Mutant of hemagglutinin protein of H3N2 subtype influenza virus and use thereof

Abstract

The disclosure relates to a mutant of hemagglutinin protein of H3N2 subtype influenza virus and use thereof. In addition, the disclosure also relates to a pharmaceutical composition (e.g., a vaccine) comprising the mutant, a method for preparing the mutant, and a method of using the mutant for prevention and/or treatment of an infection of influenza virus and/or a disease (e.g., an influenza) caused by the infection.

Claims

1. A mutant of hemagglutinin protein of H3N2 subtype influenza virus, wherein the mutant differs from a wild-type hemagglutinin protein of the H3N2 subtype influenza virus at least in that the N residue in each characteristic sequence N-X-(S or T) in the wild-type hemagglutinin protein is independently replaced with a non-N amino acid residue, so that the mutant contains no characteristic sequence N-X-(S or T); wherein, N represents asparagine, X represents any one amino acid other than proline, S represents serine, and T represents threonine; and optionally the mutant does not contain a N-terminal signal peptide and/or a transmembrane region of the wild-type hemagglutinin protein.

2. A recombinant protein, comprising the mutant according to claim 1 and an additional peptide segment, and the additional peptide segment is linked to the mutant.

3. A nucleic acid molecule, comprising or consisting of a nucleotide sequence encoding one of the following: (i) the mutant according to claim 1; and (ii) a recombinant protein comprising the mutant of (i) and an additional peptide segment linked to the mutant.

4. A vector, comprising the nucleic acid molecule according to claim 3.

5. A host cell or virus, comprising (i) the nucleic acid molecule according to claim 3; or (ii) a vector comprising the nucleic acid molecule of (i).

6. A multimer, comprising or consisting of a plurality of (i) the mutants according to claim 1; or (ii) a recombinant protein comprising the mutant of (i) and an additional peptide segment linked to the mutant.

7. A pharmaceutical composition, comprising a pharmaceutically acceptable carrier and/or excipient, and one or more of the following: (i) the mutant according to claim 1; (ii) a recombinant protein comprising the mutant of (i) and an additional peptide segment linked to the mutant; and (iii) a multimer comprising or consisting of a plurality of the mutant of (i) or a plurality of the recombinant protein of (ii); optionally, the pharmaceutical composition is a vaccine.

8. The mutant according to claim 1, wherein the N residue in each characteristic sequence N-X-(S or T) in the wild-type hemagglutinin protein is independently conservatively replaced.

9. The mutant according to claim 1, characterized by one or more of the following items: (a) the wild-type hemagglutinin protein is from A/WISCONSIN/67/2005 (H3N2) or A/HONG_KONG/4801/2014 (H3N2); (b) the wild-type hemagglutinin protein has a sequence selected from the group consisting of: SEQ ID NOs: 1 and 6; (c) the wild-type hemagglutinin protein has an amino acid sequence as shown in SEQ ID NO: 1; and, optionally, the mutant does not contain amino acids 1-10 of SEQ ID NO: 1 and/or amino acids 504-550 of SEQ ID NO: 1; (d) the wild-type hemagglutinin protein has an amino acid sequence as shown in SEQ ID NO: 6; and, optionally, the mutant does not contain amino acids 1-25 of SEQ ID NO: 6 and/or amino acids 518-565 of SEQ ID NO: 6; and (e) the mutant has an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-13; or, the mutant has an identity of at least 85%, at least 90%, at least 91%, and 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 12-13; or, the mutant has an addition, deletion or substitution of one or more amino acid residues as compared to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 12-13.

10. The recombinant protein according to claim 2, characterized by one or more of the following items: (a) the additional peptide segment is directly linked to the mutant or is linked to the mutant through a linker; (b) the additional peptide segment is linked to the N-terminus or C-terminus of the mutant; (c) the recombinant protein comprises at least 1, at least 2, at least 3, at least 5 or more additional peptide segments; and (d) the additional peptide segment is selected from the group consisting of a signal peptide, a tag peptide, a folding motif, a detectable label, and any combination thereof.

11. The recombinant protein according to claim 10, characterized by one or more of the following items: (a) the signal peptide is linked to the N-terminus of the mutant; (b) the signal peptide has an amino acid sequence as shown in SEQ ID NO: 9; (c) the folding motif is linked to the C-terminus of the mutant; and (d) the folding motif has an amino acid sequence as shown in SEQ ID NO: 10.

12. The host cell or virus according to claim 5, wherein the virus is a baculovirus.

13. The multimer according to claim 6, wherein the multimer is a trimer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 schematically illustrates the sequence mutations and N-linked glycosylation of natural HA protein (W12005-WT-HA), HA-mut1 protein, HA-mut2 protein and HA-mut3 protein used in Example 1.

(2) FIG. 2 schematically illustrates the schematic structure diagrams of the trimers formed with natural HA protein (FIG. 2A), HA-mut1 protein (FIG. 2B), HA-mut2 protein (FIG. 2C), and HA-mut3 protein (FIG. 2D) used in Example 1, respectively; in which FIG. 2A shows that the trimer formed by natural HA protein contains N-linked glycosyl chains in both the head and stem regions; FIG. 2B shows that the trimer formed by HA-mut1 protein does not contain N-linked glycosyl chain in both the head and stem regions; FIG. 2C shows that the trimer formed by HA-mut2 protein does not contain N-linked glycosyl chain in the head region, but still contains N-linked glycosyl chain in the stem region; FIG. 2D shows that the trimer formed by HA-mut3 protein does not contain N-linked glycosyl chain in the stem region, but still contains N-linked glycosyl chain in the head region.

(3) FIG. 3 shows the results of SDS-PAGE analysis of six proteins prepared in Example 1, in which FIG. 3A shows the results of SDS-PAGE analysis of natural HA protein, HA-mut3, HA-mut2 and HA-mut1 proteins; FIG. 3B shows the results of SDS-PAGE analysis of natural HA protein, HAmg protein and HAug protein.

(4) FIG. 4 shows the neutralizing activities against influenza viruses A/Wisconsin/67/2005 (H3N2 subtype) (FIG. 4A), A/Victoria/361/2011 (H3N2 subtype) (FIG. 4B), A/Beijing/32/1992 (H3N2 subtype) (FIG. 4C), A/Aichi/2/1968 (H3N2 subtype) (FIG. 4D), A/Shanghai/02/2013 (H7N9 subtype) (FIG. 4E), and A/California/04/2009 (H1N1 subtype) (FIG. 4F) of mouse sera as obtained by immunizing mice with natural HA protein, HA-mut1, HA-mut2, HA-mut3 and PBS (used as negative control) as an immunogen, respectively.

(5) FIG. 5 shows the neutralizing activities against influenza viruses A/Wisconsin/67/2005 (H3N2 subtype) (FIG. 5A), A/Victoria/361/2011 (H3N2 subtype) (FIG. 5B), A/Beijing/32/1992 (H3N2 subtype) (FIG. 5C), A/Aichi/2/1968 (H3N2 subtype) (FIG. 5D), A/Shanghai/02/2013 (H7N9 subtype) (FIG. 5E), and A/California/04/2009 (H1N1 subtype) (FIG. 5F) of mouse sera as obtained by immunizing mice with natural HA protein, HA-mut1, HAmg, HAug and PBS (used as negative controls) as an immunogen, respectively.

(6) FIG. 6 shows the changes in body weight and survival of mice immunized with natural HA protein, HA-mut1 protein, HA-mut2 protein, HA-mut3 protein or PBS (negative control) after infection with the H3N2 subtype influenza viruses A/Beijing/32/1992 (H3N2) (FIGS. 6A-6B) and A/Aichi/2/1968 (H3N2) (FIGS. 6C-6D) which are prevalent at early ages, in which FIG. 6A and FIG. 6C show the changes in body weight of each group of experimental mice, and FIGS. 6B and 6D show the survival rate of each group of experimental mice.

