Chimeric influenza vaccines

Abstract

The present invention relates to a chimeric influenza virus hemagglutinin (HA) polypeptide comprising one or more stem domain sequences fused to one or more globular head domain sequences, each of the one or more stem domain sequences 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), each of the one or more globular head domain sequences 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).

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application U.S. Application Serial No. (USSN) 62 / 022,328, filed May 8, 2020. The contents of each are incorporated herein by reference in their entirety.

[0003] SEQUENCE LISTING

[0004] This case contains a Sequence Listing submitted electronically in ASCII format, the content of which is incorporated herein by reference in its entirety. The ASCII copy, created on May 6, 2021, is named G4590-08600PCT_SeqListing.txt and has a file size of 28 kilobytes. TECHNICAL FIELD

[0005] The present application relates to chimeric influenza virus hemagglutinin (HA) polypeptides, immunogenic / vaccine compositions containing the same, and uses thereof. BACKGROUND

[0006] Traditional methods for producing influenza vaccines are to culture the virus in specific pathogen free (SPF) chicken embryonated eggs, and the methods often require more than six months for large scale production. However, some vaccine virus strains do not grow well in eggs, and people who are allergic to chicken eggs can have safety issues. New methods based on cell culture of the virus have been developed to replace the egg-based methods; but the cell culture methods still have the risk of producing potentially harmful viruses. To overcome these problems, research into alternative strategies has shown that vaccines based on recombinant HA can induce neutralizing antibodies against influenza virus infection. However, the antibodies induced by a particular influenza virus subtype can not be effective in neutralizing other influenza subtypes. In addition, because of the constant mutation of the virus, the vaccine must be updated every year.

[0007] Therefore, there is still a need to develop a universal vaccine against a broad spectrum of influenza virus strains. SUMMARY

[0008] In one aspect, the present application provides a chimeric influenza virus hemagglutinin (HA) polypeptide, the polypeptide comprising one or more stem domain sequence fused to one or more globular head domain sequence, each of the one or more stem domain sequence having at least 60% homology to a stem domain consensus sequence of H1 subtype HA (H1 HA) and / or H5 subtype HA (H5 HA), each of the one or more globular head domain sequence having at least 60% homology to a globular head consensus sequence of H1 subtype HA (H1 HA) or H5 subtype HA (H5 HA).

[0009] In some embodiments, the HA is an influenza A HA, an influenza B HA, or an influenza C HA.

[0010] In some embodiments, the homology is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0011] 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 H1+H5 HA sequence; or an N-terminal stem segment of H5 HA or a C-terminal stem segment of H1+H5 HA sequence.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] In some embodiments, one or more glycosylation sites on the HA are monoglycosylated. In another embodiment, the monoglycosylated HA has only N-acetylglucosamine (GlcNAc) at each glycosylation site.

[0016] In one embodiment, the chimeric influenza virus HA polypeptide is used as an immunogen.

[0017] In another aspect, the present application provides an immunogenic composition comprising a chimeric influenza virus HA polypeptide and an adjuvant. In one embodiment, the adjuvant is a glycolipid adjuvant.

[0018] In another aspect, the present application provides a recombinant polynucleotide comprising a nucleic acid sequence encoding a polypeptide of the present application 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.

[0019] In another aspect, the present application provides a vector comprising a recombinant polynucleotide of the present application. Also provided are host cells comprising a vector of the present application.

[0020] In another aspect, the present application provides a method of immunizing an individual against an influenza virus, the method comprising administering to the individual an effective amount of a chimeric influenza virus hemagglutinin (HA) polypeptide or immunogenic composition of the present application.

[0021] In another aspect, the present application provides a method of preventing an influenza virus disease in an individual, the method comprising administering to the individual an effective amount of a chimeric influenza virus hemagglutinin (HA) polypeptide or immunogenic composition of the present application.

[0022] In one embodiment, the methods described herein elicit CD4 + and / or CD8 + T cell immune responses.

[0023] In one embodiment, the methods described herein induce stem-specific antibodies with higher antibody-dependent cellular cytotoxicity (ADCC), better neutralization activity, and stronger cross-protection activity against H1, H3, H5, and H7 strains and subtypes.

[0024] In one embodiment, the methods described herein improve vaccine efficacy, producing more IFN-γ, IL-4, and CD8+ memory T cells. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 (A) to (I). Chimeric H5 / 1 constructs (cHA) with common H5 globular head and common H1 stem and stem-specific antibodies elicited by cHA mg immunization. (A) Constructs of exchange H1 / 5 (H1 globular head and H1+H5 [HA2] stem), exchange H5 / 1 (H5 globular head and H5+H1 [HA2] stem), and chimeric H5 / 1 (cHA: H5 globular head and H1 stem). (B) Neutralization activity against H1N1 California / 07 / 2009 and H5N1 Vietnam / 1194 / 2004 viruses. (C) Granzyme B (GrzB)-producing CD8 + T cell numbers were assessed by flow cytometry. (D-I) cHA fg and cHA mg adjuvanted with Al(OH)3 fg and cHA mg adjuvanted with C34Antibody titers in mice 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. Endpoint antibody titers were defined as the last dilution of antiserum that produced an optical density 2.5-fold greater than that produced by the negative control (pre-immune serum). Data were examined by 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.

