pigs), H9N2, H7N2, H7N3, and H10N7.

[0031] Influenza B virus is responsible for seasonal influenza and there is only one influenza B virus. Influenza B infects almost exclusively humans and is less common than influenza A. The only animal known to be susceptible to influenza B virus infection is the fur seal. This type of influenza mutates at a rate two to three times slower than type A, so it is not genetically diverse and there is only one influenza B serotype. As a result of this lack of antigenic diversity, although some immunity to influenza B is usually acquired at an early age, influenza B mutates enough that sustained immunity is not possible. Influenza type C virus can infect humans and pigs and cause serious illness or localized epidemics, but this is rarer than other types and is usually thought to cause mild illness in children.

[0032] In the present invention, the influenza A virus may be, for example, any one selected from the group consisting of H5N6, H7N9, and H9N2, but is not limited thereto.

[0033] In a specific embodiment of the present invention, a recombinant HA protein that forms a trimer was produced using HA proteins derived from influenza A viruses H5N6 and H9N2. Specifically, in order to increase the productivity of HA, amino acid sequences encoding only the ectodomain from H5N6 or H9N2 HA, excluding the transmembrane domain, i.e., amino acid sequences including the amino acid residues at positions 17 to 531 of H5N6 HA (GenBank: AJD09950.1) and the amino acid sequence including the amino acid residues at positions 19 to 524 of H9N2 HA (GenBank: AFM47147.1), were selected and used. The recombinant vector of the present invention can be used to produce a trimer-forming recombinant HA protein by selecting from a variety of amino acid sequences in the region consisting of amino acid residues 17 to 531 of H5N6 HA (GenBank: AJD09950.1), and similarly, can be used by selecting from a variety of amino acid sequences in the region consisting of amino acid residues 19 to 524 of H9N2 HA (GenBank: AFM47147.1).

[0034] In the recombinant vector of the present invention, the protein lacking the transmembrane protein portion in the influenza virus-derived HA (hemagglutinin) may include, but is not limited to, the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 18.

[0035] In the above-mentioned influenza virus-derived HA, the gene encoding the protein lacking the transmembrane protein portion may include the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 17. Specifically, the above-mentioned gene may include a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more to the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 17, respectively.

[0036] "Percent sequence identity" for a polynucleotide is ascertained by comparing a comparison region with two optimally aligned sequences, where the portion of the polynucleotide sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for the optimal alignment of the two sequences.

[0037] In the recombinant vector of the present invention, the coronin 1 may be mouse-derived coronin 1 (mCor 1) (GenBank: EDL17419.1), and the protein at the trimerization motif site may include the amino acid sequence of SEQ ID NO: 4. In the recombinant vector of the present invention, the coronin 1 (mCor 1) is linked to the C-terminus of the influenza virus-derived HA ectodomain, allowing trimers to be formed even when only the HA ectodomain is expressed.

[0038] The gene encoding the protein of the trimerization motif site of the coronin 1 (mCor 1) may include the base sequence of SEQ ID NO: 3, and specifically, the gene may include a base sequence having sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more to the base sequence of SEQ ID NO: 3.

[0039] The recombinant vector of the present invention can additionally contain a gene encoding a LysM domain protein. In the recombinant vector of the present invention, the LysM domain protein may include the amino acid sequence of SEQ ID NO: 14, and the gene encoding the LysM domain protein may include the nucleotide sequence of SEQ ID NO: 13. Specifically, the gene may include a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more to the nucleotide sequence of SEQ ID NO: 13.

[0040] In the present invention, the recombinant HA protein containing a protein lacking a transmembrane protein portion in influenza virus-derived HA and a protein containing a trimer motif portion of coronin 1 (mCor 1) is capable of promoting various immune effects and binding to bacteria such as lactococci and chitosan particles for effective delivery of antigens. The amino acid residues from 220 to 320 (total of 101 amino acid residues) of the LysM domain (GenBank: WP_011834353), which is the cell wall binding domain of a protein containing the LysM peptidoglycan-binding domain of Lactococcus lactis, were fused to the C-terminus of the trimer motif of coronin 1 (mCor1) using a linker having six amino acid residues. Subsequently, a His tag having six His residues was fused for separation and purification of the recombinant protein, and a HDEL motif was fused for ER accumulation to complete the construct tHA (Figure 1a). As a control group, a construct mHA without the trimer motif of mCor1 was constructed and compared (Figure 1b).

[0041] The recombinant HA gene constructed in this way was introduced into the plant expression vector pTEX1 to produce plant expression vectors (pTEX-tHA and pTEX-mHA, respectively). The expression vectors produced were then introduced into Agrobacterium, which was then infiltrated into the plant to induce transient expression. The expressed recombinant HA protein was expressed as a Ni-His tag. 2+ -NTA affinity column or through binding to Lactococcus using the LysM domain.

[0042] To confirm the expression of proteins in the leaf extracts of Nicotiana benthamiana transfected with these genes, Western blot analysis was performed using anti-His antibody. As shown in Figure 2a, HA-LysM-His-HDEL was identified at a position of approximately 80 kDa. This is larger than the calculated protein position, which is thought to be due to N-glycosylation of the HA protein. In addition, tHA is slightly larger than mHA, as shown in Figure 2b.