(7) FIG. 7 shows the changes in body weight and survival of mice immunized with natural HA protein, HA-mut1 protein, HAmg protein, HAug protein or PBS (negative control) after infection with H3N2 subtype influenza viruses A/Beijing/32/1992 (H3N2) (FIGS. 7A-7B) and A/Aichi/2/1968 (H3N2) (FIGS. 7C-7D) which are prevalent at early ages, in which FIG. 7A and FIG. 7C show the changes in body weight of each group of experimental mice, and FIG. 7B and FIG. 7D show the survival rate of each group of experimental mice.

(8) FIG. 8 shows the changes in body weight and survival of mice immunized with natural HA protein, HA-mut1 protein, HA-mut2 protein, HA-mut3 protein or PBS (negative control) after infection with non-H3N2 subtype influenza viruses A/Shanghai/02/2013 (H7N9) (FIGS. 8A-8B) and A/California/04/2009 (H1N1) (FIGS. 8C-8D), in which FIG. 8A and FIG. 8C show the changes in body weight of each group of experimental mice, and FIG. 8B and FIG. 8D show the survival rate of each group of experimental mice.

(9) FIG. 9 shows the changes in body weight and survival of mice immunized with natural HA protein, HA-mut1 protein, HAmg protein, HAug protein or PBS (negative control) after infection with non-H3N2 subtype influenza viruses A/Shanghai/02/2013 (H7N9) (FIGS. 9A-9B) and A/California/04/2009 (H1N1) (FIGS. 9C-9D), in which FIG. 9A and FIG. 9C show the changes in body weight of each group of experimental mice, and FIG. 9B and FIG. 9D shows the survival rate of each group of experimental mice.

(10) FIG. 10 shows the results of SDS-PAGE analysis (left panel) and Western blot analysis (right panel) of HK2014-WT-HA protein; in which lane M: molecular weight marker; lane 1: sample without being purified by Ni-NTA nickel ion chromatography column; lane 2: fraction flowing through Ni-NTA nickel ion chromatography column; lane 3: fraction being eluted with 50 mM imidazole; lane 4: fraction being eluted with 50 mM imidazole; lane 5: fraction being eluted with 250 mM imidazole; the arrow indicates the position of the protein HK2014-WT-HA of interest.

(11) FIG. 11 shows the results of SDS-PAGE analysis (left panel) and Western blot analysis (right panel) of HK2014-DG-HA protein; in which lane M: molecular weight marker; lane 1: sample without being purified with Ni-NTA nickel ion chromatography column; lane 2: fraction flowing through Ni-NTA nickel ion chromatography column; lane 3: fraction being eluted with 50 mM imidazole; lane 4: fraction being eluted with 250 mM imidazole; the arrow indicates the position of the protein HK2014-DG-HA of interest.

(12) FIG. 12 shows the results of SDS-PAGE analysis of natural HA protein HK2014-WT-HA and deglycosylated protein HK2014-HAug; in which, lane M: molecular weight marker; lane 1: purified HK2014-WT-HA; lane 2: HK2014-HAug (obtained by digesting HK2014-WT-HA with endoglycosidase F for 3 hours).

(13) FIG. 13 shows the results of ELISA analysis evaluating binding activities to influenza viruses A/Wisconsin/67/2005 (H3N2), A/Xiamen/N794/2013 (H3N2) and A/Shanghai/02/2013 (H7N9) of mouse sera obtained by immunizing mice with HK2014-WT-HA, HK2014-DG-HA and PBS (used as negative control) as an immunogen, respectively.

(14) FIG. 14 shows the results of ELISA analysis evaluating binding activities to influenza viruses A/Wisconsin/67/2005 (H3N2), A/Xiamen/N794/2013 (H3N2) and A/Shanghai/02/2013 (H7N9) of mouse sera obtained by immunizing mice with HK2014-WT-HA, HK2014-HAug and PBS (used as negative control) as an immunogen, respectively.

(15) FIG. 15 shows the changes in body weight (left panel) and survival (right panel) of each group of mice (3/group) immunized with HK2014-WT-HA, HK2014-DG-HA or PBS (used as negative control) after infection with A/Aichi/2/1968 (H3N2).

(16) FIG. 16 shows the changes in body weight (left panel) and survival (right panel) of each group of mice (3/group) immunized with HK2014-WT-HA, HK2014-DG-HA or PBS (used as negative control) after infection with A/Shanghai/059/2013 (H7N9).

(17) FIG. 17 shows the changes in body weight of each group of mice (4/group) immunized with HK2014-WT-HA, HK2014-HAug or PBS (used as negative control) after infection with A/Shanghai/059/2013 (H7N9).

SEQUENCE INFORMATION

(18) Information of the sequences involved in the invention is provided in Table 1 below.

(19) TABLE-US-00001 TABLE 1 Sequence information SEQ ID NO: Description of sequence 1 Full-length amino acid sequence of HA protein of influenza strain A/WISCONSIN/67/2005 (H3N2) 2 Amino acid sequence of WI2005-WT-HA protein 3 Amino acid sequence of HA-mut1 protein 4 Amino acid sequence of HA-mut2 protein 5 Amino acid sequence of HA-mut3 protein 6 Full-length amino acid sequence of HA protein of influenza strain A/HONG_KONG/4801/2014 (H3N2) 7 Amino acid sequence of HK2014-WT-HA protein 8 Amino acid sequence of HK2014-DG-HA protein 9 Amino acid sequence of N-terminal signal peptide MLLVNQSHQGFNKEHTSKMVAIVLYVLLAAAAHSAFA 10 Amino acid sequence of C-terminal folding motif SGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG 11 Amino acid sequence of 6*His tag: HHHHHH 12 Amino acid sequence of mutant of HA protein of influenza strain A/WISONSIN/67/2005 (H3N2) 13 Amino acid sequence of mutant of HA protein of influenza strain A/HONG_KONG/4801/2014 (H3N2) 0

SPECIFIC MODELS FOR CARRYING OUT THE PRESENT INVENTION

(20) The present invention will now be described with reference to the following examples which are intended to illustrate the present invention without limiting it.

(21) Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this application were performed by substantially referring to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F M Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Restriction enzymes were used in accordance with conditions recommended by the product manufacturers. If the specific conditions were not indicated in the examples, the conventional conditions or the conditions recommended by the manufacturers were used. If the reagents or instruments used were not specified by the manufacturer, they were all conventional products that were commercially available. Those skilled in the art know that the examples are used to illustratively describe the present invention, and are not intended to limit the scope of the present invention as claimed.

Example 1

Preparation of HA Protein of H3N2 Influenza Virus and Mutant Thereof

(22) (A) Design and Structure of HA Protein Mutant

(23) In the natural HA protein of influenza virus, the amino acid undergoing N-linked glycosylation is usually asparagine (N) in the characteristic sequence N-X-(S or T), in which N represents asparagine, X represents any one amino acid other than proline, S represents serine, and T represents threonine. In this example, the N-linked glycosylation site of HA protein was removed by mutating asparagine (N) in a characteristic sequence N-X-(S or T) in the natural HA protein to alanine (A).

