Chimeric influenza vaccine

Abstract

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

Description

Technology Field

[0001] Reference to related applications

[0002] This application claims priority to U.S. provisional application serial number 63 / 022.328 filed May 8, 2020, which is incorporated herein by reference in its entirety for all purposes.

[0003] Sequence list

[0004] The present invention contains a sequence list submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. The above ASCII copy created on May 6, 2021, is named G4590-08600PCT_SeqListing.txt and has a size of 28 kilobytes.

[0005] Field of invention

[0006] The present disclosure relates to a chimeric influenza virus hemagglutinin (HA) polypeptide, an immunogenic / vaccine composition containing the same, and the application thereof. Background Technology

[0007] The traditional method for producing influenza vaccines involves culturing the virus in Specific Pathogen-Free (SPF) incubated eggs, a process that often requires more than six months for mass production. However, some vaccine virus strains grow poorly in eggs, raising safety concerns for individuals with egg allergies. While new approaches based on viral cell culture have been developed to replace egg-based methods, these methods still carry the risk of generating potentially harmful viruses. To overcome these issues, the search for 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 generally cannot effectively neutralize other influenza subtypes. Furthermore, such vaccines must be updated annually due to the constant mutation of the virus.

[0008] Therefore, there is still a need to develop a universal vaccine against a wide range of influenza virus strains.

[0009] In one embodiment, the present disclosure provides a chimeric influenza virus hemagglutinin (HA) polypeptide comprising one or more stem domain sequences having at least 60% homology to the stem domain common sequence of subtype HA (H1 HA) and / or H5 subtype HA (H5 HA), each fused with one or more globular head domain sequences having at least 60% homology to the globular head domain consensus sequence of H1 subtype HA (H1 HA) or H5 subtype HA (H5 HA).

[0010] In some embodiments, HA is influenza A HA, influenza B HA, or influenza C HA.

[0011] In some embodiments, homology is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

[0012] In some embodiments, the stem domain sequence is an N-terminal stem segment of H1 HA or a C-terminal stem segment of H1 HA; an N-terminal stem segment of H1 HA or a C-terminal stem segment of an H1+H5 HA sequence; or an N-terminal stem segment of H5 HA or a C-terminal stem segment of an H1+H5 HA sequence.

[0013] In some embodiments, the common sequence of the stem domain 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.

[0014] In some embodiments, the common sequence of the spherical head domain of H1 HA or H5 HA includes the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO: 11.

[0015] 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.

[0016] In some embodiments, one or more glycosites on HA are monoglycosylated. In additional embodiments, the monoglycosylated HA has only N-acetylglucosamine (GlcNAc) on each glycosite.

[0017] In one embodiment, a chimeric influenza virus HA polypeptide is used as an immunogen.

[0018] In another aspect, the present disclosure provides an immunogenic composition comprising a chimeric influenza virus HA polypeptide and an adjuvant. In one embodiment, the adjuvant is a glycolipid adjuvant.

[0019] In another aspect, the present disclosure provides a recombinant polynucleotide comprising a nucleic acid sequence encoding the polypeptide of the present 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.

[0020] In another aspect, the present disclosure provides a vector comprising the recombinant polynucleotide of the present disclosure. Additionally, a host cell comprising the vector of the present disclosure is provided.

[0021] In another aspect, the present disclosure provides a method for immunizing a subject against an influenza virus, comprising administering an effective amount of the chimeric influenza virus hemagglutinin (HA) polypeptide or immunogenic composition of the present disclosure to the subject.

[0022] In another aspect, the present disclosure provides a method for preventing influenza virus disease in a subject, comprising administering an effective amount of the chimeric influenza virus hemagglutinin (HA) polypeptide or immunogenic composition of the present disclosure to the subject.

[0023] In one embodiment, the method described herein is CD4 + and / or CD8 + It triggers a T-cell immune response.

[0024] In one embodiment, the method described herein induces stem-specific antibodies having higher antibody-dependent cellular cytotoxicity (ADCC), better neutralization, and stronger cross-protective activity against H1, H3, H5, and H7 strains and subtypes.

[0025] In one embodiment, the method described herein enhances vaccine efficacy by producing more IFN-γ, IL-4, and CD8+ memory T cells. Brief explanation of the drawing