[0043] Next, to confirm the formation of trimers of the above proteins, mHA in monomer form and tHA in trimer form were mixed and gel filtration was performed. As a result, two peaks appeared as shown in Figure 3a, and the fractions (sections) corresponding to each peak were subjected to Western blotting using anti-His antibody. As shown in Figure 3b, the protein in the peak corresponding to the trimer was confirmed to be tHA, and the protein in the peak corresponding to the monomer was confirmed to be mHA protein.

[0044] In a specific embodiment of the present invention, in order to confirm the binding of the recombinant protein LysM to lactococci, a His-tagged GFP-mCor1-LysM (GFP-mCor1-LysM) construct and a His-tagged GFP-LysM (GFP-LysM) construct were prepared using GFP as a control protein, and introduced into the E. coli expression vector pRSET-A to construct an expression vector that can be expressed in E. coli. The above proteins GFP-mCor1-LysM and GFP-LysM were expressed in E. coli, separated and purified, and the degree of binding to lactococci was observed under a fluorescent microscope. As a result, it was confirmed that GFP-mCor1-LysM had higher GFP expression than GFP-LysM, as shown in Figure 4.

[0045] In another specific embodiment of the present invention, the trimerization effect of mCor1 was confirmed in the binding of HA to the lactococcus surface by LysM. To this end, the leaf tissue of Nicotiana benthamiana expressing monomeric mHA (mH5N6) and trimeric tHA (tH5N6) was crushed to obtain a total extract, which was mixed with lactococci treated with TCA to induce binding, and the lactococci were pelleted and collected, then developed by SDS / PAGE and stained with Coomassie brilliant blue. As a result, as seen in Figure 5, tHA (tH5N6) formed a trimer structure of HA, while mHA (mH5N6) did not form a trimer structure of HA, and the amount of tHA (tH5N6) that formed a trimer structure bound to lactococci increased as the amount of HA increased, while mHA hardly bound to lactococci.

[0046] The recombinant vector of the present invention may additionally include any promoter selected from the group consisting of the 35S promoter derived from cauliflower mosaic virus, the 19S RNA promoter derived from cauliflower mosaic virus, the Mac promoter, a plant ectin protein promoter, and a ubiquitin protein promoter, preferably the Mac promoter, and more preferably the MacT promoter, but is not limited to these.

[0047] The MacT promoter may be a promoter in which the 3'-terminal base A of the Mac promoter base sequence is replaced with T, and the MacT promoter may include the base sequence of SEQ ID NO: 15, and specifically, the gene may include a base sequence having sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more to the base sequence of SEQ ID NO: 15.

[0048] The recombinant vector of the present invention may additionally include an RD29B-t termination site, and the gene of the RD29B-t termination site may include the base sequence of SEQ ID NO:16, and specifically, the gene may include a base sequence having a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more to the base sequence of SEQ ID NO:16.

[0049] In the recombinant vector of the present invention, the signal sequence of BiP (chaperone binding protein) and HDEL, an endoplasmic reticulum retention signal (ER retention sequence), are inserted into the N- and C-termini of the gene encoding the recombinant protein, respectively, to induce high concentration accumulation in the ER (endoplasmic reticulum). Thus, the recombinant vector of the present invention can additionally contain a gene encoding BiP and / or a gene encoding HDEL (His-Asp-Glu-Leu) peptide, and the gene encoding BiP can contain the base sequence of SEQ ID NO: 9, and HDEL (His-Asp-Glu-Leu) can contain the base sequence of SEQ ID NO: 10.

[0050] In the present invention, the term "recombinant" refers to a cell that replicates or expresses a heterologous nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. Recombinant cells may express genes or segments of genes not found in the native form of the cell, either in sense or antisense form. Recombinant cells may also express genes found in the native state of the cell, but that have been altered and reintroduced into the cell by artificial means.

[0051] The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors. Generally, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. The important characteristics of the expression vector are that it has an origin of replication, a promoter, a marker gene and a translation control element. The recombinant expression vector and the expression vector containing the appropriate transcription / translation control signals can be constructed by methods well known to those skilled in the art. The methods include in vitro recombinant DNA techniques, DNA synthesis techniques and in vivo recombination techniques.

[0052] A preferred example of a recombinant vector of the present invention is a Ti plasmid vector, which, when present in a suitable host, can transfer a part of itself, the so-called T region, to a plant cell. Other types of Ti plasmid vectors are currently utilized to transfer hybrid DNA sequences in plant cells, or in protoplasts from which new plants can be produced that allow the hybrid DNA to be properly inserted into the plant genome. Particularly preferred forms of Ti plasmid vectors are the so-called binary vectors, as claimed in EP 0 120 516 B1 and U.S. Pat. No. 4,940,838. Other suitable vectors that can be utilized to introduce the constructs encoding the recombinant HA protein from trimer-forming influenza viruses designed in the present invention into a plant host can be selected from viral vectors, such as defective plant virus vectors, which can be derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, geminiviruses, etc. The use of such vectors is advantageous, especially when it is difficult to properly transform the plant host.