(24) The natural HA protein (WI2005-WT-HA) used in this example was the HA protein of H3N2 subtype influenza virus strain A/Wisconsin/67/2005. The HA protein of this strain contained the amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acids 1 to 10 of SEQ ID NO: 1 were of a signal peptide, the amino acids 504 to 550 were of a transmembrane region, and, there were 10 potential N-linked glycosylation sites, namely asparagines (N) at positions 22, 38, 63, 126, 133, 144, 165, 246, 285 and 483. Among these N-linked glycosylation sites, with the exception of the asparagine at position 483 that was located in the HA2 subunit of HA protein, all asparagines at other positions were located in the HA1 subunit of HA protein. In addition, in terms of spatial structure, the asparagines at positions 22, 38, 285 and 483 were located in the stem region of the HA protein trimer; while the asparagines at positions 63, 126, 133, 144, 165 and 246 were located in the head region of the HA protein trimer.

(25) Based on the above structural information, the following natural HA protein and three HA protein mutants were designed in this example (FIG. 1):

(26) (1) Natural HA protein (WI2005-WT-HA), which contained the amino acid sequence as shown in SEQ ID NO: 2, and differed from SEQ ID NO: 1 in that the amino acids 1 to 10 and 504 to 550 of SEQ ID NO: 1 were deleted, and a peptide segment (which contained sequences of SEQ ID NOs: 10 and 11, to facilitate the protein purification and trimer formation) containing a thrombin cleavage site, a folding motif, and a 6*His tag was introduced in the C-terminus of SEQ ID NO: 1. Accordingly, the trimer formed from the natural HA protein (WI2005-WT-HA) contained N-linked glycosyl chains in both the head and stem regions (FIG. 2A).

(27) (2) HA-mut1, which contained the amino acid sequence shown in SEQ ID NO: 3, and differed from the natural HA protein (WI2005-WT-HA; SEQ ID NO: 2) in that the asparagine at each of the aforementioned 10 N-linked glycosylation sites was mutated to alanine. Accordingly, the trimer formed by HA-mut1 did not contain N-linked glycosyl chain in the head and stem regions (FIG. 2B).

(28) (3) HA-mut2, which contained the amino acid sequence shown in SEQ ID NO: 4, and differed from the natural HA protein (WI2005-WT-HA; SEQ ID NO: 2) in that each of the asparagines located in the head region (i.e., at positions 63, 126, 133, 144, 165, and 246 of SEQ ID NO: 1) was mutated to alanine. Accordingly, the trimer formed by HA-mut2 did not contain N-linked glycosyl chain in the head region, but still contained N-linked glycosyl chains in the stem region (FIG. 2C).

(29) (4) HA-mut3, which contained the amino acid sequence shown in SEQ ID NO: 5, and differed from the natural HA protein (WI2005-WT-HA; SEQ ID NO: 2) in that each of the asparagines located in the stem region (i.e., at positions 22, 38, 285, and 483 of SEQ ID NO: 1) was mutated to alanine. Accordingly, the trimer formed by HA-mut3 did not contain N-linked glycosyl chain in the stem region, but still contained N-linked glycosyl chains in the head region (FIG. 2D).

(30) In addition, in order to facilitate the secretion of the protein, a nucleotide sequence encoding a signal peptide (SEQ ID NO: 9) was introduced at the 5′ end of the nucleotide sequences encoding the natural HA protein, HA-mut1 protein, HA-mut2 protein and HA-mut3 protein. The expressed signal peptide was excised during protein secretion. Therefore, the finally obtained natural HA protein, HA-mut1 protein, HA-mut2 protein and HA-mut3 protein did not contain the signal peptide, and their amino acid sequences were shown in SEQ ID NOs: 2-5.

(31) FIG. 1 schematically illustrates the sequence mutations and N-linked glycosylation of the natural HA protein, HA-mut1 protein, HA-mut2 protein and HA-mut3 protein used in Example 1 (note: the signal peptide would be excised during protein secretion). Specifically, the natural HA protein had asparagine at positions corresponding to the positions 22, 38, 63, 126, 133, 144, 165, 246, 285 and 483 of SEQ ID NO: 1, and thus could carry N-linked glycosyl chains at these positions. The HA-mut1 protein had alanine at positions corresponding to the positions 22, 38, 63, 126, 133, 144, 165, 246, 285 and 483 of SEQ ID NO: 1, and therefore no longer carried any N-linked glycosyl chains. The HA-mut2 protein had asparagine at positions corresponding to the positions 22, 38, 285 and 483 of SEQ ID NO: 1, and therefore could carry N-linked glycosyl chains at these positions; however, it had alanine at positions corresponding to the positions 63, 126, 133, 144, 165 and 246 of SEQ ID NO: 1, and therefore no longer carried any N-linked glycosyl chains at these positions. The HA-mut3 protein had asparagine at positions corresponding to the positions 63, 126, 133, 144, 165 and 246 of SEQ ID NO: 1, and therefore could carry N-linked glycosyl chains at these positions; however, it had alanine at positions corresponding to the positions 22, 38, 285 and 483 of SEQ ID NO: 1, and therefore no longer carried any N-linked glycosyl chains at these positions. In addition, in order to facilitate the secretion, purification and trimer formation of the protein, a signal peptide (which had an amino acid sequence as shown in SEQ ID NO: 9, and would be excised during protein secretion) was introduced into the N-terminus of the natural HA protein, HA-mut1 protein, HA-mut2 protein and HA-mut3 protein, respectively, and a peptide segment containing a thrombin cleavage site, a folding motif, and a 6*His tag (which contained amino acid sequences as shown in SEQ ID NOs: 10 and 11) was introduced into their C-terminus, respectively.

(32) FIG. 2 schematically illustrates the schematic structure diagrams of the trimers formed with natural HA protein (FIG. 2A), HA-mut1 protein (FIG. 2B), HA-mut2 protein (FIG. 2C), and HA-mut3 protein (FIG. 2D) used in Example 1, respectively; in which FIG. 2A shows that the trimer formed by natural HA protein contained N-linked glycosyl chains in both the head and stem regions; FIG. 2B shows that the trimer formed by HA-mut1 protein contained no N-linked glycosyl chain in both the head and stem regions; FIG. 2C shows that the trimer formed by HA-mut2 protein did not contain N-linked glycosyl chain in the head region, but still contained N-linked glycosyl chain in the stem region; FIG. 2D shows that the trimer formed by HA-mut3 protein did not contain N-linked glycosyl chain in the stem region, but still contained N-linked glycosyl chain in the head region.

(33) (B) Preparation of Transfer Plasmid

(34) The DNA sequences separately encoding natural HA protein, HA-mut1 protein, HA-mut2 protein and HA-mut3 protein (for each of them, a signal peptide (SEQ ID NO: 9) was introduced into the N-terminus, and a peptide segment containing a thrombin cleavage site, a folding motif and a 6*His tag (SEQ ID NOs: 10 and 11) was introduced into the C-terminus) were synthesized by Shanghai Sangon Biotechnology Co., Ltd, and then these DNA sequences were cloned into baculovirus transfer vector pAcGP67-B (BD Company, Catalog Number: 554757), respectively. Subsequently, the transfer vectors carrying the DNA sequences of interest were separately transformed into competent cells of E. coli DH5a and amplified. A plasmid miniprep kit (TIANprep Mini Plasmid Kit; TianGen Corporation, Catalog Number: DP103-03) was used to extract the transfer plasmid containing the DNA sequence of interest from the transformed E. coli for later use.

(35) (C) Co-Transfection

(36) 1 hour before transfection, 1×10.sup.6 insect cells (Sf9 cells, Invitrogen) were plated on a 6-well culture plate and cultured in a serum-added medium. 1 μg of the transfer plasmid prepared in step (B), 0.1 μg of baculovirus linear DNA (BD), 1 μl of liposomes (Sigma), and 100 μl of serum-free cell culture medium were mixed and allowed to stand at room temperature for 30 minutes to obtain a transfection mixture. The serum-containing medium was removed from each well and the transfection mixture was added. After 6 hours of incubation at 27° C., the transfection mixture was removed from each well, and 2 ml of CCM3-containing medium was added to each well to continue culturing the cells. As a result, the transfer plasmid carrying the DNA sequence of interest and the baculovirus linear DNA were transfected into insect cells to produce a recombinant baculovirus.