[0026] FIGS. 1a to 1i. Common H5 spherical head and common H1 stem (cHA) and cHA mg Chimeric H5 / 1 components having broad cross-protective stem-specific antibodies induced by vaccination with an immunogen (Fig. 1a) Compositions of swab H1 / 5 (H1 globular head and H1+H5[HA2] stem), swab H5 / 1 (H5 globular head and H5+H1[HA2] stem), and chimeric H5 / 1 (cHA: H5 globular head and H1 stem). (Fig. 1b) Neutralizing activity against H1N1 California / 07 / 2009 and H5N1 Vietnam / 1194 / 2004 viruses. (Fig. 1c) CD8 in splenocytes stimulated with HA (black bars) or PBS (white bars) controls for 2 days in mice vaccinated with PBS (control), HA+Alu, or HA+C34. + The number of T cell-generating granzyme B (GrzB) was evaluated by flow cytometry. (Figs. 1d to 1i) Al(OH)3-assisted cHA fg and cHA mg cHA aided by C34 fg and cHA mgAntibody titers from mice vaccinated by [the virus] were measured on day 42 by ELISA with A / California / 07 / 2009 H1N1 HA protein (Fig. 1d), A / Brisbane / 59 / 2007 H1N1 HA protein (Fig. 1e), A / Brisbane / 10 / 2007 H3N2 HA protein (Fig. 1f), A / Vietnam / 1194 / 2004 H5N1 HA protein (Fig. 1g), A / Shanghai / 2 / 2013 H7N9 HA protein (Fig. 1h) and A / Brisbane / 59 / 2007 (Bris / 07) stem HA (no. 4900) protein (Fig. 1i) as coating antigens. The endpoint antibody titer was defined as the final dilution of antiserum that produced an absorbance 2.5 times higher than the optical absorbance produced by the negative control (pre-immunization serum). Data were analyzed 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 mean ± SEM. FIGS. 2a to 2c. ADCC reporter analysis of antiserum from cHA-vaccine treated mice against target cells expressing H1N1, H3N2, or H5N1 and subtypes of HA. . Ammonium hydroxide or C34-assisted cHA fg or cHA mg Antiserum collected from mice immunized with the protein was incubated with MDCK cells infected with (Fig. 2a) H1N1 virus, (Fig. 2b) H5N1 virus, or (Fig. 2c) H3N2 virus. Subsequently, ADCC reporter analysis was performed using Jurkat effector cells expressing mouse FcγRIII, and relative luminescence units (RLU) were measured, with values ​​being mean ± SEM. ***P < 0.001. P values ​​were calculated using Prism software with two-way ANOVA. FIGS. 3a to 3e. More CD4 + and CD8 + T-cell responses and broad-spectrum neutralizing antibodies are induced in cHA with adjuvant C34 mg It granted wider cross-defense. BALB / c mice with cHA adjuvant Al(OH)3 or C34 fg and cHA mg Immunized with; cells from the spleen of immunized mice were obtained after three immunization cycles, and IFN-γ (Fig. 3a), IL-4 (Fig. 3b), and GzB (Fig. 3c)-secreting cells were measured by ELISpot assay using specific peptides. The number of spot-forming cells (SFCs) is expressed as mean ± SEM. cHA fg and cHA mg The neutralizing activity of antiserum from vaccinated mice was analyzed against (Fig. 3d) H1N1 virus and (Fig. 3e) H5N1 virus. Data are expressed as mean ± SEM. Results were calculated using Prism software with Student's t-test and two-way ANOVA; significant differences were indicated as *P < 0.05; **P < 0.01; ***P < 0.001. FIGS. 4a to 4f. Cross-protective efficacy in mice challenged with lethal doses of H1N1 and H5N1 viruses. BALB / c mice were fed cHA with the adjuvant Al(OH)3 or C34 at 2-week intervals. fg and cHA mg Immunized mice were administered three doses of [specific virus name]. Immunized mice were challenged with H1N1 A / California / 07 / 2009 (Fig. 4a), H1N1 A / New Caledonia / 1999 (Fig. 4b), H1N1 A / WSN / 1933 (Fig. 4c), H1N1 A / Solomon Islands / 03 / 2006 (Fig. 4d), H5N1 A / Vietnam / 1194 / 2004 / NIBRG14 (Fig. 4e), or H5N1 A / Turkey / 1 / 2005 / NIBRG23 (Fig. 4f), and efficacy was evaluated by recording survival rates for 14 days after infection. **P < 0.01. Significant differences in survival rates were analyzed using the log-rank (Mantel-Cox) test. FIGS. 5a to 5e. Design and preparation of chimeric HA protein(Fig. 5a) A chimeric HA was generated by constructing the designed influenza HA sequence using the common H1N1 sequence and the common H5N1 sequence pCHA5-II. The globular head domain consists of the amino acid sequence between residues C52 and C277 (H3 numbering). The stem region consists of parts of the HA1 and HA2 subunits. Protein structures were downloaded from Protein Data Bank ID codes 2IBX (VN1194 H5 HA) and 3LZG (A / California / 04 / 2009). The final images were generated by PyMol. Since the structure of the common HA is not publicly available, images of the head domain of avian flu H5 (Vietnam / 1194 / 2004) and the stem region of pandemic H1N1 (California / 07 / 2009) were used for the chimeric HA construction. (Figs. 5b to 5d) Purification of chimeric HA protein and gel-thermal and chromatographic analysis. (Fig. 5b) Purified HA protein was analyzed by SDS / PAGE. M: Molecular weight marker. Right: cHA fg , fully glycosylated cHA directly purified from HEK293T cells; (Fig. 5c) cHA mg Monoglycosylated cHA purified from HEK293S cells and digested by endoglycosidase H. (Fig. 5d) Gel filtration analysis of purified and secreted HA protein. Fully glycosylated cHA and monoglycosylated cHA from HEK293T cells existed as trimers (>200 kDa) as shown in the chromatography. The figure shows the superimposed elution profile of HEK293T cell-expressed cHA protein overlaid with a calibration standard (dotted line). (Fig. 5e) cHA measured by LC-MS / MS fg and cHA mgA schematic diagram for indicating the main glycan on the glycosite. General glycan symbols are included. Figs. 6a and 6b. Composition and purification of secreted HA (Fig. 6a) A sequence encoding the echodomain of HA was prepared in the expression vector pcDNA and transfected into HEK293T cells. The protein was engineered to contain a stabilized / trimer, a signal, a foldon, as well as a C-terminal (His)6 tag for purification. (Fig. 6b) The purified HA protein was 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. FIGS. 7a to 7f. cHA fg and cHA mg HA binding activity of antiserum from mice vaccinated with [specific agent] BALB / c mice (n=10 per group) were fed Al(OH)3 or C34-assisted cHA fg or cHA mg Immunized at 2-week intervals. Al(OH)3-assisted cHA fg and cHA mg vs. C34-assisted cHA fg and cHA mgAntibody titers from vaccine-treated mice were measured on day 28 by ELISA with the A / California / 07 / 2009 H1N1 HA protein (Fig. 7a), A / Brisbane / 59 / 2007 H1N1 HA protein (Fig. 7b), A / Brisbane / 10 / 2007 H3N2 HA protein (Fig. 7c), A / Vietnam / 1194 / 2004 H5N1 HA protein (Fig. 7d), A / Shanghai / 2 / 2013 H7N9 HA protein (Fig. 7e) and the A / Brisbane / 59 / 2007 (Bris / 07) stem HA (#4900) protein (Fig. 7f) as coating antigens. The endpoint antibody titer was defined as the highest dilution of serum required to produce an absorbance 2.5 times higher than the optical absorbance (OD) produced by the negative control (pre-immunization serum). Data were analyzed using two-way ANOVA from Prism; differences were considered statistically significant at **P < 0.01; ***P < 0.001. Data are expressed as mean ± SEM. Figs. 8a and 8b. Binding of Stork-reactive antibodies (F10 IgG) to recombinant H1, H5, and cHA (Fig. 8a) Purified F10 was analyzed by SDS / PAGE. M: Molecular weight marker. Lane 1: F10 antibody. (Fig. 8b) The binding affinity of F10 IgG and various HAs was measured using ELISA. The x-axis represents the concentration of various HA proteins, and the y-axis represents the absorbance value at OD 405 nm. FIGS. 9a to 9d. Dose-dependent effect of C34 on antibody titer.BALB / c mice (n=10 per group) were injected with 20 μg cHA supplemented with 0.5 μg, 2 μg, or 10 μg of C34 at 2-week intervals. Mouse serum was collected 2 weeks after the second (D28) and third (D42) immunizations. Antibody titers were measured by ELISA using HA proteins from H1N1 A / California / 07 / 2009 (Figs. 9a and 9c) and H5N1 Vietnam / 1194 / 2004 (Figs. 9b and 9d). P-values ​​of antibody titers were calculated using two-way ANOVA from Prism; differences were considered statistically significant at *P < 0.05 and **P < 0.01. Data are expressed as mean ± SEM. FIGS. 10a to 10c. Dose-dependent effects of C34 on antigen-specific cytokine-secreting cells BALB / c mice (n=5 per group) were injected with 20 μg of purified cHA supplemented with three different doses of C34 at 0.5, 2, and 10 μg at 2-week intervals. Splenocytes from cHA-immunized mice were obtained after the second (D28) and third (D42) immunizations. (Fig. 10a) IFN-γ and (Fig. 10b) IL4-secreting cells were analyzed by the Elispot assay. (Fig. 10c) CD8 in splenocytes + The number of T cell-generating granzyme B was measured by Elispot analysis using specific peptides. ***P < 0.001. P values ​​were calculated using Prism software via two-way ANOVA. FIGS. 11a to 11f. cHA challenged by H1N1 and H5N1 viruses at lethal doses fg or cHA mg body weight of vaccinated miceBody weight changes in immunized mice challenged with H1N1 A / California / 07 / 2009 (A), H1N1 A / New Caledonia / 1999 (B), H1N1 A / WSN / 1933 (C), H1N1 A / Solomon Islands / 03 / 2006 (D), H5N1 A / Vietnam / 1194 / 2004 (E), or H5N1 A / Turkey / 1 / 2005 (F) viruses were monitored for 14 days post-infection. Body weight changes are expressed as mean ± SEM. Specific details for implementing the invention

[0027] Practice of the present invention will utilize ordinary molecular biology, microbiology, recombinant DNA, and immunology techniques within the art, unless otherwise indicated. Such techniques are sufficiently described in the literature. References and literature [Molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (DN Glover ed., 1985); Culture Of Animal Cells (RI Freshney, 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 (JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, by Harlow and Lane s (Cold Spring Harbor Laboratory Press, 1988); and Handbook of Experimental Immunology, Volumes I-IV (DMWeir and C. C. Blackwell, eds., 1986)].

[0028] definition

[0029] The singular form used in the specification and claims includes multiple objects unless otherwise clearly indicated in the context. For example, the term "chimeric dural receptor" includes multiple chimeric dural receptors.