[0053] A second aspect of the present invention relates to a transformant transformed with the aforementioned recombinant vector. The transformant of the present invention may be a prokaryote or a eukaryote, examples of which include yeast (Saccharomyces cerevisiae), mold such as Escherichia coli, insect cells, human cells (e.g., CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cells, preferably Agrobacterium. In the case of insect cells, human cells and the like, a gene encoding a trimer-forming recombinant HA protein can be expressed using an expression vector required for expression in each type of cell.

[0054] The recombinant vector of the present invention can be delivered into a host cell by the CaCl2 method, the Hanahan method, electroporation, etc., when the host cell is a prokaryotic cell, or by microinjection, calcium phosphate coprecipitation, electroporation, liposome-mediated transfection, DEAE-dextran method, gene barmbardment, etc., when the host cell is a eukaryotic cell.

[0055] A third aspect of the present invention relates to a method for producing a recombinant HA protein from an influenza virus that forms a trimer in a plant, the method comprising the steps of: (a) producing the recombinant vector; (b) introducing the recombinant vector into a cell to produce a transformant; (c) culturing the transformant; (d) infiltrating a culture of the transformant into a plant; and (e) crushing the plant to obtain a recombinant influenza virus HA (hemagglutinin) protein that forms a trimer. In the method of the present invention for producing a trimer-forming recombinant influenza virus-derived HA protein, the above (a) and (b) are the same as described above, and therefore the description thereof will be omitted. In the method for producing a recombinant influenza virus-derived HA protein that forms a trimer of the present invention, the above step (c) can be carried out by appropriately selecting any method known in the art for producing a recombinant influenza virus-derived HA protein that forms a trimer of the present invention. In the method of producing a recombinant influenza virus-derived HA protein that forms a trimer of the present invention, the above step (d) can be carried out by infiltrating a culture of a transformant into a plant using, for example, a chemical cell method, a vacuum or a syringe infiltration method, and preferably, the plant can be infiltrated using a syringe infiltration method, but is not limited thereto.

[0056] In the method of producing a recombinant HA protein derived from an influenza virus that forms a trimer of the present invention, the plant in step (d) above may be selected from food crops including rice, wheat, barley, corn, beans, potatoes, wheat, red beans, oats, and sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Japanese melon, pumpkin, green onion, onion, and carrot; specialty crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, and rapeseed; fruit trees including apples, pears, jujubes, peaches, grapes, mandarin oranges, persimmons, plums, apricots, and bananas; and ornamental plants including roses, carnations, chrysanthemums, lilies, and tulips.

[0057] A fourth aspect of the present invention relates to a trimer-forming recombinant influenza virus HA protein produced by the above-mentioned production method, and a vaccine composition containing the same and having increased immunogenicity for preventing, ameliorating or treating influenza virus infection.

[0058] In the vaccine composition of the present invention, the trimer-forming recombinant HA protein from influenza virus can be coated on the surface of bacteria or chitosan that contain peptidoglycan in the cell wall. The bacteria that contain peptidoglycan in the cell wall can be bacteria that are generally recognized as safe (GRAS). For example, Lactococcus, Lactobacillus, Streptococcus, etc. are not limited thereto. In a specific embodiment of the present invention, the surface of Lactococcus lactis was coated with one or more different recombinant HA proteins by various methods to effectively deliver antigens.

[0059] For this purpose, the trimer-forming recombinant HA proteins from two or more different types of influenza viruses of the present invention can be coated onto the surface of bacteria or chitosan containing peptidoglycan in the cell wall by any of the following methods (i) to (iii): (i) Mixing recombinant HA proteins from two or more different influenza viruses that form the trimer, and then coating the mixture on the surface of bacteria or chitosan that contain peptidoglycan in the cell wall, or (ii) mixing recombinant HA proteins from two or more different influenza viruses that form the trimers described above by coating them on the surface of bacteria or chitosan that contain peptidoglycan in their cell walls; or (iii) Using the two methods described above (i) and (ii), recombinant HA proteins derived from two or more different types of influenza viruses are coated onto the surface of bacteria or chitosan that contain peptidoglycan in their cell walls. For example, the vaccine composition of the present invention can be produced by coating lactococci with recombinant HA proteins derived from two or more different influenza viruses that form trimers, respectively, and then mixing the lactococci coated with the different HA recombinant proteins at the same ratio (for example, when mixing lactococci coated with two different HA recombinant proteins, they may be mixed at a ratio of 1:1), or by mixing them at any suitable ratio to produce a single vaccine composition. In addition, recombinant HA proteins derived from two or more different influenza viruses can be mixed at the same ratio or any suitable ratio, and then coated on the surface of lactococci to produce a single vaccine composition. This makes it possible to produce a single vaccine composition that can effectively deliver two or more antigens at the same time. In addition, killed lactococcus cells coated with recombinant HA proteins derived from two or more different influenza viruses can be mixed with killed lactococcus cells coated with recombinant HA proteins derived from two or more different influenza viruses to produce a vaccine composition that delivers multiple antigens.