(37) (D) Production and Purification of Target Protein

(38) The obtained recombinant baculovirus was passaged to obtain a second-generation recombinant baculovirus. 15 ml of the second-generation recombinant baculovirus was added to 1200 ml of Sf9 insect cells, and cultured at 27° C. for 48 hours. The cells and culture supernatant were collected and centrifuged at 11,500 rpm for 30 minutes. After centrifugation, the supernatant was collected, which contained the recombinantly produced target protein.

(39) The supernatant containing the protein of interest was concentrated to 35 ml with an ultrafiltration concentration centrifuge tube from Millipore, adjusted to pH 7.4, and then centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected, and Ni-NTA nickel ion chromatography column (NI-sepharose 6 fast flow, GE, Catalog Number: 17-5318-04) was used to enrich and purify the protein of interest in the supernatant, in which the eluent was PBS containing 250 mM imidazole. The eluate containing the protein of interest was concentrated to 1 ml, and dialyzed into PBS buffer, and stored at 4° C. for later use. Thus, the purified natural HA protein, HA-mut1 protein, HA-mut2 protein and HA-mut3 protein were obtained (the N-terminal signal peptide was excised during the secretion process, so the obtained protein retained the folding motif and 6*His tag, but did not contain the N-terminal signal peptide).

(40) (E) Preparation of HAmg and HAug Proteins

(41) In addition, by referring to the method described in Juine-Ruey Chen et al. (Proc Natl Acad Sci, USA. 2014 Feb. 18; 111 (7): 2476-81), the natural HA protein (WI2005-WT-HA) was subjected to enzymatic treatment by using endoglycosidase H and endoglycosidase F to prepare an HA protein carrying a single glycosyl group at N-linked glycosylation site (hereinafter referred to as HAmg) and an HA protein substantially carrying no glycosyl group at N-linked glycosylation site (hereinafter referred to as HAug).

(42) It should be noted that due to the restriction of enzymatic action and the inaccessibility of some glycosylation sites, HAug inevitably still carries a small amount of glycosyl groups at N-linked glycosylation sites, which can also be confirmed by the data provided in Table 51 of Juine-Ruey Chen et al. (Ibid.). In contrast, since asparagine at each of all N-linked glycosylation sites has been replaced with alanine, the HA-mut1 protein no longer carries any N-linked glycosyl groups.

Example 2

Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis

(43) Polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the six proteins (natural HA protein, HA-mut1, HA-mut2, HA-mut3, HAmg, and HAug) prepared in Example 1. The upper gel used was 5% concentrated gel (prepared as follows: 830 μl of 30% acrylamide, 630 μl of 1M Tris (pH6.8), 50 μl of 10% SDS, 50 μl of 10% ammonium persulfate, and 5 μl of TEMED were added into 3.4 ml of water). The lower gel used was 12% separation gel (prepared as follows: 4 ml of 30% acrylamide, 2.5 ml of 1M Tris (pH8.8), 100 μl of 10% SDS, 100 μl of 10% ammonium persulfate, and 10 μl of TEMED were added to 3.3 ml of water). The electrophoresis condition used was that electrophoresis was performed at 150 V for 2 hours. After electrophoresis, the polyacrylamide gel was stained with Coomassie brilliant blue (Sigma). The experimental results are shown in FIG. 3.

(44) FIG. 3 shows the results of SDS-PAGE analysis of six proteins prepared in Example 1. FIG. 3A shows the results of SDS-PAGE analysis of natural HA protein, HA-mut3, HA-mut2, and HA-mut1 proteins; FIG. 3B shows the results of SDS-PAGE analysis of natural HA protein, HAmg protein and HAug protein. The results in FIG. 3 show that the molecular weight of natural HA protein is above 70 kD, while the molecular weights of HA-mut3, HA-mut2, HA-mut1, HAmg and HAug proteins are all significantly reduced, all below 70 kD, and the HA-mut1 protein has the smallest molecular weight.

Example 3

Evaluation of Neutralizing Activity of Antisera

(45) (A) Immune Experiment

(46) 6-Week-old, SPF-grade, female Balb/C mice were provided by the Experimental Animal Center of Xiamen University, and had a body weight of approximately 20 g. The six proteins (natural HA protein, HA-mut1, HA-mut2, HA-mut3, HAmg and HAug) prepared in Example 1 and PBS (used as negative control) were separately mixed with aluminum adjuvant in a volume ratio of 1:1, and used to immunize the mice. The immunization schedule was as follows: 6 mice were used in each group; the immunization method was intramuscular injection; the immunization dose was 5 μg protein/mouse; the injection volume was 100 μl/mouse; the immunization was performed twice with an interval of 14 days. Fourteen days after the second immunization, serum was collected from each mouse. The collected serum samples were inactivated at 56° C. for 30 minutes, and then stored at −20° C. for later use.

(47) (B) Evaluation of Neutralizing Titers of Serum Samples

(48) Neutralization titer is an important indicator for evaluating whether a serum sample has the potential to prevent and treat a disease. In this experiment, a plaque reduction neutralization test (PRNT) was used to analyze the neutralizing antibody titers of the collected serum samples. The influenza viruses used were representative strains of influenza viruses isolated at different time, from different regions and representing different subtypes (H3N2, H7N9 and H1N1), in which the specific virus strains were as follows: A/Wisconsin/67/2005 (H3N2 subtype), A/Victoria/361/2011 (H3N2 subtype), A/Beijing/32/1992 (H3N2 subtype), A/Aichi/2/1968 (H3N2 subtype), A/Shanghai/02/2013 (H7N9 subtype) and A/California/04/2009 (H1N1 subtype).

(49) 6×10.sup.5 MDCK cells were seeded in a 6-well cell culture plate. The influenza viruses used were diluted to 50 PFU/50 μl in MEM medium containing 0.5 μg/ml TPCK trypsin. Then, serially diluted serum samples were mixed with influenza viruses and incubated at 37° C. for 1 hour, and then added to a 6-well cell culture plate seeded with MDCK cells, and the incubation was continued at 37° C. for 1 hour. After incubation, the cell culture fluid was sucked out and the cells were washed twice with PBS. Then, the cell surface was covered with 0.5% agarose-containing MEM medium, and the cells were placed in a constant temperature incubator at 5% CO.sub.2 and 37° C. for 2 days. After that, the cells were stained with 2% crystal violet, and the titers of influenza viruses were determined by counting the number of plaques, and then the neutralizing activity of each serum sample was calculated. The results are shown in FIGS. 4-5.

(50) FIG. 4 shows the neutralizing activities against influenza viruses A/Wisconsin/67/2005 (H3N2 subtype) (FIG. 4A), A/Victoria/361/2011 (H3N2 subtype) (FIG. 4B), A/Beijing/32/1992 (H3N2 subtype) (FIG. 4C), A/Aichi/2/1968 (H3N2 subtype) (FIG. 4D), A/Shanghai/02/2013 (H7N9 subtype) (FIG. 4E), and A/California/04/2009 (H1N1 subtype) (FIG. 4F) of mouse sera as obtained by immunizing mice with natural HA protein, HA-mut1, HA-mut2, HA-mut3 and PBS (used as negative control) as an immunogen, respectively.

(51) As shown in FIG. 4A, for the influenza virus strain A/Wisconsin/67/2005 from which the HA protein used in this experiment was derived, the mouse sera obtained from mice immunized with natural HA protein, HA-mut1, HA-mut2 or HA-mut3 all had strong neutralizing activities, in which the sera obtained from mice immunized with natural HA protein and HA-mut1 had the highest neutralizing titer, and the serum obtained from mice immunized with HA-mut3 had the lowest neutralizing titer.

