Chimeric influenza vaccine

Abstract

The present disclosure relates to chimeric influenza virus hemagglutinin (HA) polypeptides comprising one or more stem domain sequences, each having at least 60% homology to the stem domain consensus sequence of an H1 subtype HA (H1 HA) and / or an H5 subtype HA (H5 HA), fused to one or more globular head domain sequences, each having at least 60% homology to the globular head domain consensus sequence of an H1 subtype HA (H1 HA) or an H5 subtype HA (H5 HA).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 022.328 (filed May 8, 2020), which is incorporated by reference herein in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein in its entirety. The ASCII copy was created on May 6, 2021, is named G4590-08600PCT_SeqListing.txt, and is 28 kilobytes in size.

[0003] FIELD OF THE INVENTION The present disclosure relates to chimeric influenza virus hemagglutinin (HA) polypeptides, immunogenic / vaccine compositions comprising same, and uses thereof. [Background technology]

[0004] Background of the Invention The traditional method for producing influenza vaccines involves culturing the virus in specific pathogen-free (SPF) embryonated hens' eggs, a process that often requires more than six months for mass production. However, some vaccine virus strains grow poorly in eggs, and people with allergies to chicken eggs pose safety concerns. Novel approaches based on viral cell culture have been developed to replace egg-based methods. However, cell culture methods still pose the potential risk of producing dangerous viruses. To overcome these issues, exploration of alternative strategies has demonstrated that recombinant HA-based vaccines can induce neutralizing antibodies against influenza virus infection. However, antibodies induced by specific influenza virus subtypes usually fail to effectively neutralize other influenza subtypes. Furthermore, these vaccines must be updated annually due to the constant mutation of the virus. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there remains a need to develop a universal vaccine against a wide range of influenza virus strains. [Means for solving the problem]

[0006] Summary of the Invention In one aspect, the disclosure provides a chimeric influenza virus hemagglutinin (HA) polypeptide comprising one or more stem domain sequences each having at least 60% homology to a stem domain consensus sequence of an H1 subtype HA (H1 HA) and / or an H5 subtype HA (H5 HA), fused to one or more globular head domain sequences each having at least 60% homology to a globular head domain consensus sequence of an H1 subtype HA (H1 HA) or an H5 subtype HA (H5 HA).

[0007] In some embodiments, the HA is an influenza A HA, an influenza B HA, or an influenza C HA.

[0008] In some embodiments, the homology is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

[0009] In some embodiments, the stem domain sequence is the N-terminal stem segment of an H1 HA or the C-terminal stem segment of an H1 HA; the N-terminal stem segment of an H1 HA or the C-terminal stem segment of an H1+H5 HA sequence; or the N-terminal stem segment of an H5 HA or the C-terminal stem segment of an H1+H5 HA sequence.

[0010] In some embodiments, the stem domain consensus sequence of H1 HA and / or H5 HA comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:10.

[0011] In some embodiments, the globular head domain consensus sequence of H1 HA or H5 HA comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:7, or SEQ ID NO:11.

[0012] In one embodiment, the chimeric influenza virus HA polypeptide comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:12.

[0013] In some embodiments, one or more glycosylation sites in the HA are monoglycosylated. In further embodiments, the monoglycosylated HA has only N-acetylglucosamine (GlcNAc) at each glycosylation site.

[0014] In one embodiment, the chimeric influenza virus HA polypeptide is used as an immunogen.

[0015] In another aspect, the disclosure provides an immunogenic composition comprising a chimeric influenza virus HA polypeptide and an adjuvant. In one embodiment, the adjuvant is a glycolipid adjuvant.

[0016] In another aspect, the disclosure provides a recombinant polynucleotide comprising a nucleic acid sequence encoding a polypeptide of the disclosure and, optionally, a nucleic acid sequence encoding a signal peptide. In some embodiments, the signal peptide comprises the sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

[0017] In another aspect, the present disclosure provides a vector comprising a recombinant polynucleotide of the present disclosure. Host cells comprising the vector of the present disclosure are also provided.

[0018] In another aspect, the present disclosure provides a method of immunizing a subject against influenza virus, comprising administering to the subject an effective amount of a chimeric influenza virus hemagglutinin (HA) polypeptide or immunogenic composition of the present disclosure.

[0019] In another aspect, the present disclosure provides a method of preventing influenza virus disease in a subject, comprising administering to the subject an effective amount of a chimeric influenza virus hemagglutinin (HA) polypeptide or immunogenic composition of the present disclosure.

[0020] In one embodiment, the methods described herein involve CD4 + and CD8 + Triggers a T cell immune response.

[0021] In one embodiment, the methods described herein induce stem-specific antibodies with higher antibody-dependent cellular cytotoxicity (ADCC), better neutralizing activity, and stronger cross-protective activity against H1, H3, H5, and H7 strains and subtypes.

[0022] In one embodiment, the methods described herein increase vaccine efficacy by producing more IFN-γ, IL-4, and CD8+ memory T cells. [Brief explanation of the drawings]