[0060] Therefore, the recombinant HA proteins derived from two or more different influenza viruses contained in the vaccine composition of the present invention may be derived from one or more viruses selected from the group consisting of conventionally known influenza viruses types A, B, C, Isavirus, and Thogotovirus. The influenza A virus may be, for example, any one or more viruses selected from the group consisting of H5N6, H7N9, and H9N2, but is not limited thereto.

[0061] The vaccine composition of the present invention may further comprise a cholera toxin B subunit, which can increase the immunogenicity of the recombinant HA protein of the present invention and more effectively induce an immune response. The vaccine composition of the present invention may be in the form of an injectable preparation, but is not limited thereto.

[0062] In the vaccine composition of the present invention, influenza virus infection includes acute respiratory disease caused by influenza virus type A, type B, or type C infection, or clinical symptoms and complications caused thereby. Clinical symptoms of acute respiratory disease caused by influenza virus infection include respiratory symptoms such as high fever (about 38 to 40°C), dry cough, and sore throat, and systemic symptoms such as headache, muscle pain, fatigue, weakness, and loss of appetite. The most common complications are secondary respiratory diseases, such as upper respiratory tract infections such as sinusitis and otitis media, and other neurological complications such as encephalitis, myelitis, and Guillain-Barré syndrome, transverse myelitis, myocarditis, myositis, and pneumothorax. As used herein, the terms "prevention," "amelioration," and / or "treatment" refer to any action that inhibits or delays the onset of a disease or condition, any action that improves or beneficially alters the disease or condition, and any action that slows, halts, or reverses the progression of a disease or condition.

[0063] The vaccine composition of the present invention may be comprised of an antigen, a pharma- ceutical acceptable carrier, suitable adjuvants, and other conventional substances, and is administered in an immunologically effective amount. In the present invention, the term "immunologically effective amount" means an amount sufficient to induce an immune response, but not causing side effects or a severe or excessive immune response, and the exact concentration administered will vary depending on the particular immunogen administered and can be determined by those skilled in the art using known methods to test for the generation of an immune response. It may also vary depending on the form and route of administration, the age, health and weight of the recipient, the nature and severity of symptoms, and the type and frequency of current therapy.

[0064] The carrier may be any of those known in the art, including stabilizers, diluents, and buffers. Suitable stabilizers may include carbohydrates such as sorbitol, lactose, mannitol, starch, sugar, dextran, and glucose; proteins such as albumin or casein; and the like. Suitable diluents include salts, Hanks' balanced salts, infusion solutions, and the like. Suitable buffers include alkali metal phosphates, alkali metal carbonates, and alkaline earth metal carbonates. The vaccine composition of the present invention may further include one or more selected from the group consisting of solvents, adjuvants (auxiliary agents), and excipients. The solvents include physiological saline or distilled water, the immune enhancers include Freund's incomplete or complete adjuvants, aluminum hydroxide gel, and vegetable and mineral oils, and the excipients include, but are not limited to, aluminum phosphate, aluminum hydroxide, or potassium aluminum sulfate, and may further include substances used in the manufacture of vaccines that are well known to those skilled in the art.

[0065] The vaccine compositions of the present invention may be administered via any known route of administration, including, but not limited to, oral, transdermal, intramuscular, peritoneal, intravenous, subcutaneous, or nasal, and may be administered by any device capable of transferring the active agent to target cells.

[0066] The vaccine composition of the present invention is capable of inducing a humoral or, alternatively, a cell-mediated immune response and / or a combination of the two above mentioned immune responses. A fifth aspect of the present invention relates to a method for preventing, ameliorating or treating influenza virus infection comprising the step of administering to an individual in need thereof the various forms of vaccine compositions described above.

[0067] In the method for preventing, ameliorating or treating influenza virus infection of the present invention, the term "individual" refers to all animals, including humans, that are already infected or may be infected with influenza virus. Examples include, but are not limited to, humans, dogs, cats, pigs, horses, chickens, ducks, geese, turkeys, fur seals, etc. By administering the vaccine composition of the present invention to an individual in need thereof, acute respiratory disease caused by influenza virus type A, type B or type C infection or clinical symptoms and complications caused thereby can be effectively prevented or treated. For example, the vaccine composition of the present invention can be used to treat humans infected with various influenza virus subtypes or mutant influenza viruses. In addition, the composition of the present invention can be used to treat chickens or pigs infected with various influenza virus subtypes or mutant avian influenza. The composition of the present invention can be administered in combination with a conventional influenza virus infection treatment agent and / or cholera toxin B subunit.

[0068] The term "co-administration" means that the vaccine composition of the present invention is administered to an individual in need thereof together with a conventional influenza virus infection treatment agent and / or cholera toxin B subunit. The administration of each component together means that each component is administered simultaneously, separately or sequentially to achieve the desired effect.

[0069] In the method of preventing, ameliorating or treating influenza virus infection of the present invention, the vaccine composition may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally or through an implanted reservoir. The term "parenteral" as used in this application includes, but is not limited to, subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intraspinal, intrahepatic, intralesional and intracranial injection. In particular, the composition may be administered orally, intraperitoneally or intravenously.