(52) As shown in FIG. 4B, for the H3N2 subtype virus strain A/Victoria/361/2011, which had a close evolutionary relationship with the HA protein used in this experiment, the serum obtained from mice immunized with HA-mut1 had the highest neutralizing titer (even higher than the serum obtained from mice immunized with natural HA protein), and the serum obtained from mice immunized with HA-mut3 had the lowest neutralizing titer.

(53) As shown in FIG. 4C, for the H3N2 subtype virus strain A/Beijing/32/1992, which had a farther evolutionary relationship with the HA protein used in this experiment, the serum obtained from mice immunized with HA-mut3 had the highest neutralizing titer, the serum obtained from mice immunized with HA-mut1 had the second high neutralizing titer (both were higher than the serum obtained from mice immunized with natural HA protein), and the serum obtained from mice immunized with HA-mut2 had the lowest neutralizing titer.

(54) As shown in FIG. 4D, for the H3N2 subtype virus strain A/Aichi/2/1968, which had the farthest evolutionary relationship with the HA protein used in this experiment, the serum obtained from mice immunized with HA-mut1 had the highest neutralizing titer, the serum obtained from mice immunized with HA-mut3 had the second high neutralizing titer, and the serum obtained from mice immunized with natural HA protein or HA-mut2 substantially had no neutralizing activity (no significant difference from the negative control).

(55) As shown in FIG. 4E and FIG. 4F, for the virus strains A/Shanghai/02/2013 (H7N9 subtype) and A/California/04/2009 (H1N1 subtype) that belonged to different subtypes from the HA protein used in this experiment, only the serum obtained from mice immunized with HA-mut1 had neutralizing activity, while the sera obtained from mice immunized with other proteins had substantially no neutralizing activity (no significant difference from the negative control).

(56) The results in FIG. 4 show that the serum obtained from mice immunized with HA-mut1 had the broadest spectrum of neutralizing activity, which not only can effectively neutralize multiple virus strains of H3N2 subtype (regardless of the distance of evolutionary relationship), but also can neutralize strains of other subtypes (e.g., stains of H7N9 and H1N1 subtypes). In contrast, the sera obtained from mice immunized with natural HA protein, HA-mut2 and HA-mut3 had neutralizing activity only on some strains of H3N2 subtype, and had no neutralizing activity on strains of other subtypes. Thus, HA-mut1 is particularly suitable as a broad-spectrum vaccine for inducing protective antibodies with broad-spectrum neutralizing activity in vivo.

(57) FIG. 5 shows the neutralizing activities against influenza viruses A/Wisconsin/67/2005 (H3N2 subtype) (FIG. 5A), A/Victoria/361/2011 (H3N2 subtype) (FIG. 5B), A/Beijing/32/1992 (H3N2 subtype) (FIG. 5C), A/Aichi/2/1968 (H3N2 subtype) (FIG. 5D), A/Shanghai/02/2013 (H7N9 subtype) (FIG. 5E), and A/California/04/2009 (H1N1 subtype) (FIG. 5F) of the mouse sera as obtained by immunizing mice with natural HA protein, HA-mut1, HAmg, HAug and PBS (used as negative controls) as an immunogen, respectively.

(58) As shown in FIG. 5A, for the influenza virus strain A/Wisconsin/67/2005 from which the HA protein used in this experiment was derived, the sera obtained from mice immunized with natural HA protein, HA-mut1, HAmg or HAug had strong neutralizing activity with comparable potency.

(59) As shown in FIG. 5B, for the H3N2 subtype virus strain A/Victoria/361/2011, which had a close evolutionary relationship with the HA protein used in this experiment, the serum obtained from mice immunized with HA-mut1 had the highest neutralizing titer, and the serum obtained from mice immunized with natural HA protein had the lowest neutralizing titer.

(60) As shown in FIG. 5C, for the H3N2 subtype virus strain A/Beijing/32/1992, which had a farther evolutionary relationship with the HA protein used in this experiment, the serum obtained from mice immunized with HA-mut1 had the highest neutralizing titer, and the sera obtained from mice immunized with other proteins had lower and comparable neutralizing titers between each other.

(61) As shown in FIG. 5D, for the H3N2 subtype virus strain A/Aichi/2/1968, which had the farthest evolutionary relationship with the HA protein used in this experiment, the serum obtained from mice immunized with HA-mut1 had highest neutralizing titer, the serum obtained from mice immunized with HAmg or HAug had the second high neutralizing titer (the two were comparable), while the serum obtained by mice with natural HA protein substantially had no neutralizing activity (no significant difference from the negative control).

(62) As shown in FIG. 5E, for the virus strain A/Shanghai/02/2013 (H7N9 subtype) that belonged to a different subtype from the HA protein used in this experiment, the serum obtained from mice immunized with HA-mut1 had the highest neutralizing titer, the serum obtained from mice immunized with HAmg or HAug had the second high neutralizing titer (the two were comparable), while the serum obtained by mice with natural HA protein substantially had no neutralizing activity (no significant difference from the negative control).

(63) As shown in FIG. 5F, for the virus strain A/California/04/2009 (H1N1 subtype) that belonged to a different subtype from the HA protein used in this experiment, only the serum obtained from mice immunized with HA-mut1 had neutralizing activity, while the sera obtained from mice immunized with other proteins substantially had no neutralizing activity (no significant difference from the negative control).

(64) The results in FIG. 5 show that the serum obtained from mice immunized with natural HA protein only has neutralizing activity against H3N2 subtype influenza virus; the sera obtained from mice immunized with HAmg and HAug not only can neutralize H3N2 subtype influenza virus, but also show weak neutralizing activity across HA subtypes (capable of neutralizing H7N9 subtype, but not neutralizing H1N1 subtype); the serum obtained from mice immunized with HA-mut1 has the broadest spectrum of neutralizing activity and the highest neutralizing titer, which not only can effectively neutralize multiple virus strains of H3N2 subtypes (regardless of the distance of evolutionary relationship), but also has strong neutralizing activity across HA subtypes (for example, capable of neutralizing the strains of H7N9 and H1N1 subtypes). It can be seen that HA-mut1 is particularly suitable as a broad-spectrum vaccine for inducing protective antibodies with broad-spectrum neutralizing activity in vivo.

Example 4

Evaluation of In Vivo Protective Activity

(65) The PRNT experiment in Example 3 confirmed that the neutralizing titers on the H3N2 subtype, H7N9 subtype, and H1N1 subtype virus strains of the antisera induced by the six proteins prepared in Example 1 were different, among which the antiserum induced by HA-mut1 had the broadest spectrum of neutralizing activity. In order to further verify the effect of these six proteins in inducing immune protection against influenza virus in animals, the present inventors evaluated the in vivo antiviral efficacy of these six proteins in a biosafety laboratory, based on the mouse animal models infected with influenza viruses A/Beijing/32/1992 (H3N2 subtype), A/Aichi/02/1968 (H3N2 subtype), A/Shanghai/02/2013 (H7N9 subtype) and A/California/04/2009 (H1N1 subtype). The specific scheme is as follows.

(66) Materials and Methods

(67) Animals: Balb/C mice, SPF grade, 6-8 weeks old, female, body weigh about 20 g.

(68) Vaccines: Natural HA protein, HA-mut1 protein, HA-mut2 protein, HA-mut3 protein, HAmg protein, HAug protein and PBS (used as negative control).