[0023] [Figure 1-1] Figure 1(A)-(C). Broadly cross-protective stem-specific antibodies elicited by vaccination with chimeric H5 / 1 constructs with a common H5 globular head and common H1 stem (cHA) and the cHAmg immunogen. (A) Swap H1 / 5 (H1 globular head and H1 + H5[HA2] stem), swap H5 / 1 (H5 globular head and H5 + H1[HA2] stem), and chimeric H5 / 1 (cHA:H5 globular head and H1 stem) constructs. (B) Neutralizing activity against H1N1 California / 07 / 2009 and H5N1 Vietnam / 1194 / 2004 viruses. (C) The number of granzyme B (GrzB)-producing CD8+ T cells in splenocytes stimulated with HA (black bars) or PBS (white bars) control over 2 days in mice vaccinated with PBS (control), HA+Alu, or HA+C34 was assessed by flow cytometry analysis. [Figure 1-2]Figure 1(D)-(I). Broadly cross-protective stem-specific antibodies elicited by vaccination with a chimeric H5 / 1 construct harboring a consensus H5 globular head and a consensus H1 stem (cHA) and the cHAmg immunogen. (D-I) Antibody titers from mice vaccinated with cHAfg and cHAmg adjuvanted with Al(OH)3 versus cHAfg and cHAmg adjuvanted with C34 were measured on day 42 by ELISA using A / California / 07 / 2009 H1N1 HA protein (D), A / Brisbane / 59 / 2007 H1N1 HA protein (E), A / Brisbane / 10 / 2007 H3N2 HA protein (F), A / Vietnam / 1194 / 2004 H5N1 HA protein (G), A / Shanghai / 2 / 2013 H7N9 HA protein (H), and A / Brisbane / 59 / 2007 (Bris / 07) stem HA (no. 4900) protein (I) as coating antigens. The endpoint antibody titer is defined as the final dilution of antiserum that produces an absorbance 2.5-fold higher than that produced by the negative control (pre-immune serum). Data were examined using Student's t-test and two-way ANOVA from Prism; differences were considered statistically significant at *P<0.05; **P<0.01. Data represent the mean ± SEM. [Figure 2-1] Figure 2(A)-(B). ADCC reporter assay of antisera from cHA-vaccinated mice against target cells expressing H1N1, H3N2, or H5N1 HA and subtypes. Antisera collected from mice immunized with cHAfg or cHAmg proteins adjuvanted with aluminum hydroxide or C34 were incubated with MDCK cells infected with (A) H1N1 virus or (B) H5N1 virus for 30 minutes. ADCC reporter assays were then performed using Jurkat effector cells expressing mouse FcγRIII, and relative luminescence units (RLU) were measured. Values ​​are mean ± SEM; ***P<0.001. P values ​​were calculated using two-way ANOVA using Prism software. [Figure 2-2]Figure 2(C). ADCC reporter assay of antisera from cHA-vaccinated mice against target cells expressing H1N1, H3N2, or H5N1 HA and subtypes. Antisera collected from mice immunized with cHAfg or cHAmg proteins adjuvanted with aluminum hydroxide or C34 (C) were incubated with MDCK cells infected with H3N2 virus for 30 minutes. ADCC reporter assays were then performed using Jurkat effector cells expressing mouse FcγRIII, and relative luminescence units (RLU) were measured. Values ​​are mean ± SEM; ***P<0.001. P values ​​were calculated using two-way ANOVA using Prism software. [Figure 3-1] Figure 3(A)-(C). cHAmg with the adjuvant C34 elicited more CD4+ and CD8+ T cell responses and elicited broadly neutralizing antibodies, resulting in broader cross-protection. BALB / c mice were immunized with cHAfg and cHAmg with the adjuvant Al(OH)3 or C34; cells from the spleens of immunized mice were obtained after three immunizations, and IFN-γ (A), IL-4 (B), and GzB (C)-secreting cells were measured by ELISpot assay using specific peptides. The number of spot-forming cells (SFC) is expressed as the mean ± SEM. [Figure 3-2] Figure 3(D)-(E). cHAmg with the adjuvant C34 elicited more CD4+ and CD8+ T cell responses and elicited broadly neutralizing antibodies, resulting in broader cross-protection. BALB / c mice were immunized with cHAfg and cHAmg with the adjuvant Al(OH)3 or C34. The neutralizing activity of antisera from cHAfg- and cHAmg-vaccinated mice was assayed against (D) H1N1 and (E) H5N1 viruses. Data are presented as mean ± SEM. Results were calculated using Student's t-test and two-way ANOVA using Prism software; significant differences were noted as *P<0.05; **P<0.01; and ***P<0.001. [Figure 4-1]Figure 4(A)-(C). Cross-protective efficacy in mice challenged with lethal doses of H1N1 and H5N1 viruses. BALB / c mice were immunized with three doses of cHAfg and cHAmg with the adjuvants Al(OH)3 or C34, spaced 2 weeks apart. Immunized mice were challenged with H1N1 A / California / 07 / 2009 (A), H1N1 A / New Caledonia / 1999 (B), and H1N1 A / WSN / 1933 (C), and efficacy was assessed by recording survival rates for 14 days post-infection. **P<0.01. Significant differences in survival rates were analyzed by the log-rank (Mantel-Cox) test. [Figure 4-2] Figure 4(D)-(F). Cross-protective efficacy in mice challenged with lethal doses of H1N1 and H5N1 viruses. BALB / c mice were immunized with three doses of cHAfg and cHAmg with the adjuvants Al(OH)3 or C34, spaced 2 weeks apart. Immunized mice were challenged with H1N1 A / Solomon Islands / 03 / 2006 (D), H5N1 A / Vietnam / 1194 / 2004 / NIBRG14 (E), or H5N1A / Turkey / 1 / 2005 / NIBRG23 (F), and efficacy was assessed by recording survival rates for 14 days postinfection. **P<0.01. Significant differences in survival rates were analyzed by the log-rank (Mantel-Cox) test. [Figure 5-1]Figure 5(A)-(D). Design and preparation of chimeric HA proteins. (A) The designed influenza HA sequence was constructed using the consensus H1N1 and H5N1 sequences pCHA5-II to generate chimeric HAs. The globular head domain consists of the amino acid sequence between residues C52 and C277 (H3 numbering). The stem region consists of portions of the HA1 and HA2 subunits. Protein structures were downloaded from the Protein Data Bank ID codes 2IBX (VN1194 H5 HA) and 3LZG (A / California / 04 / 2009). Final images were generated using PyMol. Because the consensus HA structure has not been published, images of the head domain of avian influenza H5 (Vietnam / 1194 / 2004) and the stem region of pandemic H1N1 (California / 07 / 2009) were used for the chimeric HA construct. (B-D) Purification and gel filtration chromatography analysis of chimeric HA proteins. (B) Purified HA proteins were analyzed by SDS / PAGE. M: molecular weight marker. Left: cHAfg, fully glycosylated cHA purified directly from HEK293T cells; (C) cHAmg, monoglycosylated cHA purified from HEK293S cells and digested with endoglycosidase H. (D) Gel filtration analysis of purified secreted HA proteins. Chromatography shows fully glycosylated cHA from HEK293T cells and monoglycosylated cHA (>200 kDa) present as a trimer. This figure shows the overlaid elution profile of cHA protein expressed by HEK293T cells overlaid with a calibration standard (dashed line). [Figure 5-2] Figure 5(E). Design and preparation of chimeric HA proteins. (E) Schematic diagram showing the major glycans at the glycosylation sites of cHAfg and cHAmg as determined by LC-MS / MS, followed by common glycan symbols. [Figure 6-1]Figure 6(A). Construction and purification of secreted HA. (A) The sequence encoding the ectodomain of HA was prepared in the expression vector pcDNA and transfected into HEK293T cells. The protein was engineered to contain a stabilization / trimerization signal, a foldon, and a C-terminal (His)6 tag for purification. [Figure 6-2] Figure 6(B). Construction and purification of secreted HA. (B) Purified HA proteins were analyzed by SDS / PAGE. M: molecular weight marker. Lane 1: H1N1 (A / Brisbane / 59 / 2007) HA protein; Lane 2: H1N1 (A / California / 07 / 2009) HA protein; Lane 3: H3N2 (Brisbane / 10 / 2007) HA protein; Lane 4: H5N1 (Vietnam / 1194 / 2004) HA protein; Lane 5: H7N9 (A / Shanghai / 2 / 2013) HA protein. [Figure 7-1] Figure 7(A)-(B). HA-binding activity of antisera from mice vaccinated with cHAfg and cHAmg. BALB / c mice (n = 10 per group) were immunized with cHAfg or cHAmg adjuvanted with Al(OH)3 or C34 at 2-week intervals. Antibody titers from mice vaccinated with cHAfg and cHAmg adjuvanted with Al(OH)3 versus cHAfg and cHAmg adjuvanted with C34 were measured on day 28 by ELISA using the A / California / 07 / 2009 H1N1 HA protein (A) or the A / Brisbane / 59 / 2007 H1N1 HA protein (B) as the coating antigen. The endpoint antibody titer was defined as the highest dilution of serum generating an optical density (OD) 2.5-fold higher than that generated by the negative control (pre-immune serum). Data were examined using two-way ANOVA from Prism; differences were considered statistically significant at **P<0.01; ***P<0.001. Data represent the mean±SEM. [Figure 7-2]Figure 7(C)-(F). HA-binding activity of antisera from mice vaccinated with cHAfg and cHAmg. BALB / c mice (n = 10 per group) were immunized with cHAfg or cHAmg adjuvanted with Al(OH)3 or C34 at 2-week intervals. Antibody titers from mice vaccinated with cHAfg and cHAmg adjuvanted with Al(OH)3 versus cHAfg and cHAmg adjuvanted with C34 were measured on day 28 by ELISA using A / Brisbane / 10 / 2007 H3N2 HA protein (C), A / Vietnam / 1194 / 2004 H5N1 HA protein (D), A / Shanghai / 2 / 2013 H7N9 HA protein (E), and A / Brisbane / 59 / 2007 (Bris / 07) stem HA (#4900) protein (F) as coating antigens. The endpoint antibody titer was defined as the highest dilution of serum that generated an optical density (OD) 2.5 times higher than that generated by the negative control (pre-immune serum). Data were examined using two-way ANOVA from Prism; differences were considered statistically significant at **P<0.01; ***P<0.001. Data represent the mean ± SEM. [Figure 8] Figure 8 (A) and (B). Binding of stalk-reactive antibody (F10 IgG) to recombinant H1, H5, and cHA. (A) Purified F10 was analyzed by SDS / PAGE. M: molecular weight marker. Lane 1: F10 antibody. (B) The binding affinity of F10 IgG to various HAs was measured using ELISA. The x-axis indicates the concentration of various HA proteins, and the y-axis indicates the absorbance value at OD405 nm. [Figure 9]Figure 9 (A)-(D). Dose-dependent effect of C34 on antibody titers. BALB / c mice (n = 10 per group) were injected with 20 μg cHA adjuvanted with 0.5 μg, 2 μg, or 10 μg C34 at 2-week intervals. Mouse sera were collected 2 weeks after the second (D28) and third (D42) immunizations. Antibody titers were measured using ELISA with HA proteins of H1N1A / California / 07 / 2009 (A and C) and H5N1 Vietnam / 1194 / 2004 (B and D). P values ​​for antibody titers were calculated using two-way ANOVA from Prism; differences were considered statistically significant at *P<0.05; **P<0.01. Data represent mean ± SEM. [Figure 10] Figure 10 (A)-(C). Dose-dependent effect of C34 on antigen-specific cytokine-secreting cells. BALB / c mice (n = 5 per group) were injected with 20 μg of purified cHA adjuvanted with three different doses of C34: 0.5, 2, and 10 μg, at 2-week intervals. Splenocytes from cHA-immunized mice were obtained 2 weeks after the second (D28) and third (D42) immunizations. (A) IFN-γ and (B) IL4-secreting cells were assessed by Elispot analysis. (C) The number of granzyme B-producing CD8+ T cells in splenocytes was determined by Elispot analysis using specific peptides. ***P<0.001. P values ​​were calculated using two-way ANOVA using Prism software. [Figure 11-1] Figure 11 (A)-(C). Body weight of cHAfg or cHAmg vaccinated mice challenged with lethal doses of H1N1 and H5N1 viruses. Body weight changes of immunized mice challenged with H1N1 A / California / 07 / 2009 (A), H1N1 A / New Caledonia / 1999 (B), and H1N1 A / WSN / 1933 (C) viruses were monitored over 14 days post-infection. Weight changes are expressed as mean ± SEM. [Figure 11-2]Figure 11 (D)-(F). Body weight of cHAfg or cHAmg vaccinated mice challenged with lethal doses of H1N1 and H5N1 viruses. Body weight changes of immunized mice challenged with H1N1 A / Solomon Islands / 03 / 2006 (D), H5N1 A / Vietnam / 1194 / 2004 (E), or H5N1 A / Turkey / 1 / 2005 (F) viruses were monitored over 14 days post-infection. Weight changes are expressed as mean ± SEM. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description of the Invention The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA and immunology which are within the skill of those in the art, and such techniques are fully exemplified by the literature. For example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed., Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (DNGlover ed., 1985); Culture Of Animal Cells (RIFreshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press,1986);B.Perbal,A Practical Guide To Molecular Cloning(1984);the treatise,Methods in Enzymology(Academic Press,Inc.,NY);Gene Transfer Vectors For Mammalian Cells(JHMiller and MPCalos eds.,1987,Cold Spring Harbor Laboratory);Methods In Enzymology,Vols.154 and 155(Wu et al.eds.), Immunochemical Methods in Cell and See Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, by Harlow and Lane (Cold Spring Harbor Laboratory Press, 1988); and Handbook of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986).