[0030] As used herein, the terms “hemagglutinin” and “HA” refer to any hemagglutinin known to a person skilled in the art. In certain embodiments, the hemagglutinin is an influenza hemagglutinin, e.g., influenza A hemagglutinin, influenza B hemagglutinin, or influenza C hemagglutinin. A typical hemagglutinin comprises domains known to a person skilled in the art, including a signal peptide, a stem domain, a globular head domain, a luminal domain, a dura mater domain, and a cytoplasmic domain.

[0031] 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 composed of one or more polypeptide chains that form a stem domain of influenza hemagglutinin. The stem domain polypeptide may be a single polypeptide chain, two polypeptide chains, or more than one polypeptide chain.

[0032] The terms used herein, “influenza virus hemagglutinin head domain polypeptide,” “influenza virus hemagglutinin head domain,” “HA spherical head domain,” and “HA head domain” refer to the spherical head domain of the influenza hemagglutinin polypeptide.

[0033] As used herein, the term "antigen" is defined as any substance capable of inducing an immune response.

[0034] As used herein, the term "immunogenicity" refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.

[0035] As used herein, the term "epitope" is defined as a part of an antigen molecule that comes into contact with the antigen binding site of an antibody or T cell receptor.

[0036] As used herein, the term “vaccine” refers to a preparation containing an antigen consisting of an entire disease-causing organism (lethal or weakened) or a component of such organism, e.g., a protein, glycoprotein, peptide, glycopeptide, glycolipid, polysaccharide, or any combination thereof, used to confer immunity against a disease caused by an organism. Vaccine preparations may be natural, synthetic, or derived by recombinant DNA technology.

[0037] As used herein, the term "antigen-specific" refers to the property of a cell population in which a supply of a specific antigen or fragment of an antigen generates specific cell proliferation.

[0038] "Effective dose" refers to the amount effective at the dosage required to achieve the desired therapeutic or preventive outcome and over a period of time.

[0039] The "therapeutic effective dose" of the substance / molecule of the present invention may vary depending on factors such as disease state, age, sex, and the individual's body weight, and the ability of the substance / molecule to induce the desired response in the individual. The therapeutic effective dose is also an amount in which any toxic or harmful effect of the substance / molecule outweighs the therapeutic benefit effect. The "prophylactic effective dose" represents an effective amount at the dosage required to achieve the desired prophylactic outcome and for the duration thereof. Typically, but not necessarily, because prophylactic administration is used on the subject before or at the time of the early stage of the disease, the prophylactic effective dose may be less than the therapeutic effective dose.

[0040] The common DNA sequence of avian influenza H5 (pCHA5-II) was used as a vaccine for administration in mice, and the results demonstrated 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 having a spherical head and a common H5 with common H1 as a stem was superior, and strong CD4 + and CD8 + It was found to induce a T-cell immune response. Interestingly, monoglycosylated cHA (cHA) having only GlcNAc on each glycosite, mgThe vaccine induced stem-specific antibodies with higher antibody-dependent cytotoxicity (ADCC), superior neutralization, and stronger cross-protective activity against H1, H3, H5, and H7 strains and subtypes. Furthermore, cHA combined with a glycolipid adjuvant designed for class switching mg The vaccine contains more IFN-γ, IL-4, and CD8 + The efficacy of the vaccine in generating memory T cells was further enhanced.

[0041] Chimeric influenza virus hemagglutinin (HA) polypeptide

[0042] This disclosure is CD4 + and CD8 + Provides a chimeric influenza virus hemagglutinin (HA) polypeptide used as an immunogen or vaccine to induce a T-cell immune response. Thus, the chimeric influenza virus HA polypeptide can prevent influenza virus disease in subjects.

[0043] The chimeric influenza virus hemagglutinin (HA) polypeptide of the present disclosure comprises one or more stem domain sequences having at least 60% homology to the stem domain common sequence of H1 subtype HA (H1 HA) and / or H5 subtype HA (H5 HA), each fused with one or more globular head domain sequences having at least 60% homology to the globular head common sequence of H1 subtype HA (H1 HA) or H5 subtype HA (H5 HA).

[0044] As used herein, the term “homology” indicates an overall relationship between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between 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 homology as measured by the alignment of matching residues are referred to as homology. Homology describes the relationship between molecules and is a qualitative term that may be based on quantitative similarity or identity. Similarity and identity are quantitative terms that define the degree of sequence matching between two comparison sequences. In some embodiments, polymer molecules are considered “homology” of each other if their sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar.

[0045] In some embodiments, the polypeptide according to the present disclosure may comprise one or more sequences having at least 60% homology with a common sequence of H1 HA or H5 HA relative to 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 an N-terminal stem segment of H1 HA or a C-terminal stem segment of an N-terminal stem segment; an N-terminal stem segment of H1 HA or a C-terminal stem segment of an H1+H5 HA sequence; or an N-terminal stem segment of H5 HA or a C-terminal stem segment of an H1+H5 HA sequence.

[0046] In some embodiments, the common sequence of the stem domain 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.

[0047] SEQ ID NO: 1 (H1 stem)

[0048] DTLCIGYHANNSDTVDTVLEKNVTVTHSVNLLEDKHNGKL

[0049] SEQ ID NO: 2 (H1 stem)

[0050] NTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAV GKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQ

[0051] SEQ ID NO: 5 (H1 stem)

[0052] DTLCIGYHANNSDTVDTVLEKNVTVTHSVNLLEDKHNGKL

[0053] SEQ ID NO: 6 (H1+H5 stem)

[0054] NTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQ FEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEARLKREEISGV

[0055] SEQ ID NO: 9 (H5 stem)

[0056] DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKL

[0057] SEQ ID NO: 10 (H5+H1 stem)

[0058] NTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWTGMVDDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGV

[0059] In one embodiment, the common sequence of the spherical head domain 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.

[0060] SEQ ID NO: 3 (H5 Old Head)

[0061] CDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPandLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGKSSFFRNVVWLIKKNSTYP TIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNC

[0062] SEQ ID NO: 7 (H1 Old Head)

[0063] CKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSY PKLSKSYINDKGKEVLVLWGIHHPSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDC

[0064] SEQ ID NO: 11 (H5 Old Head)

[0065] CDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPandLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGKSSFFRNVVWLIKKNSTYP TIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNC

[0066] 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.

[0067] SEQ ID NO: 4 (Chimeric H5 / 1)

[0068] DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANP및LCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQ

[0069] SEQ ID NO: 8 (Swap H1 / 5)

[0070] DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVPRYAFAAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGV

[0071] SEQ ID NO: 12 (Swap H5 / 1)

[0072] DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPandLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEAS SGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAY KIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITN KVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGV

[0073] In some embodiments, to improve immunogenicity, one or more glycosites on HA are monoglycosylated. Preferably, the monoglycosylated HA has only N-acetylglucosamine (GlcNAc) on each glycosite.

[0074] Chimeric influenza virus HA polypeptides can be produced by any suitable method, most of which are known to those skilled in the art. For example, proteins may be chemically synthesized or produced using recombinant DNA technology (e.g., in bacterial cells, in cell cultures (mammalian, yeast, or insect cells), in plants or plant cells, or by cell-free prokaryotic or eukaryotic-based expression systems, by other in vitro systems, etc. Accordingly, the present disclosure provides a recombinant polynucleotide comprising a nucleic acid sequence encoding the polypeptide of the present disclosure and optionally a nucleic acid sequence encoding a signal peptide. The present disclosure provides a vector comprising the recombinant polynucleotide of the present disclosure. Specific examples of the polypeptide of the present disclosure are described herein. In one embodiment, the signal peptide comprises the sequence SEQ ID NO: 13 (MEKIVLLLAIVSLVKS) or SEQ ID NO: 14 (MKAILVVLLYTFATANA). A host cell comprising the vector of the present disclosure is also provided.

[0075] Immunogenic composition

[0076] 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.