[0070] In the method for preventing, ameliorating, or treating influenza virus infection of the present invention, the vaccine composition is preferably administered two or more times. For example, after the primary vaccination, booster vaccinations can be administered 1-4 times at intervals of 1-10 weeks, which can be appropriately modified by those skilled in the art depending on the type of animal.

[0071] A sixth aspect of the present invention relates to the use of the trimer-forming recombinant influenza virus-derived HA protein of the present invention in the manufacture of a medicament for the prevention, amelioration or treatment of influenza virus infection.

[0072] In the following, preferred examples are shown to aid in understanding the present invention. However, the following examples are provided to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples. EXAMPLES

[0073] Design of a recombinant hemagglutinin (HA) gene derived from the influenza virus surface protein that forms a trimer To express H5N6 or H9N2 HA in a soluble form in the ER lumen, we isolated H5N6 HA (AJD09950.1) (amino acid positions 17-531) and H9N2 HA (AFM47147.1) (amino acid positions 19-524) that lack the transmembrane domain and ER targeting leader sequence. The 5' end of the HA was fused to the ER targeting signal from Arabidopsis thaliana protein BiP. In addition, the first of the three repeats of the lactococcus binding domain LysM from AcmA of Lactococcus lactis was fused to the C-terminus of HA using a linker. Then, a Hisx6 tag and an ER retention motif, HDEL, were sequentially fused to the C-terminus of LysM to construct mHA for purification of HA and high accumulation in the ER (Fig. 1a). To induce trimer formation of the recombinant HA protein thus produced, a motif inducing the formation of a homogenous trimer from a protein called mouse m-coronin 1 was added between HA and LysM to construct tHA (Fig. 1b). macT was used for expression, and the end of Rd29b was used as a terminator. These were confirmed to show high transcription efficiency. The base sequences used in the experiment are shown in Table 1 below.

[0074] [Table 1] TIFF0007627447000002.tif243151TIFF0007627447000003.tif222151 EXAMPLES

[0075] Expression and characterization of recombinant hemagglutinin (HA) gene from influenza virus surface protein forming trimer mHA and tHA were expressed through transient expression in the leaves of 4-5 week-old Nicotiana benthamiana plants using vacuum infiltration. Infiltrated leaves were harvested 3, 5, and 7 days post infiltration (dpi), respectively, ground completely in liquid nitrogen, and dissolved in 3 volumes of buffer. Total soluble proteins from infiltrated leaf extracts were resolved by SDS-PAGE followed by Western blot analysis. As seen in Figure 2a, the HA recombinant proteins showed clear bands at approximately 85 kDa (mH5N6) and 90 kDa (tH5N6), which corresponded to the sizes predicted by anti-His antibodies. As seen in Figure 2b, the same bands were also observed when the same membrane was stained with Coomassie Brilliant Blue (CBB). The expression levels of these recombinant proteins were estimated to be approximately 100 μg / g fresh weight from the infiltrated leaves, judging from the band intensities. EXAMPLES

[0076] Confirmation of trimeric structure formation of recombinant hemagglutinin (HA) gene derived from influenza virus surface protein that forms trimers The HA protein on the virus surface exists as a homotrimer and shows high stability and immunogenicity. On the other hand, recombinant HA tends to be expressed as aggregates or monomers depending on the expression system. To mimic the HA trimer present on the virus surface, mouse coronin 1-1A (mCor1, GenBank: EDL17419.1 32 amino acids), a motif that forms a coiled coil structure and homotrimers, was added to the C-terminus of HA as shown in Figure 1b. These genes were introduced into Nicotiana benthamiana to induce expression, and total soluble protein from 5 dpi leaf tissue and Ni 2+The purified mHA and tHA were purified by -NTA affinity column chromatography. The separated and purified mHA and tHA were quantified based on a BSA standard curve. To confirm whether mCor1 induces HA trimerization, approximately 10 μg of mHA and 10 μg of tHA were mixed with PBS to a total volume of 1 mL and analyzed by size exclusion chromatography (SEC). As shown in Figure 3a and Figure 3c, the mHA and tHA mixture showed two peaks in the 280 nm absorption spectrum. The fractions containing these two peaks were analyzed by Western blotting using anti-His antibody, and the results are shown in Figure 3b and Figure 3d, respectively. As shown in the SEC analysis results in Figure 3a and Figure 3c and the Western blotting results in Figure 3b and Figure 3d, tHA containing mCor1 (i.e., tH5N6 and tH9N2) eluted before mHA without mCor1 (i.e., mH5N6 and mH9N2). This confirmed that mCor1 induces HA trimerization. EXAMPLES