(69) Immunization scheme: The natural HA protein, HA-mut1 protein, HA-mut2 protein, HA-mut3 protein, HAmg protein, HAug protein and PBS negative control were separately mixed with aluminum adjuvant in a volume ratio of 1:1 and used for immunization of mice. Six mice were used in each group, and immunized by intramuscular injection; the immunization dose was 5 μg protein/mouse, and the injection volume was 100 μl/mouse. The immunization was performed twice with an interval of 14 days between the two immunizations. Fourteen days after the second immunization, the mice were challenged with viruses. The following influenza virus strains were used:

(70) mouse adaptive strain of H3N2 subtype influenza virus: A/Beijing/32/1992;

(71) mouse adaptive strain of H3N2 subtype influenza virus: A/Aichi/02/1968;

(72) mouse adaptive strain of H7N9 subtype influenza virus: A/Shanghai/02/2013;

(73) mouse adaptive strain of H1N1 subtype influenza virus: A/California/04/2009.

(74) Anesthetic: Isoflorane.

(75) Animal grouping: mice were sent to the biosafety laboratory one day in advance, grouped as 6 mice in one cage, and the weight of each mouse was recorded.

(76) Virus infection: The challenge dose of each virus was 25 times the median lethal dose (LD.sub.50), and the virus inoculation volume was 50 μl/mouse. Before inoculation, the mice were anesthetized with isophorane, and then the mice were inoculated with viruses through nasal cavity.

(77) Observations: The changes in body weight and survival of mice were recorded daily from 1 to 14 days after virus infection. The experimental results are shown in FIGS. 6-9.

(78) FIG. 6 shows the changes in weight and survival of mice immunized with natural HA protein, HA-mut1 protein, HA-mut2 protein, HA-mut3 protein or PBS (negative control) after infection with the H3N2 subtype influenza viruses A/Beijing/32/1992 (H3N2) (FIGS. 6A-6B) and A/Aichi/2/1968 (H3N2) (FIGS. 6C-6D) which are prevalent at early ages, in which FIG. 6A and FIG. 6C show the changes in body weight of each group of experimental mice, and FIG. 6B and FIG. 6D show the survival rate of each group of experimental mice. The results of FIGS. 6A-6B show that the mice immunized with HA-mut1 or HA-mut3, after being infected with a lethal dose of virus A/Beijing/32/1992, began to recover body weight after the day 7, and the mouse survival rate was 100% at the end of experiment; however, the mice immunized with natural HA protein, HA-mut2 or PBS all continuously lost body weight and all died before the end of experiment. This result indicates that HA-mut1 and HA-mut3 have complete protection and can be used as vaccines against A/Beijing/32/1992. The results of FIGS. 6C-6D show that after the mice immunized with HA-mut1 were infected with a lethal dose of virus A/Aichi/2/1968, their body weight began to recover after the day 4, and the mouse survival rate was 100% at the end of the experiment; HA-mut3 has partial protection to the mice infected with a lethal dose of virus A/Aichi/2/1968, and the mouse survival rate was 33.3% at the end of the experiment; however, the mice immunized with natural HA protein, HA-mut2 or PBS all continuously lost body weight and all died before the end of the experiment. This result indicates that HA-mut1 has full protection and can be used as a vaccine against A/Aichi/2/1968.

(79) FIG. 7 shows the changes in weight and survival of mice immunized with natural HA protein, HA-mut1 protein, HAmg protein, HAug protein or PBS (negative control) after infection with H3N2 subtype influenza viruses A/Beijing/32/1992 (H3N2) (FIGS. 7A-7B) and A/Aichi/2/1968 (H3N2) (FIGS. 7C-7D), in which FIG. 7A and FIG. 7C show the changes in body weight of each group of experimental mice, and FIG. 7B and FIG. 7D show the survival rate of each group of experimental mice. The results of FIGS. 7A-7B show that after the mice immunized with HA-mut1 protein, HAmg protein or HAug protein were infected with a lethal dose of virus A/Beijing/32/1992, their body weight began to recover after the day 7 (the mice immunized with HA-mut1 showed the best weight recovery effect), and the mouse survival rate was 100% at the end of the experiment; however, the mice immunized with natural HA protein or PBS all continuously lost body weight and all died before the end of the experiment. This result indicates that HA-mut1 protein, HAmg protein and HAug protein have complete protection and can be used as vaccines against A/Beijing/32/1992. The results of FIGS. 7C-7D show that after the mice immunized with HA-mut1, HAmg or HAug were infected with a lethal dose of virus A/Aichi/2/1968, their body weight began to recover after the day 4 or 5 (the mice immunized with HA-mut1 showed the best weight recovery effect), and the mouse survival rate was 100% at the end of the experiment; however, the mice immunized with natural HA protein or PBS all continuously lost body weight and all died before the end of the experiment. This result indicates that HA-mut1 protein, HAmg protein and HAug protein have complete protection and can be used as vaccines against A/Aichi/2/1968.

(80) FIG. 8 shows the changes in weight and survival of mice immunized with natural HA protein, HA-mut1 protein, HA-mut2 protein, HA-mut3 protein or PBS (negative control) after infection with non-H3N2 subtype influenza viruses A/Shanghai/02/2013 (H7N9) (FIGS. 8A-8B) and A/California/04/2009 (H1N1) (FIGS. 8C-8D), in which FIG. 8A and FIG. 8C show the changes in body weight of each group of experimental mice, and FIG. 8B and FIG. 8D show the survival rate of each group of experimental mice. The results of FIGS. 8A-8B show that after the mice immunized with HA-mut1 were infected with a lethal dose of virus A/Shanghai/02/2013 (H7N9), their body weight began to recover after the day 6, and the mouse survival rate was 100% at the end of the experiment; however, the mice immunized with natural HA protein, HA-mut2, HA-mut3 or PBS all continuously lost body weight and all died before the end of the experiment. This result indicates that HA-mut1 has complete protection and can be used as a vaccine against A/Shanghai/02/2013. The results of FIGS. 8C-8D show that, after the mice immunized with HA-mut1 were infected with a lethal dose of virus A/California/04/2009 (H1N1), their body weight remained stable after the day 8 and did not decrease anymore, and the mouse survival rate was 66.7% at the end of experiment; however, the mice immunized with natural HA protein, HA-mut2, HA-mut3 or PBS all continuously lost body weight and all died before the end of the experiment. This result indicates that HA-mut1 has a strong in vivo protective effect against influenza virus A/California/04/2009 (H1N1).

(81) FIG. 9 shows the changes in weight and survival of mice immunized with natural HA protein, HA-mut1 protein, HAmg protein, HAug protein or PBS (negative control) after infection with non-H3N2 subtype influenza viruses A/Shanghai/02/2013 (H7N9) (FIGS. 9A-9B) and A/California/04/2009 (H1N1) (FIGS. 9C-9D), in which FIGS. 9A and 9C show the changes in body weight of each group of experimental mice, and FIG. 9B and FIG. 9D shows the survival rate of each group of experimental mice. The results of FIGS. 9A-9B show that after the mice immunized with HA-mut1 protein or HAug protein were infected with a lethal dose of virus A/Shanghai/02/2013 (H7N9), their body weight began to recover after the day 6 or 7 (the mice immunized with HA-mut1 showed the best weight recovery effect), and the mouse survival rate was 100% at the end of the experiment; however, the mice immunized with natural HA protein, HAmg protein or PBS all continuously lost body weight and all died before the end of the experiment. This result indicates that HA-mut1 protein and HAug protein have complete protection and can be used as vaccines against A/Shanghai/02/2013 (H7N9). The results of FIGS. 9C-9D show that after the mice immunized with HA-mut1 were infected with a lethal dose of the virus A/California/04/2009 (H1N1), their body weight remained stable after the day 8 and did not decrease any more, and the mice survival rate was 66.7% at the end of experiment; however, the mice immunized with natural HA protein, HAmg, HAug or PBS all continuously lost body weight and all died before the end of the experiment. This result indicates that HA-mut1 has a strong in vivo protective effect against influenza virus A/California/04/2009 (H1N1).