[0025] definition As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. For example, the term "chimeric transmembrane receptor" includes a plurality of chimeric transmembrane receptors.

[0026] As used herein, the terms "hemagglutinin" and "HA" refer to any hemagglutinin known to those of skill in the art. In certain embodiments, the hemagglutinin is an influenza hemagglutinin, e.g., influenza A hemagglutinin, influenza B hemagglutinin, or influenza C hemagglutinin. Exemplary hemagglutinins contain domains known to those of skill in the art, including a signal peptide, a stem domain, a globular head domain, a luminal domain, a transmembrane domain, and a cytoplasmic domain.

[0027] As used herein, the terms "stem domain polypeptide," "HA stem domain," "influenza virus hemagglutinin stem domain polypeptide," and "HA stalk domain" refer to a polypeptide comprising or consisting of one or more polypeptide chains that make up the A stem domain of influenza hemagglutinin. A domain polypeptide may be one polypeptide chain, two polypeptide chains, or more polypeptide chains.

[0028] As used herein, the terms "influenza virus hemagglutinin head domain polypeptide," "influenza virus hemagglutinin head domain," "HA globular head domain," and "HA head domain" refer to the globular head domain of an influenza hemagglutinin polypeptide.

[0029] As used herein, the term "antigen" is defined as any substance that is capable of eliciting an immune response.

[0030] As used herein, the term "immunogenicity" refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.

[0031] As used herein, the term "epitope" is defined as the portion of an antigen molecule that makes contact with the antigen-binding site of an antibody or T-cell receptor.

[0032] As used herein, the term "vaccine" refers to an antigen-containing preparation consisting of a whole disease-causing organism (killed or weakened) or a component part of such an organism, such as a protein, glycoprotein, peptide, glycopeptide, glycolipid, polysaccharide, or any combination thereof, that confers immunity to the disease caused by the organism. Vaccine preparations may be natural, synthetic, or obtained by recombinant DNA technology.

[0033] As used herein, the term "antigen-specific" refers to the property of a cell population to present a particular antigen, or fragment of an antigen, resulting in specific cell proliferation.

[0034] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic effect.

[0035] A "therapeutically effective amount" of a substance / molecule of the invention can vary according to factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the substance / molecule to elicit a desired response in that individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the substance / molecule. A "prophylactically effective amount" refers to an amount effective, at dosages for periods of time necessary, to achieve the desired prophylactic effect. Typically, but not necessarily, a prophylactically effective amount will be less than the therapeutically effective amount because a prophylactic dose is used in subjects prior to contracting the disease or at an earlier stage of the disease.

[0036] A consensus DNA sequence of avian influenza H5 (pCHA5-II) was used as a vaccine for administration in mice, and the results showed broad protection against various H5 subtypes (Chen, MW et al. Broadly neutralizing DNA vaccine with specific mutation alters the antigenicity and sugar-binding activities of influenza hemagglutinin. Proc. Natl Acad. Sci. USA 108, 3510-3515 (2011)). This disclosure reports the design and evaluation of various chimeric vaccines based on the most common avian influenza H5 and human influenza H1 sequences. Among these constructs, a chimeric HA (cHA) vaccine with consensus H5 as the globular head and consensus H1 as the stem was the best, and showed strong CD4 + and CD8 + It has been shown that monoglycosylated cHA (cHAmg) vaccines, which have only GlcNAc at each glycosylation site, induced more stem-specific antibodies with higher antibody-dependent cellular cytotoxicity (ADCC), better neutralizing activity against H1, H3, H5, and H7 strains and subtypes, and stronger cross-protective activity. Furthermore, cHAmg vaccines combined with glycolipid adjuvants designed for class switching further increased vaccine efficacy and induced more IFN-γ, IL-4, and CD8. + This was accompanied by memory T cell production.

[0037] Chimeric influenza virus hemagglutinin (HA) polypeptides The present disclosure relates to CD4 + and CD8 + Chimeric influenza virus hemagglutinin (HA) polypeptides are provided for use as immunogens to elicit T cell immune responses or as vaccines, and thus can prevent influenza virus disease in a subject.

[0038] Chimeric influenza virus hemagglutinin (HA) polypeptides of the present disclosure comprise one or more stem domain sequences each having at least 60% homology to the stem domain consensus sequence of an H1 subtype HA (H1 HA) and / or an H5 subtype HA (H5 HA), fused to one or more globular head domain sequences each having at least 60% homology to the globular head consensus sequence of an H1 subtype HA (H1 HA) or an H5 subtype HA (H5 HA).

[0039] As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. Polymer molecules (e.g., nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules) that share a threshold level of similarity or identity, as determined by alignment of matching residues, are said to be homologous. Homology is a qualitative term that refers to the relationship between molecules and can be based on quantitative similarity or identity. Similarity and identity are quantitative terms that define the degree of sequence matching between two compared sequences. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar.

[0040] In some embodiments, polypeptides according to the present disclosure may comprise one or more sequences having at least 60% homology to the consensus sequence of H1 HA or H5 HA across known human and avian influenza virus strains. In some embodiments, the homology is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the stem domain sequence is the N-terminal stem segment of an H1 HA or the C-terminal stem segment of an H1 HA; the N-terminal stem segment of an H1 HA or the C-terminal stem segment of an H1+H5 HA sequence; or the N-terminal stem segment of an H5 HA or the C-terminal stem segment of an H1+H5 HA sequence.

[0041] In some embodiments, the H1 HA and / or H5 HA stem domain consensus sequence comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:10.

[0042] Sequence number 1 (H1 stem) DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKL

[0043] Sequence number 2 (H1 stem) NTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAV GKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQ

[0044] Sequence number 5 (H1 stem) DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKL

[0045] Sequence number 6 (H1+H5 stem) NTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQ FEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGV

[0046] SEQ ID NO: 9 (H5 stem) DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKL

[0047] Sequence number 10 (H5+H1 stem) NTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGRNSPQRERRRKKR GLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGV

[0048] In one embodiment, the H1 HA or H5 HA globular head domain consensus sequence comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:7 or SEQ ID NO:11.

[0049] Sequence number 3 (H5 globular head) CDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGKSSFFRNVVWLIKKNSTY PTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNC

[0050] Sequence number 7 (H1 spherical head) CKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSY PKLSKSYINDKGKEVLVLWGIHHPSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDC

[0051] Sequence number 11 (H5 globular head) CDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGKSSFFRNVVWLIKKNSTY PTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNC

[0052] In one embodiment, the chimeric influenza virus HA polypeptide comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:12.