[0077] Confirmation of peptidoglycan binding ability of a recombinant influenza virus surface protein hemagglutinin (HA) gene that forms a trimer AcmA, the main autolysin of Lactococcus lactis phage MG1363, has a domain called LysM that appears in the third repeat at the C-terminus, and the third repeat of this LysM has the ability to bind to peptidoglycan, a component of the cell wall of lactococci. Three repeats are necessary for optimal activation against peptidoglycan. However, when it contains three repeats, the domain length is too long, so only one LysM was fused to the C-terminus of GFP for confirmation. As a result, as confirmed in Figures 4a and 4b, GFP-LysM did not bind well to lactococci. To this, the trimerization motif of mouse coronin 1A (mCor-1A), which was used to form HA trimers, was added to construct GFP-mCOr1-LysM, and after inducing it to form a trimer like the trimer in HA, it was confirmed whether it would bind to lactococci. As a result, as shown in Figures 4a and 4b, a significantly increased GFP signal was confirmed in lactococci compared to GFP-LysM. This suggests that the trimerization motif of mCor1A induces the formation of GFP trimers, and that the GFP trimers thus formed have three LysM motifs, which explains why GFP binds well to Lactococcus. These results suggest that tHA binds well to Lactococcus. EXAMPLES

[0078] Confirmation of maximum binding amount of recombinant hemagglutinin (HA) gene derived from influenza virus surface protein that forms trimers Both recombinant HA proteins, mHA and tHA, were highly expressed in Nicotiana benthamiana. Infiltrated leaves were completely pulverized with liquid nitrogen and dissolved in 10 volumes of PBS buffer containing 0.5% Triton X-100, 1 mM EDTA, and 25% glycerol. Total soluble protein was collected from the leaves, and an amount of total soluble protein equivalent to 200 mg to 2 g of leaves was incubated with TCA-pretreated L. lactis (L. lactics) at 37°C for 1 hour, and then washed three times with PBS buffer. Lactococci were precipitated from each sample, heated in SDS buffer, and then run on SDS-PAGE with various amounts of BSA, and stained with Coomassie Brilliant Blue to confirm HA. As a result, as seen in Figure 5, tHA with the trimer motif mCor1 increased in binding to L. lactis as the amount of HA increased, while mHA hardly bound to L. lactis. The maximum binding amount of trimer tHA was estimated to be about 1.7 μg / 1 ml of Lactococcus lactis (OD = 1) (Figure 5 ). EXAMPLES

[0079] Production of killed Lactococcus cells and method for coating said killed cells with a trimer-forming recombinant influenza virus surface protein-derived hemagglutinin (HA) protein Lactococcus (Korea Center for Microorganisms; KCCM No. 43146) was cultured to an OD600 of 1.0, and the culture was collected by centrifugation to pellet the cells, which were then resuspended in the same volume of 10% trichloroacetic acid (TCA) and treated at 100°C for 10 min. To remove TCA, the cells were pelleted by centrifugation, washed three times with PBS, and the pellet was resuspended to prepare killed Lactococcus cells. Various amounts of total soluble protein extracts made using a buffer (PBS pH = 7.5, 1 mM EDTA, Triton X-100 cocktail, and 25% glycerol) were added and incubated at 37°C for 1 h. Killed Lactococcus cells were pelleted by centrifugation at 12,000 rpm for 5 min, and washed three times with PBS buffer for protein extraction. EXAMPLES

[0080] Confirmation of immunogenicity of trimer-forming antigens in mice The recombinant tH5N6, tH5N6 coated with killed Lactococcus cells, tH9N2, and tH9N2 vaccines coated with killed Lactococcus cells produced in Examples 2 and 6 were intraperitoneally injected twice at two-week intervals into 6-week-old female C57BL / 6 mice (Orient Bio, Korea). As a control group, PBS and killed Lactococcus cells were administered. Test vaccines were produced by adding the above recombinant vaccine with or without adjuvant in the composition shown in Table 2 below.

[0081] [Table 2] To analyze the humoral immune response induced by the test vaccine administered above, mouse serum was separated on week 4, which was the second week after the administration of the second vaccine on week 0 (before immunization), and the formation of antigen-specific antibodies was analyzed by ELISA to determine the antibody titer. The total IgG antibody titer was confirmed by the following method.

[0082] Specifically, the purified recombinant antigen was coated on a 96-well microplate at a concentration of 50ng / well, and then 200μl of PBST buffer (NaCl 137mM, KCl 2.7mM, Na2HPO4 10mM, KH2PO4 1.8mM, Tween 20 1%) containing 3% skim milk was added to prevent non-specific binding, and the reaction was allowed to proceed for 2 hours. The microplate was washed, and serum diluted with PBST solution containing 3% skim milk was added to each well in sequence, and the reaction was allowed to proceed on a microplate shaker at room temperature for 1 hour. After washing three times with 200μl PBST buffer, anti-mouse IgGHRP (horseradish peroxidase, KPL, USA, Bethyl.) was added as a secondary antibody, and the reaction was allowed to proceed under the same conditions for 2 hours. The reacted microplate was washed, and the color-developing reagent TMB (3,3',5,5'-tetramethylbenzidine) and ferroxidase substrate (KPL, USA) were added and reacted at room temperature for 10 minutes. The color-developing reaction was stopped using 0.18M H2SO4 resting solution, and the OD was measured at 450 nm using an ELISA reader. The antibody titer was defined as the reciprocal of the antibody dilution ratio that showed an OD value twice that of the negative control group. As a result, as confirmed in Figure 6b, tHA coated on the surface of lactococci without an adjuvant showed the same antibody induction effect as that containing an adjuvant. This suggests that the antigen coated on lactococci can strongly induce immunity without an adjuvant, and that killed lactococci can be a strong adjuvant in intraperitoneal and intramuscular administration, which may be applicable to cost-effective vaccination. EXAMPLES