(82) The above experimental results show that HA-mut1 protein as a vaccine can effectively prevent influenza virus infections of H3N2 subtypes (regardless of the distance of evolutionary relationship), H7N9 subtypes and H1N1 subtypes, and diseases caused thereby, and thus can be used as an effective, broad-spectrum vaccine against multiple subtypes of influenza viruses.

Example 5

Preparation and Analysis of H3N2 Influenza Virus HA Protein and its Mutants

(83) In this example, the N-linked glycosylation site of HA protein was removed by mutation of asparagine (N) in the characteristic sequence N-X-(S or T) in natural HA protein to glutamine (Q).

(84) The natural HA protein (HK2014-WT-HA) used in this example was the HA protein of H3N2 subtype influenza virus strain A/HONG_KONG/4801/2014 (H3N2). The HA protein of this strain contained the amino acid sequence shown in SEQ ID NO: 6, wherein the amino acids 1 to 25 of SEQ ID NO: 6 were of a signal peptide, and the amino acids 518 to 565 were of a transmembrane region, and it had 11 potential N-linked glycosylation sites, i.e., asparagines (N) at positions 37, 53, 60, 78, 137, 141, 148, 180, 261, 300 and 498.

(85) Based on the above structural information, the natural HA protein HK2014-WT-HA and its mutant HK2014-DG-HA were designed in this example:

(86) (1) Natural HA protein (HK2014-WT-HA), which contained the amino acid sequence shown in SEQ ID NO: 7, and which differed from SEQ ID NO: 6 in that the amino acids 1 to 25 and 518 to 565 of SEQ ID NO: 6 were deleted, and a peptide segment containing a thrombin cleavage site, a folding motif, and a 6*His tag (which contained the sequences of SEQ ID NO: 10 and 11 to facilitate protein purification and trimer formation) was introduced into the C-terminus of SEQ ID NO: 6. Accordingly, the trimer formed by the natural HA protein (HK2014-WT-HA) contained N-linked glycosyl chains in both the head and stem regions.

(87) (2) Mutant HK2014-DG-HA, which contained the amino acid sequence shown in SEQ ID NO: 8, and which differed from the natural HA protein (HK2014-WT-HA; SEQ ID NO: 7) in that the asparagine (N) at each of the aforementioned 11 N-linked glycosylation sites was mutated to glutamine (Q). Accordingly, the trimer formed by the mutant HK2014-DG-HA did not contain N-linked glycosyl chain in both the head and stem regions.

(88) In addition, in order to facilitate the secretion of protein, a nucleoside sequence encoding a signal peptide (SEQ ID NO: 9) was introduced at the 5′ end of the nucleotide sequence encoding the natural HA protein HK2014-WT-HA and the mutant protein HK2014-DG-HA. The expressed signal peptide would be excised during protein secretion. Therefore, neither the finally obtained natural HA protein HK2014-WT-HA nor its mutant HK2014-DG-HA contained a signal peptide, and their amino acid sequences were shown in SEQ ID NOs: 7-8.

(89) The DNA sequences separately encoding the natural protein HK2014-WT-HA and mutant protein HK2014-DG-HA (for each of them, a signal peptide (SEQ ID NO: 9) was introduced into the N-terminus, and a peptide segment (SEQ ID NOs: 10 and 11) containing a thrombin cleavage site, a folding motif and a 6*His tag was introduced into the C-terminus) were cloned into a baculovirus transfer vector pAcGP67-B (BD Company, Catalog Number: 554757), respectively. Subsequently, the transfer vectors carrying the DNA sequences of interest were transformed into competent cells of E. coli DH5a and amplified. A plasmid miniprep kit (TIANprep Mini Plasmid Kit; TianGen Corporation, Catalog Number: DP103-03) was used to extract the transfer plasmids containing the DNA sequences of interest from the transformed E. coli for later use.

(90) Subsequently, as described in Example 1, a recombinant baculovirus containing the DNA sequence of interest was constructed using the transfer plasmid prepared as described above, and cultured in Sf9 insect cells. After the culture, the cells and the culture supernatant were collected and centrifuged at 11,500 rpm for 30 minutes. After centrifugation, the supernatant was collected, which contained the recombinantly produced target protein. Then, as described in Example 1, the proteins of interest, i.e., HK2014-WT-HA and HK2014-DG-HA (the N-terminal signal peptide was excised during the secretion process, so the obtained proteins retained the folding motif and 6*His tag, but did not contain the N-terminal signal peptide), in the supernatant was enriched and purified by Ni-NTA nickel ion chromatography column (NI-sepharose 6 fast flow, GE, Catalog Number: 17-5318-04) using PBS containing imidazole (50 mM or 250 mM) as an eluent.

(91) In addition, by referring to the method described in Juine-Ruey Chen et al. (Proc Natl Acad Sci, USA. 2014 Feb. 18; 111 (7): 2476-81), the natural HA protein (HK2014-WT-HA) obtained as above was subjected to enzymatic treatment by using endoglycosidase F to prepare a deglycosylated HA protein (hereinafter referred to as HK2014-HAug) which did not substantially carry glycosyl group at all N-linked glycosylation sites.

(92) SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (the used antibody was: HRP-conjugated 6*His, His-Tag Antibody, Proteintech, Catalog Number: HRP-66005) were used to analyze the above prepared 3 proteins (HK2014-WT-HA, HK2014-DG-HA and HK2014-HAug). The experimental results are shown in FIGS. 10-12.

(93) FIG. 10 shows the results of SDS-PAGE analysis (left panel) and Western blot analysis (right panel) of HK2014-WT-HA protein; in which lane M: molecular weight marker; lane 1: sample without being purified by Ni-NTA nickel ion chromatography column; lane 2: fraction flowing through Ni-NTA nickel ion chromatography column; lane 3: fraction being eluted with 50 mM imidazole; lane 4: fraction being eluted with 50 mM imidazole; lane 5: fraction being eluted with 250 mM imidazole; the arrow indicates the position of the protein of interest, HK2014-WT-HA.

(94) FIG. 11 shows the results of SDS-PAGE analysis (left panel) and Western blot analysis (right panel) of HK2014-DG-HA protein; in which lane M: molecular weight marker; lane 1: sample without being purified by Ni-NTA nickel ion chromatography column; lane 2: fraction flowing through Ni-NTA nickel ion chromatography column; lane 3: fraction being eluted with 50 mM imidazole; lane 4: fraction being eluted with 250 mM imidazole; the arrow indicates the position of the protein of interest, HK2014-DG-HA.

(95) The results of FIGS. 10-11 show that the proteins HK2014-WT-HA and HK2014-DG-HA were mainly contained in the fraction eluted with 250 mM imidazole; and that the molecular weight of HK2014-WT-HA was above 70KD, the molecular weight of HK2014-DG-HA decreased in some extent. These results indicate that the glycosylation modification in HK2014-DG-HA was effectively removed.

(96) FIG. 12 shows the results of SDS-PAGE analysis of the natural HA protein HK2014-WT-HA and the deglycosylated protein HK2014-HAug; in which, lane M: molecular weight marker; lane 1: purified HK2014-WT-HA; lane 2: HK2014-HAug (obtained by digesting HK2014-WT-HA with endoglycosidase F for 3 hours).

(97) The results of FIG. 12 show that the molecular weight of HK2014-WT-HA was above 70KD, and the molecular weight of HK2014-HAug decreased in some extent. These results indicate that the glycosylation modification in HK2014-HAug was effectively removed.