[0077] The chimeric influenza virus HA polypeptide of the present disclosure may be formulated or administered in combination with one or more pharmaceutically acceptable excipients. The immunogenic / vaccine composition may be sterile, pyrogen-free, or both sterile and pyrogen-free. General considerations for the formulation and / or manufacture of pharmaceutical preparations, e.g., vaccine compositions, can be found, for example, in the literature [Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005] (the entirety incorporated herein by reference).

[0078] The immunogenic composition is administered in a manner compatible with the dosage form and in an amount that is therapeutically effective, protective, and immunogenic. The amount administered depends on the subject being treated, including the capacity of the individual’s immune system to synthesize antibodies and, if necessary, generate a cell-mediated immune response. The exact amount of the active ingredient to be administered is subject to the judgment of the practitioner. However, a suitable dosage range can be easily determined by a person skilled in the art. The regimen suitable for the initial and additional doses may also vary and may include subsequent doses following the initial dose. The dosage of the vaccine may also depend on the route of administration and varies depending on the size of the host.

[0079] The formulations of the vaccine compositions described herein may be manufactured by any method known in the pharmaceutical field or subsequently developed. Generally, such manufacturing methods include the steps of associating an active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, as necessary and / or preferably, dividing the product into desired single- or multiple-dose units, / or molding, / or packaging.

[0080] apply

[0081] Cytotoxic T lymphocytes (CTLs) have been known for quite some time to be capable of providing an immune response against influenza virus strains. Recent studies have shown that CTL responses in humans can be induced by multiple epitopes.

[0082] Methods for preventing influenza virus disease in humans and other mammals are provided herein. Additionally, methods for inducing an immune response in a subject against an influenza virus are provided. Such methods comprise administering an effective amount of the chimeric influenza virus HA polypeptide or immunogenic composition / vaccine of the present disclosure to a subject to induce an immune response specific to influenza virus strains (e.g., H1, H3, H5, and H7 strains and subtypes) in the subject. Preferably, such methods involve CD4 + and CD8 + It can induce a T-cell immune response. More preferably, such a method induces stem-specific antibodies having higher antibody-dependent cytotoxicity (ADCC), superior neutralization, and stronger cross-protective activity against H1, H3, H5, and H7 strains and subtypes. Such a method also induces more IFN-γ, IL-4, and CD8 + It improves vaccine efficacy by inducing the production of memory T cells.

[0083] Antibody titers in subjects increase after vaccination. In exemplary embodiments, the immune composition or vaccine of the present disclosure is used to provide prophylactic protection against influenza. Prophylactic protection against influenza can be achieved after administration of the vaccine or combination vaccine of the present disclosure. The vaccine (including the combination vaccine) may be administered once, twice, three times, four times, or more, but administering the vaccine once (optional, followed by a single booster) appears sufficient. Accordingly, administration may need to be adjusted.

[0084] The prophylactic effective dose is the therapeutic effective dose that provides protection against the influenza virus at a clinically acceptable level. In some embodiments, the therapeutic effective dose is the dose listed in the package insert for the vaccine.

[0085] The chimeric influenza virus HA polypeptide or immunogenic composition / vaccine of the present disclosure may be administered by any route that produces a therapeutically effective result. These include, but are not limited to, intramuscular, and / or subcutaneous administration. In some embodiments, the chimeric influenza virus HA polypeptide or immunogenic composition / vaccine of the present disclosure may be administered intramuscularly or desquamously, similar to the administration of inactivated vaccines known in the art.

[0086] The application of the present invention is not limited to the details of the arrangement of components and structures described in the following detailed description or illustrated in the drawings. The present invention may have other embodiments and may be implemented or performed in various ways.

[0087] Examples

[0088] method

[0089] Vaccine and Plasmid CompositionAll 102-length HA sequences from H1N1 viruses available from 2009 to 2013 were downloaded from the NCBI database and aligned using the ClustalW algorithm in the BioEdit program. Common H1 sequences were generated by selecting most conserved amino acids at each position. Common hemagglutinin H5 (pCHA5-II) sequences were generated as previously described. The nucleotide sequences of common hemagglutinin H5 (pCHA5-II) and common H1 were cloned into a pcDNA expression vector, and the resulting plasmids were used as templates for the construction of swap and chimeric HA. Swap H1 / 5 consists of H1 as HA1 (amino acids 1–327 of SEQ ID NO: 8) and H5 as HA2 (amino acids 328–503 of SEQ ID NO: 8), generating H1 as a spherical head and H1+H5 (HA2) stems. Swap H5 / 1 consists of H5 as HA1 (amino acids 1–330 of SEQ ID NO: 12) and H1 as HA2 (amino acids 331–506 of SEQ ID NO: 12), forming H5 as a globular head and an H5+H1(HA2) stem. In the case of the chimeric H5 / 1 construct, the globular head domain consists of the amino acid sequence between residues C42 and C274 (H3 numbering) of SEQ ID NO: 4, and the stem region consists of parts of the HA1 and HA2 subunits (amino acids 1–41 of SEQ ID NO: 4 and 4 and 275–511 of SEQ ID NO: 4). The dural domain was replaced at the C-terminus of HA with the bacteriophage T4 fibritin foldon trimerization sequence, the thrombin cleavage site, and additional residues from the (His)6-tag.Both DNA sequences of the common HA were optimized for expression using human-preferred codons, various regions were amplified by PCR, and subsequently cloned into a pcDNA vector for expression. Furthermore, HA genes from seasonal influenza viruses H1N1 Brisbane / 59 / 2007, pandemic H1N1 California / 07 / 2009, H3N2 Brisbane / 10 / 2007, H7N9 A / Shanghai / 2 / 2013, and avian flu H5N1 Vietnam / 1194 / 2004 were also optimized, synthesized, and cloned into a pcDNA expression vector. The sequences were verified by DNA sequencing and prepared for high-quality protein expression and purification.

[0090] Expression of recombinant secreted HA from expressed cellsHuman 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 instructions. In summary, 293T or 293S cells at 80% confluency were transfected with Mirus TransIT®-LT1 (Mirus Bio) transfection reagent using a 3:1 ratio of reagent to plasmid DNA. The TransIT®-LT1 reagent was diluted in Opti-MEM (Gibco), and the mixture was incubated at room temperature for 5–20 minutes. Plasmid DNA was added to the solution, thoroughly mixed, and then incubated for 15–30 minutes. Before transfection, the cells were replaced with fresh DMEM (Gibco) medium supplemented with 10% fetal bovine serum. The TransIT®-LT1 reagent / DNA complex was added to the cells and incubated at 37°C for 48 hours. The expression of hemagglutinins was confirmed by immunoblotting using anti-(his)6 antibody (Qiagen) or specific anti-hemagglutinin antibodies and horseradish peroxidase (HRP)-conjugated secondary antibody (PerkinElmer).

[0091] Purification of recombinant secreted hemagglutininsFor expression in human 293T cells, high-quality pcDNA containing the gene of interest was prepared and transfected into cells with Mirus TransIT®-LT1 (Mirus Bio). 48 hours after transfection, the medium was collected, and the cells were purified by centrifugation at 1,000 xg 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 proteins were washed with a 25 to 50 mM imidazole gradient in 20 mM Tris-HCl pH 8.0 and 300 mM NaCl (Buffer A). Next, the HA protein was eluted in a 100 to 300 mM imidazole gradient in Buffer A. The purified HA protein was concentrated in an Amicon Ultrafiltration Unit (MW30K cutoff) (Millipore) in PBS, pH 7.4. Purity was monitored using SDS-PAGE, and the protein was confirmed by Western blot using anti-(his)6 antibody (Qiagen) or specific anti-hemagglutinin antibodies and mustard bean peroxidase-conjugated secondary antibody (PerkinElmer). Finally, the trimer morphology of the HA protein was obtained using a size-exclusion column, a Superdex 200 Increase 10 / 300 GL gel filtration column (GE Healthcare).