[0053] SEQ ID NO: 4 (chimera H5 / 1) DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQ

[0054] Sequence number 8 (Swap H1 / 5) DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGV

[0055] Sequence number 12 (Swap H5 / 1) DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHE ASSGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYA YKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGRNSPQRERRRKKRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKIT NKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGV

[0056] In some embodiments, one or more glycosylation sites on HA are monoglycosylated to increase immunogenicity. Preferably, monoglycosylated HA has only N-acetylglucosamine (GlcNAc) at each glycosylation site.

[0057] Chimeric influenza virus HA polypeptides can be produced by any suitable method. Many such methods are known to those of skill in the art. For example, proteins can be chemically synthesized or produced using recombinant DNA technology (e.g., in bacterial cells, in cell culture (mammalian cells, yeast cells, or insect cells), in plants or plant cells, or by cell-free prokaryotic or eukaryotic-based expression systems, other in vitro systems, etc.). Accordingly, the present disclosure provides recombinant polynucleotides comprising a nucleic acid sequence encoding a polypeptide of the present disclosure and, optionally, a nucleic acid sequence encoding a signal peptide. The present disclosure provides vectors comprising recombinant polynucleotides of the present disclosure. Embodiments of polypeptides of the present disclosure are described herein. In one embodiment, the signal peptide comprises the sequence of SEQ ID NO: 13 (MEKIVLLLAIVSLVKS) or SEQ ID NO: 14 (MKAILVVLLYTFATANA). Host cells comprising vectors of the present disclosure are also provided.

[0058] immunogenic composition The immunogenic composition preferably comprises at least one pharmaceutically acceptable carrier and / or adjuvant. In one embodiment, the adjuvant is a glycolipid adjuvant. Examples of adjuvants include, but are not limited to, Al(OH)3, AlPO4, C34, squalene, and QS21.

[0059] The chimeric influenza virus HA polypeptides of the present disclosure can be formulated or administered in combination with one or more pharmaceutically acceptable excipients. Immunogenic / vaccine compositions can be sterile, pyrogen-free, or both sterile and pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceuticals, e.g., vaccine compositions, can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0060] Immunogenic compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, protective, and immunogenic. The amount administered will depend on the subject being treated, including, for example, the capacity of the individual's immune system to synthesize antibodies and, if necessary, generate a cell-mediated immune response. Precise amounts of active ingredient required for administration depend on the judgment of the clinician. However, appropriate dosage ranges can be readily determined by one skilled in the art. Suitable regimens for initial and booster doses also vary, but can include an initial administration followed by subsequent administrations. The dosage of the vaccine will also depend on the route of administration and may vary according to the size of the host.

[0061] Formulations of the vaccine compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparatory methods include the step of bringing into association the active ingredient with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping, and / or packaging the product into the desired single- or multi-dose unit.

[0062] Purpose It has been known for some time that cytotoxic T lymphocytes (CTLs) can mount immune responses against influenza virus strains. Recent studies have shown that CTL responses in humans can be directed against multiple epitopes.

[0063] Provided herein are methods for preventing influenza virus disease in humans and other mammals. Also provided are methods for raising an immune response to influenza virus in a subject. The methods comprise inducing an immune response specific to influenza virus strains (e.g., H1, H3, H5, and H7 strains and subtypes) in a subject by administering to the subject an effective amount of a chimeric influenza virus HA polypeptide or immunogenic composition / vaccine of the present disclosure. Preferably, the methods involve ...+ and CD8 + This method elicits a T cell immune response. More preferably, this method induces stem-specific antibodies with higher antibody-dependent cellular cytotoxicity (ADCC), better neutralizing activity, and stronger cross-protective activity against H1, H3, H5, and H7 strains and subtypes. This method also increases vaccine efficacy and is accompanied by increased IFN-γ, IL-4, and CD8+ memory T cell production.

[0064] The antibody titer in the subject increases after vaccination. In an exemplary embodiment, the immune composition or vaccine of the present disclosure is used to provide preventive protection against influenza. Preventive protection against influenza can be achieved after administration of the vaccine or combination vaccine of the present disclosure. Vaccines (including combination vaccines) can be administered once, twice, three times, four times, or more times, although one vaccine administration is likely to be sufficient (optionally followed by one booster). Thus, dosing may need to be adjusted.

[0065] A prophylactically effective dose is a therapeutically effective dose that protects against influenza virus at a clinically acceptable level. In some embodiments, the therapeutically effective dose is the dose listed on the package insert for the vaccine.

[0066] The chimeric influenza virus HA polypeptides or immunogenic compositions / vaccines of the present disclosure can be administered by any route that produces a therapeutically effective result, including, but not limited to, intradermal, intramuscular, and / or subcutaneous administration. In some embodiments, the chimeric influenza virus HA polypeptides or immunogenic compositions / vaccines of the present disclosure can be administered intramuscularly or intradermally, similar to the administration of inactivated vaccines known in the art.

[0067] The invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings, as the invention is capable of other embodiments and of being practiced or carried out in various ways. [Example]

[0068] method Vaccine and Plasmid Construction. A total of 102 full-length HA sequences from H1N1 viruses available from early 2009 to 2013 were downloaded from the NCBI database and aligned using the ClustalW algorithm in the BioEdit program. The most conserved amino acid at each position was selected to create a consensus H1 sequence. The consensus hemagglutinin H5 (pCHA5-II) sequence was generated as previously described. The nucleotide sequences of consensus hemagglutinin H5 (pCHA5-II) and consensus H1 were cloned into the pcDNA expression vector, and the resulting plasmid was used as a template for swap and chimeric HA construction. Swap H1 / 5 is composed of H1, which is HA1 (amino acids 1-327 of SEQ ID NO:8), and H5, which is HA2 (amino acids 328-503 of SEQ ID NO:8), resulting in a globular head H1 and an H1+H5(HA2) stem. Swap H5 / 1 is composed of H5, which is HA1 (amino acids 1-330 of SEQ ID NO:12), and H1, which is HA2 (amino acids 331-506 of SEQ ID NO:12), resulting in a globular head, H5, and an H5+H1 (HA2) stem. For the chimeric H5 / 1 construct, the globular head domain consists of the amino acid sequence between residues C42 and C274 of SEQ ID NO:4 (H3 numbering), and the stem region consists of portions of the HA1 and HA2 subunits (amino acids 1-41 of SEQ ID NO:4 and 275-511 of SEQ ID NO:4). The transmembrane domain was replaced with additional residues from the bacteriophage T4 fibritin foldon trimerization sequence, a thrombin cleavage site, and a (His)6-tag at the C-terminus of HA. Both consensus HA DNA sequences were optimized for expression using human-preferred codons, and various regions were amplified by PCR and subsequently cloned into the pcDNA vector for expression.Furthermore, the HA genes from influenza viruses seasonal H1N1 Brisbane / 59 / 2007, pandemic H1N1 California / 07 / 2009, H3N2 Brisbane / 10 / 2007, H7N9 A / Shanghai / 2 / 2013, and avian influenza H5N1 Vietnam / 1194 / 2004 were also optimized, synthesized, and cloned into pcDNA expression vectors. The sequences were confirmed by DNA sequencing and prepared in high quality for protein expression and purification.

[0069] Expression of recombinant secreted HA from expressing cells. Human epithelial kidney (HEK) 293T and HEK293S cells were routinely maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco). For transient transfection, 293T or 293S cells were seeded in 10 cm dishes (Nunc, Roskilde, Denmark), and all procedures were performed according to the manufacturer's protocol. Briefly, 293T or 293S cells at 80% confluency were transfected in a Mirus TransIT (R) Transfection was performed with TransIT-LT1 (Mirus Bio) transfection reagent at a ratio of 3:1 between reagent and plasmid DNA. (R) The LT1 reagent was diluted with Opti-MEM (Gibco) and the mixture was incubated at room temperature for 5-20 minutes. Plasmid DNA was added to this solution and, after thorough mixing, the mixture was incubated for 15-30 minutes. Prior to transfection, the cells were replaced with fresh DMEM (Gibco) medium supplemented with 10% fetal bovine serum. (R) The -LT1 reagent / DNA complex was added to the cells and incubated for 48 hours at 37° C. Expression of hemagglutinin was confirmed by immunoblotting using an anti-(his)6 antibody (Qiagen) or a specific antihemagglutinin antibody and a horseradish peroxidase (HRP)-conjugated secondary antibody (PerkinElmer).