Example 6

Evaluation of Immunogenicity of H3N2 Influenza Virus HA Protein and its Mutants

(98) The proteins HK2014-WT-HA, HK2014-DG-HA and HK2014-HAug prepared in Example 5 were separately mixed with Freund's adjuvant to prepare immunogens, which were then used to immunize 6-8 week-old Balb/C female mice (body weight about 20 g). The immunization procedure was as follows: subcutaneous immunization 3 times with an interval of 14 days for each immunization. Fourteen days after the third immunization, mouse sera were collected, and the collected serum samples were inactivated at 56° C. for 30 minutes, and then stored at −20° C. for later use.

(99) ELISA assay was used to evaluate whether the mouse serum samples collected above had specific binding activity to the three influenza viruses A/Wisconsin/67/2005 (H3N2), A/Xiamen/N794/2013 (H3N2) and A/Shanghai/02/2013 (H7N9). Briefly, Elisa plates were coated with 100 μl of different types of influenza viruses (128HA), and then gradient-diluted mouse serum was added to the virus-coated plates and incubated at 37° C. for 1 hour. Subsequently, 1:5000 diluted GAM-HRP (provided by the National Engineering Center of Xiamen University) was added and incubated at 37° C. for 30 min. After incubation, the plates were washed, added with chromogenic solution A&B (provided by Beijing Wantai Company) and developed for 15 minutes, and then the chromogenic reaction was stopped with a stop solution. Finally, the absorbance of each well was read using a microplate reader, and the specific binding activity of mouse serum to virus was calculated. The ELISA results are shown in FIGS. 13-14.

(100) FIG. 13 shows the results of ELISA analysis evaluating binding activities to influenza viruses A/Wisconsin/67/2005 (H3N2), A/Xiamen/N794/2013 (H3N2) and A/Shanghai/02/2013 (H7N9) of mouse sera obtained by immunizing mice with HK2014-WT-HA, HK2014-DG-HA and PBS (used as negative control) as an immunogen, respectively.

(101) The results in FIG. 13 show that the mouse sera obtained from mice immunized with HK2014-WT-HA and HK2014-DG-HA all showed comparable levels of reaction titers to the three influenza viruses (A/Wisconsin/67/2005 (H3N2), A/Xiamen/N794/2013 (H3N2), A/Shanghai/02/2013 (H7N9)). The results show that HK2014-WT-HA and HK2014-DG-HA both have good immunogenicity, can trigger normal immune response in mice, induce the body to produce specific antibodies, and these specific antibodies can recognize and bind to a variety of influenza viruses.

(102) FIG. 14 shows the results of ELISA analysis evaluating binding activities to influenza viruses A/Wisconsin/67/2005 (H3N2), A/Xiamen/N794/2013 (H3N2) and A/Shanghai/02/2013 (H7N9) of mouse sera obtained by immunizing mice with HK2014-WT-HA, HK2014-HAug and PBS (used as negative control) as an immunogen, respectively.

(103) The results in FIG. 14 show that the mouse sera obtained from mice immunized with HK2014-WT-HA and HK2014-HAug all showed comparable levels of reaction titers to the three influenza viruses (A/Wisconsin/67/2005 (H3N2), A/Xiamen/N794/2013 (H3N2), A/Shanghai/02/2013 (H7N9)). The results show that HK2014-WT-HA and HK2014-HAug both have good immunogenicity, can trigger normal immune response in mice, induce the body to produce specific antibodies, and these specific antibodies can recognize and bind a variety of influenza virus.

Example 7

Evaluation of Immuno-Protective Properties of H3N2 Influenza Virus HA Protein and its Mutants

(104) To further verify the immuno-protective effect of the proteins prepared in Example 5 against influenza virus in animals, the following experiments were performed.

(105) The proteins HK2014-WT-HA, HK2014-DG-HA and HK2014-HAug prepared in Example 5 were mixed with Freund's adjuvant to prepare the immunogens, which were then used to immunize 6-8 week-old Balb/C female mice (body weight about 20 g). The immunization procedure was as follows: subcutaneous immunization 3 times with an interval of 14 days for each immunization. Fourteen days after the third immunization, the mice of each group were challenged with influenza viruses, and the influenza virus strains used were: H3N2 virus strain A/Aichi/2/1968 (H3N2) which was prevalent at a time far away from the epidemic year of the immunogen, and H7N9 virus strain A/Shanghai/059/2013 (H7N9) prevalent in recent years, and both of them were lethal strains. After challenge, the body weight and survival rate of each group of mice were observed and recorded, and the potencies of the prepared proteins in protecting mice against the infection of lethal viruses were evaluated. The experimental results are shown in FIGS. 15-17.

(106) FIG. 15 shows the changes in body weight (left panel) and survival (right panel) of each group of mice (3/group) immunized with HK2014-WT-HA, HK2014-DG-HA or PBS (used as negative control) after infection with A/Aichi/2/1968 (H3N2). The experimental results in FIG. 15 show that after the mice immunized with HK2014-WT-HA were infected with a lethal dose of virus A/Aichi/2/1968 (H3N2), one mouse died on the day 5, and the body weight of the remaining mice began to recover on the day 6, and the mouse survival rate was 66% at the end of the experiment; after the mice immunized with HK2014-DG-HA were infected with a lethal dose of virus A/Aichi/2/1968 (H3N2), the body weight of all mice began to recover on the day 5, and the mouse survival rate was 100% at the end of the experiment; while all mice in the negative control group died on the day 8 after infection with the virus. This result shows that compared with HK2014-WT-HA, HK2014-DG-HA has better protection effect against virus A/Aichi/2/1968 (H3N2).

(107) FIG. 16 shows the changes in body weight (left panel) and survival (right panel) of each group of mice (3/group) immunized with HK2014-WT-HA, HK2014-DG-HA or PBS (used as negative control) after infection with A/Shanghai/059/2013 (H7N9). The experimental results in FIG. 16 show that after the mice immunized with HK2014-WT-HA were infected with a lethal dose of virus A/Shanghai/059/2013 (H7N9), all the mice continuously lost body weight, and the mouse survival rate on the day 9 after challenge was 0%; after the mice immunized with HK2014-DG-HA were infected with a lethal dose of the virus A/Shanghai/059/2013 (H7N9), one mouse began to recover body weight on the day 8, and the mouse survival rate was 33% at the end of the experiment. This result shows that HK2014-WT-HA does not have protection effect against the influenza virus A/Shanghai/059/2013 (H7N9); in contrast, HK2014-DG-HA shows a certain protection effect (broad-spectrum protection across subtypes) against virus A/Shanghai/059/2013 (H7N9).

(108) FIG. 17 shows the changes in body weight of each group of mice (4/group) immunized with HK2014-WT-HA, HK2014-HAug or PBS (used as negative control) after infection with A/Shanghai/059/2013 (H7N9). The experimental results in FIG. 17 show that, after the mice immunized with HK2014-WT-HA, HK2014-HAug or PBS were infected with a lethal dose of virus A/Shanghai/059/2013 (H7N9), the body weight of all mice continuously decreased, and the mouse survival rate was all 0% on the day 9 after challenge. This result shows that neither HK2014-WT-HA nor HK2014-HAug has protective effect against virus A/Shanghai/059/2013 (H7N9).

(109) From the above results, it can be seen that HK2014-DG-HA is more suitable as an influenza vaccine than HK2014-WT-HA and HK2014-HAug, which can resist the infection of influenza viruses of H3N2 subtype (regardless of the distance of evolutionary relationship) and H7N9 subtype, showing a broad-spectrum protection across subtypes and better protection.

(110) Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that according to all the teachings that have been disclosed, various modifications and changes can be made to the details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.