[0092] Preparation of mono-glycosylated HA protein . Using N-acetylglucosaminyltransferase I deficient HEK293S cells high-mannose glycans 31 HA containing [the substance] was generated. Purified HA protein from HEK293S cells was treated with Endo H (NEB) overnight at 20°C to produce monoglycosylated HA mgIt was produced. The protein to Endo H ratio was 3 to 1 (w / v) for HA. Subsequently, Endo H and mono-glycosylated HA proteins were separated using a Superdex 200 Increase 10 / 300 GL gel filtration column (GE Healthcare). HA mg Proteins were concentrated using an Amicon Ultrafiltration Unit (MW30K cutoff) in PBS, pH 7.4, and confirmed by SDS-PAGE and LC-MS / MS analysis.

[0093] Confirmation of N-linked glycosylation on HA protein10 micrograms of protein were processed on SDS-PAGE and prepared for digestion in the gel. The desired protein band was cut with a sharp scalpel, diced into 1 mm pieces, and placed in a 1.3 ml Eppendorf tube. After washing twice for 3 minutes with 500 μl of 25 mM ammonium bicarbonate in 50% ACN (acetonitrile), the gel pieces were dried using a SpeedVac Thermo evaporator. The dried samples were reduced by the addition of 100 μl of 50 mM dithiothreitol (DTT) in 25 mM ammonium bicarbonate (pH 8.5) at 37°C for 1 hour, and then centrifuged at 10,000 g for 1 minute. The solution was removed, and the gel sample was subjected to an alkylation step by adding 100 μl of 100 mM iodoacetamide (IAA) in 25 mM ammonium bicarbonate (pH 8.5), followed by incubation in a dark room at room temperature for 1 hour. After washing with 500 μl of 50% acetonitrile and 500 μl of 100% acetonitrile in 25 mM ammonium bicarbonate (pH 8.5), 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). Next, the gel samples were 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 samples. The samples were sonicated for 10 seconds, followed by a 10-second pause. Fixation was repeated 10 times. The supernatant containing the peptide mixture was removed from the sample tube and transferred to a new tube. The procedure was repeated 2 times.The combined supernatant was dried in a SpeedVac concentrator, and LC-MS / MS analysis was performed.

[0094] Endotoxin measurement . Endotoxin levels Pierce ® LAL Colorimetric Endotoxin Quantification Kit (Pierce ® Measurements were performed using the LAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific). Protein samples were diluted 10, 20, 100, and 1000-fold, and endotoxin standards were prepared at 10, 5, 2.5, 1.25, 0.63, 0.31, 0.15, and 0 ng / ml. After the microplates were equilibrated under heat shock at 37°C for 10 minutes, the protein samples or standards were mixed with Limulus Amebocye Lasate (LAL) Pyrochrome reagent (final volume 100 μl) (1:1) in endotoxin-free wells at 37°C for 10 minutes. 100 μl of substrate solution was added to each well, and the plates were incubated at 37°C for 6 minutes. The reaction was stopped by adding 50 μl of stopping reagent (25% acetic acid). The absorbance of the wells was measured using a SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA). A standard curve was obtained by blotting absorbance versus the corresponding concentration of the standard. The endotoxin concentration of the samples was measured using the standard curve. The endotoxin values ​​of all purified proteins were < 0.5 ng / ml.

[0095] Mouse antivirus processing Adjuvant C34 was chemically synthesized as described and dissolved in DMSO. Magnetic BALB / c mice aged 6 to 8 weeks (n=10 per group) were saturated with 20 μg of purified chimeric HA in PBS, pH 7.4. fg or HA mgIntramuscular immunization with protein was performed and mixed with 50 μg of ammonium hydroxide (Alum; Sigma) or 2 μg of C34. 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 or third immunization. Blood was incubated at 37°C for 30 minutes, and serum was collected by centrifugation at 12,000 rpm for 10 minutes. HA-specific antibodies in the serum collected from vaccinated mice were analyzed by enzyme-linked immunosorbent assay (ELISA) and neutralization assay.

[0096] Measurement of HA-specific antibodies by ELISAHA-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 diluted in ELISA coating buffer, 100 mM sodium bicarbonate (pH 8.8), at a concentration of 5 μg / ml per well, and covered with a plastic sealer overnight at 4°C. The plate was blocked with 1% BSA in TBST (137 mM NaCl, 20 mM Tris-base, 0.05% Tween 20, pH 7.4) for 1 hour at 37°C and washed 3 times with TBST, after which the plate was incubated with 200 μl of mouse serum in a 2-fold series of dilutions for 2 hours at 37°C. After removing the serum and washing the plate 6 times, HA-specific IgG was monitored using 200 μl of secondary HRP-labeled anti-mouse antibody (1:8000) (PerkinElmer, Waltham, MA, USA). After incubation at 37°C for 1 hour, the plate was washed 6 times with TBST and developed with 100 μl of Super Aquablue ELISA substrate (eBioscience, San Diego, CA, USA) for 1 minute. The reaction was stopped by the addition of 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 was defined as the highest serum dilution that produced an absorbance 2.5 times higher than the optical absorbance (OD) produced by the negative control (pre-immunization serum). The background endpoint antibody titer was assigned as less than 1:50.

[0097] Harvesting of bone marrow-derived dendritic cells The GM-CSF-cultured bone marrow-derived dendritic cells (BMDCs) were prepared as previously described. In summary, a bone marrow single-cell suspension was added to RBC lysis to remove red blood cells (RBCs). The remaining cells were cultured in 10 ml of RPMI 1640 supplemented with 20 ng / mL rat GM-CSF (eBioscience), 10% FBS (BenchMark), 50 μM 2-ME, 100 units / mL penicillin, and 100 μg / mL streptomycin. The cells were plated into individual petri dishes, with 2 x 10⁶ 6 The final cell density of the cell / Petri dish was achieved. On day 3, the culture was supplemented by adding 10 ml of fresh culture medium containing 20 ng / mL rat GM-CSF, and on day 6, it was refilled with half a volume of complete culture medium as described above. On day 8, immature BMDCs were harvested by gently pipetting non-adherent cells, and 10 cells 6 Replatting was performed at a density of / ml. For CD8+ T cell analysis, immature BMDCs were co-cultured with CD8+ T cells and chimeric HA protein (0.1 mg / 100 μL wells) for 48 hours. The number of Granzyme B generating CD8+ T cells was measured by flow cytometry after washing.

[0098] Enzyme-linked immunospot (ELISpot) analysisELISPOT 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 for 30 minutes in RPMI-1640 supplemented with 10% fetal bovine serum (Gibco). For the detection of IFN-γ, IL-4, and Granzyme B-secreting cells from chimeric-immunized mice, splenocytes were collected and restimulated with specific peptides from HA at 37°C in 5% CO2 for 24 hours at a rate of 5 x 10⁶ cells per well. 5 Cells were cultured in [location unclear]. Cells were removed and incubated with biotinylated anti-mouse IFN-γ, IL-4 (Mabtech AB), or Granzyme B (R&D Systems) specific antibodies. Plates were washed five times prior to the addition of streptavidin-ALP conjugate 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 triple wells.

[0099] Neutralization analysis . 100 TCID of the virus 50 The culture supernatant containing was mixed with an equal volume of a series of 2-fold diluted serums and incubated at 37°C for 1 hour. Subsequently, the mixture was 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 measured based on the amount of ATP present. The neutralizing activity of the serum was measured as the maximum dilution factor that significantly protects cells from virus-induced lethality.