[0083] Comparative analysis of the inhibitory effect of recombinant influenza virus surface protein hemagglutinin (HA) coated on the surface of lactococci and its water-soluble trimer on hemagglutination PBS was used as a control, and tH9N2 (HA trimer of H9N2), tH5N6 (HA trimer of H5N6), Lactococcus killed cells, Lact.-tH9N2 (HA trimer of H9N2 coated on the surface of Lactococcus), and Lact.-tH5N6 (HA trimer of H5N6 coated on the surface of Lactococcus) were used as antigens diluted in a factor of 2 to perform hemagglutination inhibition experiments and analyze hemagglutination. 25 μl of dilution buffer was added to the first well to just before the last well of a U-bottom microplate. 25 μl of the pretreated sample was added to the first well with dilution buffer, and then diluted 2-fold in 25 μl increments up to just before the last well. At this time, 25 μl of the pretreated sample was added to the last well to check for non-specific reactions of the sample. 25 μl of each antigen diluted to 8 HA units was added to the first well to just before the last well, sealed, and incubated at room temperature for 45 minutes. 25μl of 1% chicken hemocytes was added to each well and incubated at room temperature for 1 hour before reading. As shown in Figures 7a and 7b, the antigen-coated lactococci, i.e., Lact.-tH9N2 coated with H9N2 HA trimer and Lact.-tH5N6 coated with H5N6 HA trimer, were much more active in the hemagglutination inhibition assay. EXAMPLES

[0084] Confirmation of antigenicity of Lactococcus surface-coated trimers for poultry Six-week-old chickens (Namdeok SPF, Republic of Korea) were divided into groups of five and administered PBS, killed lactococcal cells, soluble H5N6 HA trimer, and lactococcal surface-coated H5N6 HA trimer as antigens via a single intramuscular injection, and antibody production was confirmed. Similarly, H9N2 HA was used as an antigen, and the chickens were injected into the muscle in the same manner as above, and antibody production was confirmed. The composition of the vaccine used in this example is shown in Table 3 below. At this time, 1x107 EID50 of H9N2 virus was injected into the control group to confirm antibody production.

[0085] [Table 3] To analyze the humoral immune response induced by the administered test vaccine, chicken serum was isolated at 2, 3, and 4 weeks after immunization, and the formation of antigen-specific antibodies was analyzed by ELISA to determine the antibody titer. The total IgG antibody titer was confirmed by the following method.

[0086] Specifically, the purified recombinant antigen was coated on a 96-well microplate at a concentration of 100 ng / well, and then 1% bovine serum albumin was added to prevent non-specific binding and reacted for 1 hour. The microplate was washed, and serially diluted serum was added to each well and reacted at 37°C for 2 hours, and anti-mouse IgGHRP (horseradish peroxidase, KPL, USA) was added as a secondary antibody and reacted under the same conditions for 1 hour. The reacted microplate was washed, and a color-developing reagent TMB (3,3',5,5'-tetramethylbenzidine) and a ferroxidase substrate (KPL, USA) were added and reacted at room temperature for 10 minutes, after which the color-developing reaction was stopped using a static solution, and the OD was measured at 450 nm using an ELISA reader. The antibody titer was defined as the reciprocal of the antibody dilution ratio that showed an OD value twice that of the negative control group. As can be seen in Figures 8a and 8b, in chickens, the antigen coated on the surface of lactococci induced a much stronger immune response than that injected in soluble form. In particular, 2 μg of H9N2 HA trimer coated on the surface of lactococci induced a much stronger immune response than that injected in soluble form against 1×10 H9N2 virus. 7 It was confirmed that this had a much stronger immune effect than that of injections of EID50. EXAMPLES

[0087] Induction of immunity was confirmed after coating killed lactococci with CTB (cholera toxin B subunit), soluble H5N6 HA trimer, and soluble H9N2 HA trimer. Killed lactococci were coated with CTB (cholera toxin B subunit), soluble H5N6 HA trimer, and soluble H9N2 HA trimer, and then the immunogenicity was confirmed by intraperitoneal injection of the antigen into mice (strain: BALB / c; animal size: 5 weeks old, female, 20 g; animal purchase source: Semtaco, Republic of Korea; number of animals used: 3 per group). iLact coated with CTB (1 μg) was used as the control group, and iLact-tH5N6 (0.1 μg) + iLact-tH9N2 (0.1 μg), iLact-tH5N6 (0.5 μg) + iLact-tH9N2 (0.5 μg), CTB (1 μg) + iLact-tH5N6 (0.1 μg) + iLact-tH9N2 (0.1 μg), or CTB (1 μg) + iLact-tH5N6 (0.5 μg) + iLact-tH9N2 (0.5 μg) were produced as vaccine compositions, and were intraperitoneally injected twice at two-week intervals, after which blood was collected two weeks later and antibodies present in the blood were confirmed by ELISA. At that time, 20 ng each of H5N6 and H9N2 antigens were coated on the ELISA plate. As a result, as shown in FIG. 9, it was confirmed that when CTB was injected intraperitoneally simultaneously with tHA antigen, it had the effect of enhancing the immunogenicity of HA. EXAMPLES