[0070] Purification of recombinant secreted hemagglutinin. For expression in human 293T cells, high-quality pcDNA carrying the gene of interest was prepared and purified using Mirus TransIT. (R) Cells were transfected with HA-LT1 (MirusBio). 48 hours after transfection, the medium was collected and clarified by centrifugation at 1,000 × g for 10 minutes. The supernatant was purified using a Ni-NTA (nickel-nitrilotriacetic acid) affinity column (GE Healthcare). The supernatant was loaded onto a Ni-NTA affinity column pre-equilibrated in 20 mM Tris-HCl pH 8.0 and 300 mM NaCl. Unbound protein was washed with a 25-50 mM imidazole gradient in 20 mM Tris-HCl pH 8.0 and 300 mM NaCl (Buffer A). HA protein was then eluted with a 100-300 mM imidazole gradient in Buffer A. The purified HA protein was concentrated into PBS, pH 7.4, using an Amicon Ultrafiltration Unit (MW 30K cutoff) (Millipore). Purity was monitored using SDS-PAGE, and the protein was confirmed by Western blot using an anti-(his)6 antibody (Qiagen) or a specific antihemagglutinin antibody and a horseradish peroxidase-conjugated secondary antibody (PerkinElmer). Finally, the trimeric form of the HA protein was obtained by using a size-exclusion column, Superdex 200 Increase 10 / 300 GL gel filtration column (GE Healthcare).

[0071] Preparation of monoglycosylated HA protein. HEK293S cells deficient in N-acetylglucosaminyltransferase I were cultured to express high-mannose glycans. 31 Purified HA protein from HEK293S cells was treated with Endo H (NEB) overnight at 20°C to produce monoglycosylated HA. mgThe protein to Endo H ratio for HA was 3 to 1 (w / v). Endo H and monoglycosylated HA proteins were then separated using a Superdex 200 Increase 10 / 300 GL gel filtration column (GE Healthcare). mg Proteins were concentrated in PBS, pH 7.4, by an Amicon Ultrafiltration Unit (MW 30K cutoff) (Millipore) and confirmed by SDS-PAGE and LC-MS / MS analysis.

[0072] Identification of N-linked glycosylation of HA protein. Ten micrograms of protein were run on SDS-PAGE and prepared for gel digestion. The desired protein band was excised with a sharp scalpel, diced into 1 mm pieces, and placed in a 1.3 ml Eppendorf tube. After washing twice with 500 μl of 25 mM ammonium bicarbonate in 50% ACN (acetonitrile) for 3 min, the gel pieces were dried using a SpeedVac evaporator (Thermo). The dried sample was reduced by adding 100 μl of 50 mM dithiothreitol (DTT) in 25 mM ammonium bicarbonate (pH 8.5) at 37°C for 1 h, followed by centrifugation at 10,000 g for 1 min. The solution was removed, and the gel sample was subjected to the alkylation step by adding 100 μl of 100 mM iodoacetamide (IAA) in 25 mM ammonium bicarbonate (pH 8.5) and incubated for 1 hour at room temperature in the dark. After washing with 500 μl of 50% acetonitrile in 25 mM ammonium bicarbonate (pH 8.5) and 500 μl of 100% acetonitrile, the sample was centrifuged at 10,000 g for 1 minute, and the supernatant was completely removed. The gel sample was dried in a SpeedVac evaporator and redissolved in 200 μl of 25 mM ammonium bicarbonate (pH 8.5). The gel sample was then treated overnight with 0.5 μg trypsin (Promega, Madison, WI, USA) and 1 μg chymotrypsin (Promega, Madison, WI, USA). After overnight digestion, 100 μl of 50% acetonitrile in 5% TFA was added to the sample. The sample was sonicated for 10 seconds, then stopped for 10 seconds. This process was repeated 10 times. The supernatant containing the peptide mixture was removed from the sample tube and transferred to a new tube. This procedure was repeated twice. The combined supernatant was dried in a SpeedVac concentrator and processed for LC-MS / MS analysis.

[0073] Endotoxin measurement. Endotoxin levels were measured using Pierce (R)Protein samples were determined using the LAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific). Protein samples were diluted 10x, 20x, 100x, and 1000x, while endotoxin standards were prepared at 10, 5, 2.5, 1.25, 0.63, 0.31, 0.15, and 0 ng / ml. After equilibrating the microplate in a heating block for 10 minutes at 37°C, protein samples or standards were mixed 1:1 with Limulus AmebocyeLasate (LAL) Pyrochrome reagent (final volume 100 μl) in endotoxin-free wells for 10 minutes at 37°C. 100 μl of substrate solution was added to each well, and the plate was incubated at 37°C for 6 minutes. The reaction was stopped by adding 50 μl of stop reagent (25% acetic acid). The absorbance of the wells was measured at 405 nm using a SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA). A standard curve was obtained by plotting the absorbance versus the concentration of the corresponding standard. The standard curve was used to determine the endotoxin concentration of the samples. The endotoxin values ​​of all purified proteins were less than 0.5 ng / ml.

[0074] Mouse vaccination. The adjuvant C34 was chemically synthesized as described and dissolved in DMSO. Female 6- to 8-week-old BALB / c mice (n = 10 per group) were vaccinated with 20 μg of purified chimeric HA mixed with 50 μg of aluminum hydroxide (alum; Sigma) or 2 μg of C34 in PBS. fg or HA mg Mice were immunized intramuscularly with the protein (pH 7.4). Control mice were injected with phosphate-buffered saline (PBS). Three vaccinations were administered at 2-week intervals. Blood was collected 14 days after the second and third immunizations. The blood was incubated at 37°C for 30 minutes and centrifuged at 1,2000 rpm for 10 minutes to collect serum. HA-specific antibodies in the serum collected from vaccinated mice were assessed by enzyme-linked immunosorbent assay (ELISA) and neutralization assay.

[0075] Determination of HA-specific antibodies by ELISA. HA-specific antibody titers were detected by ELISA using H1N1 A / Brisbane / 59 / 2007, H1N1 A / California / 07 / 2009, H3N2 Brisbane / 10 / 2007, H7N9 A / Shanghai / 2 / 2013, and H5N1 Vietnam / 1194 / 2004 HA proteins as substrates. 96-well ELISA plates (Greiner bio-one, Frickenhausen, Germany) were coated with 100 μl of protein per well diluted at 5 μg / ml in ELISA coating buffer, 100 mM sodium bicarbonate (pH 8.8), and covered with a plastic sealer overnight at 4°C. Plates were blocked with 1% BSA in TBST (137 mM NaCl, 20 mM Tris-base, 0.05% Tween 20, pH 7.4) for 1 h at 37°C, washed three times with TBST, and incubated with 200 μl of two-fold serially diluted mouse serum for 2 h at 37°C. After removing the serum and washing the plate six times, HA-specific IgG was monitored using 200 μl of a secondary HRP-labeled anti-mouse antibody (1:8000) (PerkinElmer, Waltham, MA, USA). After 1 h of incubation at 37°C, the plates were washed six times with TBST and developed with 100 μl of Super Aquablue ELISA substrate (eBioscience, San Diego, CA, USA) for 1 min. The reaction was stopped by adding 100 μl of 0.625 M oxalic acid. The absorbance of the wells was measured at 405 nm using a SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA). The endpoint antibody titer that generated an absorbance (OD) 2.5-fold higher than that generated by the negative control (pre-immune serum) was defined as the highest dilution of serum. A background endpoint antibody titer was defined as less than 1:50.

[0076] Recovery of bone marrow-derived dendritic cells. GM-CSF-cultured bone marrow-derived dendritic cells (BMDCs) were prepared as previously described. Briefly, bone marrow mononuclear suspensions were subjected to RBC lysis to remove red blood cells (RBCs). The remaining cells were cultured in RPMI 1640 supplemented with 10 ml of 20 ng / mL mouse GM-CSF (eBioscience), 10% FBS (BenchMark), 50 μM 2-ME, 100 units / mL penicillin, and 100 μg / mL streptomycin. Cells were plated in individual dishes at 2 × 10 6 A final cell density of 10 cells / dish was achieved. Cultures were replenished by adding 10 ml of fresh culture medium supplemented with 20 ng / mL mouse GM-CSF on day 3 and refreshed with half the volume of the complete culture medium described above on day 6. On day 8, immature BMDCs were harvested by gently pipetting to collect non-adherent cells and then cultured at 10 6 The immature BMDCs were replated at a density of 100 / ml. For CD8+ T cell assays, immature BMDCs were cocultured with CD8+ T cells and chimeric HA protein (0.1 mg / well in 100 μL) for 48 hours. The number of granzyme B-producing CD8+ T cells was determined by flow cytometry analysis after washing.