[0100] Microneutralization assay . 100 TCID 50Infection medium containing the virus (DMEM supplemented with 0.3% BSA, 2 μg / ml TPCK-trypsin) was mixed with an equal volume of a series of 2-fold serum dilutions and incubated at 37°C for 1 hour. Subsequently, the mixture was added to each well of a 96-well plate containing MDCK cells (1.5 x 10⁶ per well). 4 The cells were added and incubated at 37°C for 16–20 hours. The cells were washed with PBS, fixed in an acetone / methanol solution (vol / vol 1:1), and blocked with 5% skim milk. After incubation at 37°C for 1 hour, the wells were washed 6 times with PBST, and viral titers were monitored using 100 μl of mAb against influenza A NP (1:2500). After incubation at 37°C for 1 hour, the wells were washed 6 times with PBST, and 100 μl of secondary HRP-labeled anti-rabbit antibody (1:5000) (PerkinElmer, Waltham, MA, USA) was added. After incubation at 37°C for 1 hour, the wells were washed 6 times with PBST and developed for 1 minute with 50 μl of 1-Step Ultra TMB substrate (Thermo). The reaction was stopped by the addition of 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).

[0101] Antibody-dependent cell-mediated cytotoxicity reporter analysis MDCK cells in each well of a 96-well flat-bottom plate (1 x 10⁶ per well) 4 The cells were incubated at 37°C for 24 hours. The next day, 1 x 10⁶ 4MDCK cells were infected with the influenza virus for 24 hours at a multiplicity of infection of 1. Subsequently, the medium was replaced with Roswell Park Memorial Institute (RPMI) medium 1640 supplemented with 4% low IgG serum, a series of antiserum dilutions from chimeric HA protein-vaccine-treated mice were added, and the mixture was 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 a 1:5 ratio of target cells to effector cells was added to the infected MDCK cells. After incubation at 37°C for 6 hours, the assay plates were removed from the 37°C incubator and equilibrated at ambient temperature for 15 minutes, after which Bio-Glo™ Luciferase Assay Buffer (Promega) was added in a 1:1 ratio. Luminescence was measured on a CLARIOstar plate reader.

[0102] Virus Challenge Experiment . After 3 doses of vaccination at 2-week intervals, 10 LD50 of immunized mice 2 weeks later 50Intranasal challenge was administered with H1N1 California / 07 / 2009, H1N1 A / New Caledonia / 1999, H1N1 A / WSN / 1933, H1N1 A / Solomon Islands / 03 / 2006, and re-revolutionary H5N1 virus A / Vietnam / 1194 / 2004 / NIBRG14 and H5N1 A / Turkey / 1 / 2005 / NIBRG23 (viral doses inducing 50% mouse lethality). After infection, mice were observed daily for 14 days, and survival rates and body weights were recorded. A percentage of body weight was calculated for each individual animal per group by comparing daily body weight to pre-challenge body weight, and mice that lost more than 25% of their initial body weight were sacrificed and scored as dead. The mouse study was approved by the Institutional Animal Care and Use Committee of Academia Sinica. All animal experiments were performed under biosafety level 3 enhanced conditions.

[0103] Expression and Purification of Recombinant F10 AntibodyFreeStyle™ 293F cells were transfected into serum-free controlled FreeStyle™ 293F cells using the plasmid polyethyleneimine encoding the F10 antibody and cultured in FreeStyle™ 293 expression medium (Gibco) in a 125 ml sterile Erlenmeyer flask rotated at 135 rpm on an orbital shaker platform. The supernatant was collected 72 hours after transfection, and the cells were purified by centrifuging at 1,000 xg for 10 minutes. The supernatant was pre-equilibrated with 5 column volumes (CV) of phosphate-buffered saline (PBS) wash buffer (pH 7.0) and then washed with 5 CV of wash buffer. The F10 antibody was eluted with 0.2 M glycine buffer (pH 2.5), and the fractions were collected into a tube containing 0.5 mL of 1 M Tris-HCl pH 9.0 for neutralization. Purification was monitored using SDS-PAGE.

[0104] Statistical analysis To evaluate the immune response, the animal experiments used were repeated at least 3 times (n=5 per group), and the virus challenge study was performed at least 2 times (n=10 per group). The response of each mouse was counted as individual data points for statistical analysis. Data obtained from the 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.

[0105] Example 1: Preparation and Characterization of Monoglycosylated Chimeric HA

[0106] To design a universal vaccine, the first objective was to obtain a vaccine with broad protection against influenza A virus group 1 (where H1 and H5 are major subtypes, and H2, H6, and H9 are minor). Therefore, a common H1 sequence was generated using HA sequences from H1N1 viruses available from early 2009 to 2013. Subsequently, the common H5 and common H1 were used as templates for vaccine design. During influenza virus replication, the HA precursor (HA0) is proteolytically degraded into two subunits, HA1 and HA2; the HA1 subunit possesses a 5-N-acetylneuraminic acid (sialic acid) binding site, and the HA2 subunit causes viral fusion with the host cell membrane (Fig. 5a). On the other hand, based on its three-dimensional (3D) structure, HA can be divided into two structural domains: a globular head and a stem. The stem region contains the HA2 domain, N-terminal residues 36–50, and a short stretch of the C-terminus of the HA1 domain. Accordingly, vaccines based on various combinations of domains from H1 and H5 were designed. First, for comparison, swab H1 / 5 (H1 globular head and [H1+H5(HA2) stem]), swab H5 / 1 (H5 globular head and [H5+H1(HA2) stem]), and chimeric H5 / 1 (H5 globular head and H1 stem) were generated (Figs. 1a and 5a). The results indicated that immunity with common H1N1 and swab H1 / 5 did not induce cross-protective activity, whereas swab H5 / 1 and chimeric H5 / 1 induced cross-neutralizing activity against H1N1 and H5N1 viruses (Fig. 1b). Next, this cross-protection CD8 + We investigated whether it was contributed from the T cell response and found that Granzyme B was secreted in greater amounts in chimeric H5 / 1-immunized mice, which suggests that the chimeric H5 / 1 vaccine has a stronger CD8 compared to the swab H5 / 1 vaccine.+ This suggests that a T cell response was induced (Fig. 1c).

[0107] Example 2: Effect of Glycosylation of Chimeric H5 / 1 (cHA) on Immunological Response

[0108] To explore the immunogenicity of chimeric H5 / 1 (cHA) vaccines having different glycosylation states, monoglycosylated cHA (cHA mg ) and fully glycosylated cHA (cHA fg ) The vaccines were compared (Fig. 5). It is known that Endo-H is specific to high mannose but not to complex-type glycans. HA glycoprotein expressed in HEK293S cells, which are deficient in N-acetylglucosaminyltransferase I and produce glycoproteins containing high-mannose-type N-glycans, was treated with Endo-H to cleave the N-glycan into a single GlcNAc residue. cHA mg To produce cHA, cHA was generated from human cells (HEK293S), and purified cHA containing high-mannose glycans was treated with Endo-H to remove the outer portion of the N-glycan, thereby producing HA having only a single N-acetylglucosamine (GlcNAc) connected to the asparagine residue of each glycosite. After Endo-H treatment, the mixture was passed through gel filtration to produce trimer cHA mg Endo-H was isolated from. After concentration, cHA mg The protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS / PAGE) and liquid chromatography-serial mass spectrometry (LC-MS / MS) to confirm its purity and glycan composition (Fig. 5c). Since influenza HA exists as a trimer on the surface of the virus, gel filtration was performed to obtain cHA fg and cHA mgIt was confirmed that it existed as a trimer (>200 kDa) (Fig. 5d). In addition, another fully glycosylated cHA from human cells (HEK293T) for comparison fg Generated (Fig. 5b), and cell cultured to ~6 mg / L cHA fg It generated.