[0088] Lactococcus was coated with H5N6 HA and H9N2 HA separately, and then mixed with lactococcus to confirm its immune induction ability. After coating the surface of lactococci with two different types of HA, they were mixed and injected into mice to confirm the immunogenicity of each antigen. To this end, 0.1 μg, 0.5 μg, or 1.0 μg of H5N6 tHA was coated on the surface of lactococci to produce iLact-tHA. H5N6 Similarly, 0.1 μg, 0.5 μg, or 1.0 μg of H9N2 tHA was coated on the surface of lactococci to prepare iLact-tHA. H9N2 After preparing the vaccine composition (iLact-tHA) in a 1:1 ratio, H5N6 + iLact-tHA H9N2The prepared composition was intraperitoneally injected into mice at two-week intervals to induce immune responses, and after the second injection, blood was collected two weeks later to measure the amount of antibodies. H5N6 and tHA H9N2 After coating with 50 ng of these two antigens, the amount of antibody bound to each antigen was measured by the same method as described in Example 7. As a result, as shown in Figure 10, strong immunogenicity was confirmed for both antigens. EXAMPLES

[0089] Lactococcus was simultaneously coated with H5N6 HA and H9N2 HA, and the immune-inducing ability of this was confirmed. Two antigens, H5N6 tHA (tHA H5N6 ) and H9N2 tHA (tHA H9N2 ) were mixed at a ratio of 1:1 and coated on the surface of lactococci, followed by [iLact(tHA H5N6 + tHA H9N2 )] was intraperitoneally injected into mice at two-week intervals, and after the second immune injection, blood was collected two weeks later to confirm antibody formation via ELISA. For ELISA, 50 ng of the antigen used was coated, the blood was diluted at an appropriate ratio, and the amount of antibodies present in the blood was measured in the same manner as described in [Example 7]. As shown in Figure 11, it was confirmed that the vaccine composition produced by the method of this example induced a strong immune response against both of the two antigens. Therefore, it was confirmed that Lactococcus spp. reacted with two types of antigens (tHA H5N6 and tHA H9N2 ) can be transmitted simultaneously.

Claims

1. (i) a gene encoding a hemagglutinin (HA) protein derived from influenza A virus H5N6 or H9N2 that lacks a transmembrane protein portion and a protein portion encoding an endoplasmic reticulum (ER) targeting leader sequence; (ii) a gene encoding a protein at the trimerization motif site of mouse coronin 1, wherein the mouse coronin 1 comprises the amino acid sequence represented by SEQ ID NO: 19; and (iii) A gene encoding a LysM domain protein consisting of the amino acid sequence of SEQ ID NO: 14 A recombinant vector for producing a trimer-forming recombinant HA protein derived from an influenza virus, comprising:

2. The recombinant vector of claim 1, wherein the influenza virus-derived HA protein lacking the transmembrane protein portion and the protein portion encoding the ER targeting leader sequence comprises the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO:

18.

3. The recombinant vector described in claim 1, wherein the gene encoding a protein in which the influenza virus-derived HA lacks a transmembrane protein portion and a protein portion encoding an ER targeting leader sequence comprises the base sequence of SEQ ID NO: 1 or the base sequence of SEQ ID NO:

17.

4. The recombinant vector of claim 1 , wherein the mouse coronin 1 trimerization motif region protein comprises the amino acid sequence of SEQ ID NO:

4.

5. The recombinant vector according to claim 1 , wherein the gene encoding the protein of the trimerization motif region of mouse coronin 1 comprises the base sequence of SEQ ID NO:

3.

6. The recombinant vector according to claim 1 , wherein the gene encoding the LysM domain protein comprises the base sequence of SEQ ID NO:

13.

7. A transformant transformed with the recombinant vector according to any one of claims 1 to 6.

8. (a) producing the recombinant vector of claim 1; (b) introducing the recombinant vector into Agrobacterium to produce a transformant; (c) culturing the transformant; (d) infiltrating a tobacco plant with the culture of the transformant; and (e) grinding the tobacco to obtain a trimer-forming recombinant HA (hemagglutinin) protein derived from influenza virus; 1. A method for producing a recombinant HA protein derived from an influenza virus that forms a trimer from a tobacco plant, comprising:

9. A trimer-forming recombinant HA protein derived from influenza virus produced by the method of claim 8.

10. A vaccine composition having increased immunogenicity for preventing or treating influenza virus infection, comprising killed lactococcus cells coated with a recombinant HA protein derived from an influenza virus that forms a trimer as described in claim 9.

11. The vaccine composition of claim 10, wherein the trimer-forming recombinant HA proteins derived from influenza viruses include recombinant HA proteins derived from influenza A viruses H5N6 and H9N2 that lack a transmembrane protein portion and a protein portion encoding an endoplasmic reticulum (ER) targeting leader sequence.

12. 12. The vaccine composition of claim 10 or 11, further comprising a cholera toxin B subunit.

13. The vaccine composition according to claim 10 or 11, wherein the vaccine composition is in an injectable form.

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