[0077] Enzyme-linked immunospot (ELISpot) assay. ELISPOT plates were coated with anti-mouse IFN-γ, IL-4 (Mabtech AB, Stockholm, Sweden) or granzyme B (R&D Systems) according to the manufacturer's instructions. Plates were washed four times and incubated with RPMI-1640 supplemented with 10% fetal bovine serum (Gibco) for 30 minutes. For detection of IFN-γ, IL-4 and granzyme B secreting cells from chimera-immunized mice, splenocytes were harvested and 5 × 10 per well. 5The cells were cultured with specific peptides derived from HA for restimulation at 37°C in 5% CO2 for 24 hours. The cells were removed and incubated with biotinylated anti-mouse IFN-γ, IL-4 (Mabtech AB), or granzyme B (R&D Systems) specific antibodies. After washing the plate five times, streptavidin-ALP conjugate was added and developed with ready-to-use BCIP / NPT substrate. After drying, the number of resulting spots was analyzed using an Immune Spot Reader (Cellular Technology Ltd.). Data were obtained from triplicate wells.

[0078] Neutralization assay: 100 TCID 50 Virus-containing culture supernatant was mixed with an equal volume of two-fold serially diluted serum and incubated at 37°C for 1 hour. The mixture was then added to MDCK cells in each well of a 96-well plate and incubated at 37°C for 3 days. 30 μl of CellTiter-Glo (Promega) was added to the cells, and the number of viable cells was determined based on quantitation of ATP present. The neutralizing activity of the serum was determined as the highest dilution at which cells were significantly protected from virus-induced death.

[0079] Microneutralization assay: 100 TCID of virus 50 Infection medium (DMEM supplemented with 0.3% BSA, 2 μg / ml TPCK-trypsin) containing 1000 μg / ml of sera was mixed with equal volumes of two-fold serial dilutions of serum and incubated for 1 hour at 37°C. This mixture was then transferred to MDCK cells (1.5 × 10 per well) in each well of a 96-well plate. 4The wells were washed six times with PBST, and 100 μl of a secondary HRP-conjugated anti-rabbit antibody (1:5000) (PerkinElmer, Waltham, MA, USA) was added. After 1 h of incubation at 37°C, the wells were washed six times with PBST and developed with 50 μl of 1Step Ultra TMB substrate (Thermo) for 1 min. The reaction was stopped by adding 50 μl of 1 M H2SO4. The absorbance of the wells was measured at 450 nm using a SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA).

[0080] Antibody-dependent cell-mediated cytotoxicity reporter assay. MDCK cells (1 × 10 per well) were cultured in each well of a 96-well flat-bottom plate. 4 The cells were incubated at 37°C for 24 hours. The next day, 1 x 10 4MDCK cells were infected with influenza virus at a multiplicity of infection (MOI) of 1 for 24 hours. The medium was then replaced with Roswell Park Memorial Institute (RPMI) medium 1640 supplemented with 4% low IgG serum, after which serial dilutions of antisera from mice vaccinated with chimeric HA protein were added and incubated at 37°C for 30 minutes. Jurkat effector cells expressing mouse FcγRIII (Promega) were suspended in RPMI 1640 medium containing 4% low IgG FBS and added to the infected MDCK cells at a target cell:effector cell ratio of 1:5. After incubation at 37°C for 6 hours, the assay plate was removed from the 37°C incubator and equilibrated to ambient temperature for 15 minutes before being analyzed by Bio-Glo. TM Luciferase Assay Buffer (Promega) was added at a ratio of 1:1. Luminescence was measured in a CLARIOstar plate reader.

[0081] Virus challenge experiments. Two weeks after three vaccinations at 2-week intervals, immunized mice were challenged with 10 LD of H1N1 California / 07 / 2009, H1N1 A / New Caledonia / 1999, H1N1 A / WSN / 1933, H1N1 A / Solomon Islands / 03 / 2006, and reassortant H5N1 viruses A / Vietnam / 1194 / 2004 / NIBRG14 and H5N1A / Turkey / 1 / 2005 / NIBRG23. 50 Mice were challenged intranasally with 100 mg of 1000 ribosomal RNA (a viral dose resulting in the death of 50% of mice). After infection, mice were observed daily for 14 days, and survival and body weight were recorded. Body weight percentage was calculated for each animal per group by comparing daily body weight with pre-challenge body weight, and mice that lost more than 25% of their initial weight were sacrificed and scored as dead. Mouse studies were approved by the Institutional Animal Care and Use Committee of Academia Sinica. All animal experiments were performed under extended biosafety level-3 conditions.

[0082] Expression and purification of recombinant F10 antibody. Plasmids encoding the F10 antibody were transfected into serum-free, conditioned FreeStyle™ 293F cells using polyethyleneimine and cultured in FreeStyle™ 293 expression medium (Gibco) in 125 ml sterile Erlenmeyer flasks rotating at 135 rpm on an orbital shaker platform. Supernatants were collected 72 hours after transfection, and cells were clarified by centrifugation at 1,000 × g for 10 minutes. The supernatant was loaded onto a Protein-A column (GE Healthcare) pre-equilibrated with 5 column volumes (CV) of phosphate-buffered saline (PBS) wash buffer (pH 7.0) followed by 5 CV of wash buffer. F10 antibody was eluted with 0.2 M glycine buffer (pH 2.5), and fractions were collected into tubes containing 0.5 mL of 1 M Tris-HCl pH 9.0 for neutralization. Purification was monitored using SDS-PAGE.

[0083] Statistical analysis. Animal experiments used to evaluate immune responses were repeated at least three times (n = 5 per group), and virus challenge studies were performed at least twice (n = 10 per group). For statistical analysis, the response of each mouse was counted as an individual data point. Data obtained from animal studies were examined using two-way ANOVA from Prism; data were expressed as mean ± SEM, and differences were considered significant at *P < 0.05; **P < 0.01; ***P < 0.001.

[0084] Example 1 Preparation and characterization of monoglycosylated chimeric HA. To design a universal vaccine, we first aimed to obtain a vaccine with broad protection against influenza A virus group 1 (H1 and H5 are the major subtypes, while H2, H6, and H9 are minor subtypes). Therefore, we created a consensus H1 sequence using HA sequences from H1N1 viruses available from early 2009 to 2013. The consensus H5 and H1 were then used as templates for vaccine design. During influenza virus replication, the HA precursor (HA0) is proteolytically cleaved into two subunits, HA1 and HA2; the HA1 subunit carries the 5-N-acetylneuraminic acid (sialic acid) binding site, and the HA2 subunit is responsible for viral fusion with the host cell membrane (Figure 5A). On the other hand, HA can be divided into two structural domains, a globular head and a stem, based on its three-dimensional (3D) structure. The stem region includes the HA2 domain, the N-terminal 36-50 residues, and a short C-terminal stretch of the HA1 domain. Therefore, we designed vaccines based on various combinations of domains from H1 and H5. For comparison, we first created swap H1 / 5 (H1 globular head and [H1 + H5(HA2) stem]), swap H5 / 1 (H5 globular head and [H5 + H1(HA2) stem]), and chimeric H5 / 1 (H5 globular head and H1 stem) (Fig. 1A and Fig. 5A). The results showed that immunization with common H1N1 and swap H1 / 5 did not induce cross-protective activity, but swap H5 / 1 and chimeric H5 / 1 elicited cross-neutralizing activity against H1N1 and H5N1 viruses (Fig. 1B). We then investigated whether this cross-protection was mediated by CD8 + We investigated whether this was due to a T cell response and found that granzyme B was secreted at a higher level in chimeric H5 / 1-immunized mice, suggesting that the chimeric H5 / 1 vaccine induced a stronger CD8 T cell response compared to the swap H5 / 1 vaccine. + This suggests that it induces a T cell response.