[0109] Recombinant cHA fg and cHA mg The N-linked glycosylation sites and glycan profiles of were analyzed by LC-MS / MS showing 7 glycosylation sites (N28, N40, N171, N182, N292, N303, and N497); cHA fg The N-glycans of were generally of the complex type, and cHA mg It can be obtained as a single glycoform having only GlcNAc at each of its N-glycosylation sites in ~99% (Fig. 5e and Table 1).

[0110] Table 1: N-linked glycan structures of fully-glycosylated and mono-glycosylated proteins analyzed by LC-MS / MS

[0111]

[0112]

[0113] Example 3: Fully glycosylated chimeric H5 / 1 (cHA fg ) and monoglycosylated chimeric H5 / 1 (cHA mg Cross-reactivity of antiserum from mice immunized with )

[0114] To evaluate the antibody binding activity induced by cHA constructs, BALB / c mice were administered 20 μg of cHA supplemented with Al(OH)3 or C34, analogs of α-galactosylceramide (α-GalCer). fg or cHA mgIntramuscular immunization with protein was performed. Mice were immunized at 0, 2, and 4 weeks, and HA-induced serum was obtained at 28 and 42 days and measured using enzyme-linked immunosorbent assay (ELISA) with various recombinant HAs (Fig. 6). Compared to the maximum dilution of antiserum after two immunizations, three immunizations actually produced antiserums with higher HA-specific antibody titers (Figs. 1d to 1i and Fig. 7), and cHA mg Vaccination by cHA fg It induced a superior antibody response compared to (Figs. 1d, 1e, and 1g). In addition, cHA mg The antiserum from showed slightly better binding to H3 and H7 HA proteins (Figs. 1f and 1h), and no significant difference was observed between Al(OH)3 and C34 adjuvants. These data indicate that the cHA vaccine can induce cross-reactive antibodies that recognize HA from H1N1, H3N2, H5N1, as well as H7N9 strains.

[0115] F10 is a known broad-spectrum neutralizing IgG antibody against the target stem of HA, which is highly conserved among various subtypes of the influenza virus. To compare the binding of F10 with that of recombinant H1, H5, and cHA, the binding activity of F10 to various HAs was measured, and the results revealed that F10 can bind to H1, H5, and cHA proteins (Fig. 8). To investigate whether F10-like antibodies were induced by cHA vaccination, the binding of cHA-induced serum to HA stem number 4900 was measured using ELISA. The results showed that cHA mg It was shown that the vaccine could induce higher stem-specific antibody titers than cHAfg (Figs. 1i and 7f), and superior results were observed with the C34-assisted cHA vaccine (Fig. 1i), which induces more stem-specific antibodies.

[0116] Example 4: Strong CD4 against H1, H3, and H5 viruses as well as their subtypes + and CD8 + cHA that induces T-cell responses, antibody-dependent effector function, and neutralizing activity mg and vaccination of mice by adjuvant C34

[0117] In addition to antibody-mediated neutralization, Fc-mediated effector function also plays a crucial role in defense against influenza infection. Therefore, we investigated whether antibodies can induce Fc receptor-mediated immune responses. A mouse-modulated ADCC assay was performed using Jurkat effector cells expressing FcγRIII, and cHA fg - and cHA mg ADCC activity of serum from immunized mice was evaluated (Fig. 2). As expected, cHA fg - or cHA mg - Vaccinated mice 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, Al(OH)3-assisted cHA mg Superior ADCC activity was observed in the group (Fig. 2b), and similar results were observed in 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).

[0118] To evaluate the role of antigen-specific cytokine-secreting cells in cHA-immunized mice, splenocytes were collected after 2 and 3 immunizations, and IFN-γ, IL-4, and granzyme B (GzB)-secreting cells were identified by enzyme-linked immunosorbent spot (ELISpot) analysis using specific peptides from HA for stimulation. As shown in Fig. 3, Al(OH)3-assisted cHA fg and cHA mg The vaccine generated similar levels of cytokine-secreting cells. However,

[0119] More CD4 + / IFN-γ + Th1 cells (Fig. 3A), CD4 + / IL-4 + Th2 (Fig. 3B), and CD8 + GzB-secreting cells (Fig. 3c) cHA assisted by C34 rather than Al(OH)3 mg It was induced by vaccination. These results were caused by C34-assisted cHA mg Ga cHA fg More CD4 compared to + T Helper response and stronger CD8 + It can stimulate cytotoxic effects.

[0120] To evaluate the dose dependence of C34 on antibody titers and cell-mediated immunity, mice were administered cHA assisted by three different doses of C34: 0.5, 2, and 10 μg. fg Intramuscular immunization was performed. The result was cHA assisted by 2 μg of C34. fg It was indicated that induced stronger titers after 2 or 3 immunizations than with 0.5 and 10 μg of C34 (Fig. 9). In addition, cHA assisted by 2 μg of C34 fgThe vaccine induced more IFN-γ than that produced by 0.5 and 10 μg of C34 (Fig. 10a), and 2 and 10 μg of C34 induced more IL-4 after three immunizations than that produced by 0.5 μg of C34 (Fig. 10b). On the other hand, after two or three immunizations, cHA fg CD8 when the vaccine was adjuvanted with 0.5, 2, or 10 μg of C34 + There was no difference in the increase in GzB-secreting cells (Fig. 10c). Based on these observations, 2 μg of C34 was used throughout the experiment.

[0121] The neutralizing activity of cHA-induced antiserum was further investigated. cHA mg The antiserum from the vaccination was found to possess superior neutralizing activity against homogeneous H1N1 A / California / 07 / 2009 (Fig. 3d) and homogeneous H5N1 NIBRG14 (A / Vietnam / 1194 / 2004), NIBRG23 (A / Turkey / 1 / 2005), RG5 (A / Anhui / 1 / 2005), or RG2 (A / Indonesia / 5 / 2005) (Fig. 3e). In addition, cHA mg Antiserum from mice vaccinated with [the substance] exhibits significant neutralizing activity against heterologous viruses H1N1 A / Brisbane / 59 / 2007, A / New Caledonia / 20 / 1999, and A / Solomon Islands / 3 / 2006 (Fig. 3d). Antiserum from cHA-immunized mice was clearly able to block infection by H1N1 and H5N1 viruses, and cHA mg The neutralization activity of is generally cHA fg It was superior, and in particular, superior against heterogeneous viruses.

[0122] Example 5: cHA providing cross-defense against H1N1 and H5N1 as well as their subtypes in challenge research mg Vaccination of mice with / C34

[0123] cHA mgTo analyze whether vaccination provides broad cross-protective immunity against various H1N1 and H5N1 viruses, vaccinated mice were challenged by intranasal inoculation with lethal doses of multiple H1N1 and H5N1 viruses, and the efficacy of vaccine protection was evaluated for 14 days by measuring survival rate and changes in body weight (Figs. 4 and 11). In the case of mice challenged with the H1N1 A / California / 07 / 2009 virus, all cHA vaccines provided 100% protection (Fig. 4a). In addition, C34-assisted cHA mg Mice immunized with [the drug] exhibited minimal mass loss compared to cHAfg (Fig. 11a). Mice immunized only with C34-assisted cHAfg were conferred 30% protection against the A / New Caledonia / 1999 challenge, whereas C34-assisted cHAfg mg The vaccine provided 90% protection against the cross-strain A / New Caledonia / 1999 virus, and similar results were observed with Al(OH)3-assisted cHA vaccination (Fig. 4b). In the case of mice challenged with the cross-strain A / WSN / 1933 virus, all mice immunized with Al(OH)3-assisted cHA survived; however, only the C34-assisted cHA fg Mice immunized with conferred 80% protection (Fig. 4c). A lethal challenge was also performed with A / Solomon Islands / 03 / 2006. All mice immunized with Al(OH)3-assisted cHA exhibited lower protection; however, C34-assisted cHA mgMice immunized with [the compound] exhibited superior protection against the cross-strain A / Solomon Islands / 03 / 2006 virus (Fig. 4d). In the case of mice challenged with H5N1 NIBRG14 (A / Vietnam / 1194 / 2004) and NIBRG23 (A / Turkey / 1 / 2005), all immunized mice survived (Figs. 4e and 4f). Changes in body weight after viral challenge were also evaluated (Fig. 11). The data showed that cHA was effective in inducing significant protective immunity against various H1N1 and H5N1 viruses, and cHA mg cHA fg It was found to provide a wider cross-defense capability compared to.