[0085] Example 2 Effect of glycosylation on the immune response of chimeric H5 / 1(cHA) To investigate the immunogenicity of chimeric H5 / 1 (cHA) vaccines with different glycosylation states, monoglycosylated cHA (cHA mg ) and fully glycosylated cHA (cHA fg ) vaccines were compared (Figure 5). Endo-H is specific for high mannose, but not complex glycans. HA glycoproteins expressed in HEK293S cells, which are deficient in N-acetylglucosaminyltransferase I and produce glycoproteins with high mannose N-glycans, were treated with Endo-H to cleave the N-glycans to a single GlcNAc residue. cHA mg To produce cHA, cHA was produced from human cells (HEK293S), and purified cHA with high-mannose glycans was treated with Endo-H to remove the outer portion of the N-glycans and generate HA with only one N-acetylglucosamine (GlcNAc) attached to an asparagine residue at each glycosylation site. After Endo-H treatment, the mixture was gel-filtered, and Endo-H was used to separate trimeric cHA. mg After concentration, cHA mg The protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS / PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis to confirm purity and glycan composition (Figure 5C). Because influenza HA exists as a trimer on the viral surface, gel filtration was performed to isolate cHA. fg and cHA mg We confirmed that cHA exists as a trimer (over 200 kDa) (Fig. 5D). For comparison, we also analyzed another fully glycosylated cHA derived from human cells (HEK293T). fg (Figure 5B). This cell culture produced cHA at a yield of approximately 6 mg / L. fg This resulted in...

[0086] Recombinant cHA fg and cHA mgThe N-linked glycosylation sites and glycan profile of cHA were analyzed by LC-MS / MS, which showed seven glycosylation sites (N28, N40, N171, N182, N292, N303, and N497); fg The N-glycans of cHA are mostly complex type. mg Approximately 99% of the glycoforms can be obtained as a single glycoform with only GlcNAc at each of the N-glycosylation sites (Figure 5E and Table 1).

[0087] [Table 1] TIFF0007783193000002.tif199166

[0088] Example 3 Fully glycosylated chimeric H5 / 1 (cHA fg ) and monoglycosylated chimeric H5 / 1 (cHA mg Cross-reactivity of antisera from mice immunized with To evaluate the binding activity of antibodies elicited by the cHA constructs, BALB / c mice were immunized intramuscularly with 20 μg of cHAfg or cHAmg protein adjuvanted with Al(OH)3 or C34 (an analog of α-galactosylceramide (α-GalCer)). The mice were immunized at 0, 2, and 4 weeks, and HA-induced sera were obtained on days 28 and 42 and measured using enzyme-linked immunosorbent assays (ELISAs) with various recombinant HAs (Figure 6). Compared with the maximum dilution of antisera after two immunizations, three immunizations indeed produced antisera with higher titers of HA-specific antibodies (Figures 1D-I and 7). Furthermore, cHA mg Vaccination with cHA fg cHA induced better antibody responses than cHA (Fig. 1D, E, and G). mgAntisera from the HA vaccine showed slightly better binding to H3 and H7 HA proteins (Fig. 1F and H), and no significant differences were observed between the Al(OH)3 and C34 adjuvants. These data indicate that the cHA vaccine was able to elicit cross-reactive antibodies that recognized HA from H1N1, H3N2, H5N1, and H7N9 strains.

[0089] F10 is a broadly neutralizing IgG antibody known to target the HA stem region, which is highly conserved among various influenza virus subtypes. To compare the binding of F10 to recombinant H1, H5, and cHA, the binding avidity of F10 to various HAs was measured. The results showed that F10 could bind to H1, H5, and cHA proteins (Figure 8). To examine whether F10-like antibodies are elicited by cHA vaccination, the binding of serum induced by cHA to HA stem number 4900 was measured using ELISA. The results indicated that F10-like antibodies were elicited by cHA vaccination. mg The vaccine is fg These results show that the cHA vaccine can induce higher stem-specific antibody titers than the C34 adjuvant (Figure 1I and Figure 7F), and better results were observed with the cHA vaccine adjuvanted with C34 (Figure 1I), which induced more stem-specific antibodies.

[0090] Example 4 cHA mg Vaccination of mice with the adjuvant C34 induced strong CD4 responses against H1, H3, and H5 viruses and their subtypes. + and CD8 + Induce T cell responses, antibody-dependent effector functions, and neutralizing activity In addition to antibody-mediated neutralization, Fc-mediated effector function also plays an important role in protection against influenza infection. Therefore, we tested whether antibodies induce Fc receptor-mediated immune responses. A mouse-adapted ADCC assay was performed using Jurkat effector cells expressing FcγRIII to evaluate the ADCC activity of sera from mice immunized with cHAfg and cHAmg (Figure 2). As expected, cHA fg or cHA mg Sera from mice vaccinated with cHA induced comparable levels of ADCC activity against H5N1 NIBRG14 (A / Vietnam / 1194 / 2004), NIBRG23 (A / Turkey / 1 / 2005), RG5 (A / Anhui / 1 / 2005), or RG2 (A / Indonesia / 5 / 2005) viruses. Interestingly, better ADCC activity was observed with cHA vaccinated with Al(OH)3. mg (Fig. 2B), and similar results were observed in experiments against H1N1 A / California / 07 / 2009, A / Brisbane / 59 / 2007, A / Solomon Islands / 3 / 2006, A / New Caledonia / 20 / 1999 (Fig. 2A), H3N2 A / Wisconsin / 67 / 2005, and A / Victoria / 361 / 2011 viruses (Fig. 2C).

[0091] To evaluate the role of antigen-specific cytokine-secreting cells in cHA-immunized mice, splenocytes were collected after the second and third immunizations, and IFN-γ, IL-4, and granzyme B (GzB)-secreting cells were evaluated by enzyme-linked immunosorbent spot (ELISpot) assay using specific peptides derived from HA for stimulation. As shown in Figure 3, cHA adjuvanted with Al(OH)3 fg and cHA mg The vaccine produced similar levels of cytokine-secreting cells, but more CD4 + / IFN-γ + Th1 cells (Fig. 3A), CD4 + / IL-4+ Th2 (Figure 3B) and CD8 + GzB-secreting cells (Fig. 3C) were more sensitive to cHA adjuvanted with C34 than with Al(OH)3. mg These results were consistent with those of the C34-adjuvanted cHA vaccine. mg However, cHA fg More CD4 compared to + T-helper responses and stronger CD8 + It was confirmed that it stimulated cytotoxic effects.

[0092] To assess the dose-dependence of C34 on antibody titers and cell-mediated immunity, mice were treated with three different doses of C34-adjuvanted cHA: 0.5, 2, and 10 μg. fg The results showed that after two or three immunizations, 2 μg of cHA adjuvanted with C34 was fg The results showed that cHA adjuvanted with 2 μg of C34 induced higher titers than 0.5 and 10 μg of C34 after three immunizations (Figure 9). fg The vaccine induced more IFN-γ than 0.5 and 10 μg of C34 (FIG. 10A), and 2 and 10 μg of C34 induced more IL-4 than 0.5 μg of C34 (FIG. 10B). fg After two and three immunizations, CD8 + There was no difference in terms of expansion in GzB-secreting cells (FIG. 10C). Based on these observations, 2 μg of C34 was used throughout the experiment.

[0093] The neutralizing activity of the antisera induced by cHA was further investigated. mgAntisera from the vaccinated individuals showed better neutralizing activity against the homologous virus H1N1 A / California / 07 / 2009 (Fig. 3D) and the heterologous viruses H5N1 NIBRG14 (A / Vietnam / 1194 / 2004), NIBRG23 (A / Turkey / 1 / 2005), RG5 (A / Anhui / 1 / 2005), or RG2 (A / Indonesia / 5 / 2005) (Fig. 3E). mg Antisera from mice vaccinated with cHA showed significant neutralizing activity against heterologous viruses H1N1 A / Brisbane / 59 / 2007, A / New Caledonia / 20 / 1999, and A / Solomon Islands / 3 / 2006 (Fig. 3D). Antisera from cHA-immunized mice were clearly able to block infection with H1N1 and H5N1 viruses, and cHA mg The neutralizing activity of cHA is generally fg higher than that, especially for heterologous viruses.

[0094] Example 5 In a challenge study, cHA mg Vaccination of mice with / C34 provides cross-protection against H1N1 and H5N1 and their subtypes cHA mg To assess whether vaccination provides broadly cross-protective immunity against various H1N1 and H5N1 viruses, vaccinated mice were challenged intranasally with lethal doses of multiple H1N1 and H5N1 viruses, and the efficacy of vaccine protection was assessed over 14 days by recording survival and weight change (Figures 4 and 11). For mice challenged with the H1N1 A / California / 07 / 2009 virus, all cHA vaccines provided 100% protection (Figure 4A). Furthermore, the cHA vaccines adjuvanted with C34 provided 100% protection. mg Mice immunized with cHA fg cHA with C34 as an adjuvant showed minimal weight loss compared to cHA with C34 as an adjuvant (Figure 11A). fgMice immunized with cHA adjuvanted with C34 were only 30% protected against A / New Caledonia / 1999 challenge; mg The vaccine provided 90% protection against the cross-strain A / New Caledonia / 1999 virus, and similar results were observed with cHA vaccination using Al(OH)3 as an adjuvant (Figure 4B). For mice challenged with the cross-strain A / WSN / 1933 virus, all mice immunized with cHA using Al(OH)3 as an adjuvant survived; however, cHA using C34 as an adjuvant did not. fg Mice immunized with cHA obtained only 80% protection (Figure 4C). A lethal challenge with A / Solomon Islands / 03 / 2006 was also performed. All mice immunized with cHA adjuvanted with Al(OH)3 showed lower protection; however, mice immunized with cHA adjuvanted with C34 showed lower protection. mg Mice immunized with cHA showed better protection against the cross-strain A / Solomon Islands / 03 / 2006 virus (Fig. 4D). For mice challenged with H5N1 NIBRG14 (A / Vietnam / 1194 / 2004) and NIBRG23 (A / Turkey / 1 / 2005), all immunized mice survived (Fig. 4E and F). Body weight change after survival challenge was also assessed (Fig. 11). The data demonstrate that cHA is effective in eliciting significant protective immunity against various H1N1 and H5N1 viruses, and that cHA mg However, cHA fg It has been shown to provide broader cross-protective capabilities compared to

[0095] There is currently interest in developing a universal influenza vaccine to provide protection against multiple strains and subtypes of influenza virus. Epitopes used for universal vaccine development include the highly conserved ectodomain of M2, including the 24 non-glycosylated amino acids, the nucleoprotein NP, and various HA constructs that target the HA-stem region or block viral entry and induce higher titers of broadly neutralizing antibodies. For example, a soluble trimeric HA (mini-HA) vaccine with rearranged stem subunits has been shown to fully protect mice from lethal challenge with heterologous and different subtype viruses, and chimeric HA vaccination with DNA prime-protein boost and exposure to the same stem region and various foreign head domains has been shown to elicit broadly protective stem-specific antibodies. However, this result is limited to CD8 + This indicates that T cells did not play a key role in cross-protective activity. DNA vaccines are promising but are still in the early stages of development. In this study, a cHA construct expressing the common H5 in the globular head and the common H1 in the stem region was designed to mimic the actual situation of influenza viruses transmitted from avian viruses to humans. Fully glycosylated cHA fg and monoglycosylated cHA mg Both were prepared for comparison, and the results were mg The vaccine is + and CD8 + Through T cell responses (Fig. 3A-C), higher titers of cross-reactive antibodies against H1, H3, H5, and H7 subtypes were elicited (Fig. 1D-H).

[0096] It has been shown that glycosylation of HA plays an important role in regulating protein folding and stability, as well as its biological activity, including protecting antigenic sites from neutralizing antibodies, thereby reducing immunogenicity. Furthermore, hyperglycosylated HA evolves to mask antigenic sites in the hypervariable head domain, thereby redirecting the immune response to the conserved stem region. In our results, cHA mg The neutralizing activity of the antiserum was fg The antisera were significantly superior to those induced by cHA, particularly against heterologous H1N1 A / Brisbane / 59 / 2007, A / Solomon Islands / 03 / 2006, and A / New Caledonia / 20 / 1999 (Fig. 3D). mg The broader neutralizing activity of the vaccine is probably due to the induction of many antibody variants, as previously reported. IgG is the predominant antibody present in mice and is the major subtype of HA-specific antibody with high avidity for the FcγRIII receptor on immune cells, which induces ADCC. We found that the cHA mg We showed that immunization with IFN-γ induced higher ADCC and induced more stem-specific antibodies with better protective activity (Figures 1I and 2). This is consistent with studies showing that ADCC is necessary for influenza protection in vivo. Aluminum hydroxide (alum) is known to stimulate Th2 responses and is approved for use as a vaccine adjuvant by the FDA; however, its mode of action has not been fully studied. The glycolipid C34 is a ligand for and presented by CD1d on dendritic cells, interacting with receptors on invariant natural killer T (iNKT) cells, stimulating iNKT cells to produce Th1 cytokines (e.g., IFN-γ) with adjuvant effects and Th2 cytokines (e.g., IL-4) with class-switching activity. Our results suggest that IFN-γ (a Th1 cytokine), IL-4 (a Th2 cytokine)-secreting cells and granzyme B-producing CD8 +The number of T cells was significantly higher with cHA adjuvanted with C34 than with Al(OH)3. mg Immunization with HIV significantly increased the expression of HIV-1 (Fig. 3A-C).

[0097] In summary, there is currently interest in developing next-generation influenza vaccines with broadly protective immune responses, and some promising results have been reported that put the development of a universal vaccine within reach. In an effort toward this goal, in this study, the inventors have successfully demonstrated proof-of-principle that a monoglycosylated cHA vaccine with a common H5 head and common H1 stem is an effective influenza vaccine that exhibits broad protective activity against heterologous influenza viruses (including H1, H3, H5, and H7 viruses and subtypes in neutralization studies, and H1N1, H5N1, and subtypes in challenge studies). Given this success in developing a broadly protective vaccine against different strains and subtypes of influenza A virus, the inventors aim to use the strategy developed in this study to design a broader universal vaccine against influenza A and B viruses.

Claims

1. 1. A chimeric influenza virus hemagglutinin (HA) polypeptide comprising one or more stem domain sequences each having at least 90% identity to a stem domain consensus sequence of an H1 subtype HA (H1 HA) and / or an H5 subtype HA (H5 HA), fused to one or more globular head domain sequences each having at least 90% identity to a globular head domain consensus sequence of an H1 subtype HA (H1 HA) or an H5 subtype HA (H5 HA), A chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide comprises the amino acid sequence of SEQ ID NO:

4.

2. The polypeptide of claim 1 , wherein the HA is influenza A HA, influenza B HA, or influenza C HA.

3. 2. The polypeptide of claim 1, wherein the stem domain sequence is an N-terminal stem segment of an H1 HA or a C-terminal stem segment of an H1 HA; an N-terminal stem segment of an H1 HA or a C-terminal stem segment of an H1+H5 HA sequence; or an N-terminal stem segment of an H5 HA or a C-terminal stem segment of an H1+H5 HA sequence.

4. The polypeptide of claim 1, wherein one or more glycosylation sites in the HA are monoglycosylated, and the monoglycosylated HA has only N-acetylglucosamine (GlcNAc) at each glycosylation site.

5. The polypeptide of claim 1 , wherein the chimeric influenza virus HA polypeptide is used as an immunogen.

6. An immunogenic composition comprising the chimeric influenza virus HA polypeptide of any one of claims 1 to 5 and an adjuvant.

7. The immunogenic composition of claim 6 , wherein the adjuvant is a glycolipid adjuvant.

8. A pharmaceutical composition for immunizing a subject against influenza virus or preventing influenza virus disease, comprising an effective amount of a chimeric influenza virus hemagglutinin (HA) polypeptide of any one of claims 1 to 5 or the immunogenic composition of claim 6.

9. The immune system is CD4 ＋ and CD8 ＋ The pharmaceutical composition of claim 8, which elicits a T cell immune response.

10. The pharmaceutical composition of claim 8, wherein immunization induces stem-specific antibodies with higher antibody-dependent cellular cytotoxicity (ADCC), better neutralizing activity, and stronger cross-protective activity against H1, H3, H5, and H7 strains and subtypes.

11. Immunity increases with more IFN-γ, IL-4 and CD8 ＋ The pharmaceutical composition of claim 8, which enhances vaccine efficacy by generating memory T cells.

12. A recombinant polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of claims 1 to 5.

13. 13. The recombinant polynucleotide of claim 12, further comprising a nucleic acid sequence encoding a signal peptide, wherein the signal peptide comprises the sequence of SEQ ID NO: 13 or SEQ ID NO:

14.

14. A vector comprising the recombinant polynucleotide of claim 13.

15. A host cell comprising the vector of claim 14.

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