[0124] The development of universal influenza vaccines to provide defense against multiple strains and subtypes of the influenza virus is currently of interest, and the epitopes used for the development of universal vaccines include a highly conserved ectodomain of M2 containing 24 non-glycosylated amino acids, a nucleoprotein NP, and a Dayana HA component that has been shown to target the HA-stem region or block viral entry by inducing higher titers of broad-spectrum neutralizing antibodies. For example, soluble trimer HA (mini-HA) vaccines with rearranged stem subunits were found to completely protect mice from lethal challenges posed by heterologous and heterosubtypic viruses, and chimeric HA vaccination via DNA prime-protein boost and exposure to the same stem region and divergent exotic head domain was found to induce broad protective stem-specific antibodies. However, the results were CD8 +This indicated that T cells did not play a major role in cross-protective activity. Although DNA vaccines are promising, they are in the early stages of development. In this study, a cHA construct expressing common H5 in the globular head and common H1 in the stem region was designed to mimic the actual state of the influenza virus transmitted from avian viruses to humans. Fully glycosylated cHA fg and monoglycosylated cHA mg Both were prepared for comparison, and the result was cHA mg The vaccine is CD4 + and CD8 + It was shown that it induced higher titers of cross-reactive antibodies against H1, H3, H5, and H7 subtypes (Figs. 1d to 1h) through T cell responses (Figs. 3a to 3c).

[0125] Glycosylation of HA has been found to play a significant role in regulating its biological activities, including protein folding and stability, and reducing immunogenicity by shielding the antigenic site from neutralizing antibodies. Furthermore, hyperglycosylated HA has evolved to shield the antigenic site in the highly variable head domain, and consequently, the immune response was re-induced toward the conserved stem region. In the results of the inventors of the present invention, cHA mg The neutralizing activity of the antiserum, particularly against heterologous H1N1 A / Brisbane / 59 / 2007, A / Solomon Islands / 03 / 2006, and A / New Caledonia / 20 / 1999 (Fig. 3d), cHA fg - It was significantly superior compared to the induced antiserum. cHA mgThe broader neutralizing activity of the vaccine is possibly attributed to its induction of more antibody variants as previously reported. IgG is the dominant antibody present in mice and is a major subtype of HA-specific antibody having high activity against FcγRIII receptors on immune cells for inducing ADCC. Consistent with studies indicating that ADCC is necessary for in vivo influenza defense, the inventors of the present invention [indicated] cHA mg It was confirmed that immunity induced by [the substance] resulted in higher ADCC and more stem-specific derivatives having superior protective activity (Figs. 1i and 2). Ammonium hydroxide (Alum) is known to stimulate Th2 responses and has been approved by the FDA for use as a vaccine adjuvant; however, its mechanism of action has not been well studied. Glycolipid C34 is a ligand for CD1d on dendritic cells, expressed therein, which interacts with receptors on invariant natural killer T (iNKT) cells to induce stimulation of iNKT cells to produce Th1 cytokines with adjuvant effects (e.g., IFN-γ) and Th2 cytokines with class-switch activity (e.g., IL-4). In the results of the inventors of the present invention, IFN-γ (Th1 cytokine), IL-4 (Th2 cytokine)-secreting cells, and granzyme B-producing CD8 + The number of T cells is cHA assisted by C34 rather than Al(OH)3. mg It was significantly increased by immunity (Figs. 3a to 3c).

[0126] In summary, the development of next-generation influenza vaccines with a broad protective immune response is a current area of ​​interest, and some promising results have been reported that bring the development of universal vaccines within feasible range. In efforts toward this objective, the inventors of the present invention have successfully demonstrated in this study evidence that a monoglycosylated cHA vaccine having a common H5 head and a common H1 stem is an effective influenza vaccine exhibiting broad protective activity against heterogeneous influenza viruses, including H1, H3, H5, and H7 viruses and subtypes in neutralization studies and H1N1, H5N1, and subtypes in challenge studies. With the success in developing broad protective vaccines against different strains and subtypes of influenza A viruses, the inventors of the present invention aim to utilize the strategy developed in this study to design broader universal vaccines against influenza A and B viruses.

Claims

Claim 1 A chimeric influenza virus hemagglutinin (HA) polypeptide comprising one or more stem domain sequences of H1 subtype HA (H1 HA) and / or H5 subtype HA (H5 HA) fused with one or more globular head domain sequences of H1 subtype HA (H1 HA) or H5 subtype HA (H5 HA), wherein the chimeric influenza virus hemagglutinin (HA) polypeptide comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO:

12. Claim 2 A chimeric influenza virus hemagglutinin (HA) polypeptide according to claim 1, wherein HA is influenza A HA, influenza B HA, or influenza C HA. Claim 3 delete Claim 4 A chimeric influenza virus hemagglutinin (HA) polypeptide according to claim 1, wherein the stem domain sequence is an N-terminal stem segment of H1 HA or a C-terminal stem segment of H1 HA; an N-terminal stem segment of H1 HA or a C-terminal stem segment of an H1+H5 HA sequence; or an N-terminal stem segment of H5 HA or a C-terminal stem segment of an H1+H5 HA sequence. Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 A chimeric influenza virus hemagglutinin (HA) polypeptide according to claim 1, wherein one or more glycosites on the HA are monoglycosylated, and the monoglycosylated HA has only N-acetylglucosamine (GlcNAc) on each glycosite. Claim 9 The chimeric influenza virus hemagglutinin (HA) polypeptide of claim 1, wherein the chimeric influenza virus HA polypeptide is used as an immunogen. Claim 10 An immunogenic composition comprising a chimeric influenza virus HA polypeptide and an adjuvant of any one of claims 1, 2, 4, 8, and 9. Claim 11 Immunogenic composition of claim 10, wherein the adjuvant is a glycolipid adjuvant. Claim 12 A pharmaceutical composition for immunizing a subject against an influenza virus or preventing an influenza virus disease, comprising an effective amount of the chimeric influenza virus hemagglutinin (HA) polypeptide of any one of claims 1, 2, 4, 8, and 9 for the subject. Claim 13 In claim 12, the immune is CD4 + and CD8 + A pharmaceutical composition that induces a T-cell immune response. Claim 14 A pharmaceutical composition according to claim 12, wherein the immune induces stem-specific antibodies having higher antibody-dependent cellular cytotoxicity (ADCC), superior neutralization, and stronger cross-protective activity against H1, H3, H5, and H7 strains and subtypes. Claim 15 A pharmaceutical composition according to claim 12, wherein the immune induces elevated levels of IFN-γ, IL-4, and CD8+ memory T cells compared to the pre-immunization baseline immune response. Claim 16 A recombinant polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1, 2, 4, 8, and 9, and optionally a nucleic acid sequence encoding a signal peptide. Claim 17 A recombinant polynucleotide according to claim 16, wherein the signal peptide comprises the sequence of SEQ ID NO: 13 or SEQ ID NO:

14. Claim 18 A vector comprising the recombinant polynucleotide of claim 17. Claim 19 A host cell comprising the vector of claim 18.

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