[0026] Figure 2 (A) to (C). ADCC reporter assay of antisera from cHA-vaccinated mice against target cells expressing HA of H1N1, H3N2, or H5N1 and subtypes. Antisera collected from mice immunized with cHA fg or cHA mg protein adjuvanted with Al(OH)3 or C34 were incubated with MDCK cells infected with (A) H1N1 virus, (B) H5N1 virus, or (C) H3N2 virus for 30 min. Subsequently, ADCC reporter assay was performed using Jurkat effector cells expressing mouse FcyRIII, and relative luminescent units (RLU) were measured, and values are mean ± SEM. ***P < 0.001. P values were calculated using two-way ANOVA with Prism software.

[0027] Figure 3 (A) to (E). More CD4 + and CD8 + T cell responses and broadly neutralizing antibodies were elicited by cHA mg adjuvanted with C34 to generate broader cross-protection. BALB / c mice were immunized with cHA fg and cHA mg adjuvanted with Al(OH)3 or C34; cells from the spleen of immunized mice were obtained after three immunizations, and cells secreting IFN-γ (A), IL-4 (B), and GzB (C) were determined by ELISpot analysis using specific peptides. The number of spot-forming cells (SFC) is represented as mean ± SEM. Analysis from cHA-vaccinated mice.fg and cHA mg of antisera from mice immunized with (D) H1N1 virus and (E) H5N1 virus. Data presented as mean ± SEM. Results were calculated using Student's t-test and two-way ANOVA with Prism software; significant differences marked as *P < 0.05; **P < 0.01; ***P < 0.001.

[0028] Figure 4 (A) to (F). Cross-protection efficacy in mice challenged with a lethal dose of H1N1 and H5N1 viruses. Mice were immunized with three doses of cHA fg and cHA mg BALB / c mice were immunized. Immunized mice were 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 / NIBRG14 (E) or H5N1 A / Turkey / 1 / 2005 / NIBRG23 (F) and efficacy was assessed by recording survival post-infection for 14 days. **P < 0.01. Significant differences in survival were analyzed by log-rank (Mantel-Cox) test.

[0029] Figure 5 (A) to (E). Design and preparation of chimeric HA proteins. (A) Designed influenza HA sequences are constructed using a common H1N1 sequence and a common H5N1 sequence pCHA5-II to generate chimeric HA. The globular head domain is composed of the amino acid sequence between residues C52 and C277 (H3 numbering). The stem region is composed 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). Final images were generated with PyMol. Since the structure of the common HA is not yet 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) Chimeric HA protein purification and gel filtration chromatography analysis. (B) Purified HA proteins were analyzed by SDS / PAGE. M: molecular weight marker. Left: cHA fg , i.e. fully glycosylated cHA purified directly from HEK293T cells; (C) cHA mgi.e. mono-glycosylated cHA purified from HEK293S cells and digested with endoglycosidase H. (D) Gel filtration analysis of purified secreted HA proteins. Fully glycosylated cHA and mono-glycosylated cHA from HEK293T cells exist as trimers (> 200 kDa) as shown in the chromatogram. This graph represents the overlay elution profile of cHA proteins expressed from HEK293T cells overlaid with the calibration standard (dotted line). (E) Mapping of the major glycosites on cHA fg and cHA mg schematically. The general glycan symbols are shown next.

[0030] Figure 6 (A) and (B). Secreted HA constructs and purification. (A) Sequences encoding the HA ectodomain were prepared in the expression vector pcDNA and transfected into HEK293T cells. The proteins were engineered to contain a stable / trimerization signal, a foldon, and a C-terminal (His)6 tag for purification. (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.

[0031] Figure 7 (A) to (F). HA binding activity of antisera from mice inoculated with cHA fg and cHA mg BALB / c mice (n = 10 per group) were immunized with cHA fg or cHA mg with Al(OH)3 or C34 adjuvant at two-week intervals. cHA fg and cHA mg with Al(OH)3 adjuvant fg and cHA mgAntibody titers in mice were measured on day 28 by ELISA using A / California / 07 / 2009 H1N1 HA protein (A), A / Brisbane / 59 / 2007 H1N1 HA protein (B), 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 (No. 4900) protein (F) as coating antigens. Endpoint antibody titers were defined as the highest dilution of serum that produced an optical density (OD) that was 2.5-fold higher than that produced by the negative control (pre-immune serum). Data were examined by two-way ANOVA using Prism; differences were considered statistically significant, **P < 0.01; ***P < 0.001. Data represent mean ± SEM.

[0032] Figure 8 (A) and (B). Binding of 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) Binding affinity of F10 IgG to various HAs was measured by using ELISA. The x-axis shows the concentration of various HA proteins and the y-axis shows the absorbance value at OD405 nm.

[0033] Figure 9 (A) to (D). Dose-dependent effect of C34 on antibody titers. BALB / c mice (n = 10 per group) were injected with 20 μg of cHA adjuvanted with 0.5 μg, 2 μg, or 10 μg of C34 at two-week intervals. Mouse sera were collected two weeks after the second (D28) and third (D42) immunizations. Antibody titers were measured by using ELISA with HA protein of H1N1 A / California / 07 / 2009 (A and C) and HA protein of H5N1 Vietnam / 1194 / 2004 (B and D). P values of antibody titers were calculated by using two-way ANOVA from Prism; differences were considered statistically significant, *P < 0.05; **P < 0.01. Data represent mean ± SEM.

[0034] Figure 10(A) to (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 0.5 μg, 2 μg and 10 μg of C34 at two-week intervals. Splenocytes from cHA-immunized mice were obtained after the second (D28) and third (D42) immunizations. (A) IFN-γ and (B) IL4-secreting cells were assessed by Elispot analysis. (C) Granulysin-producing CD8 T cells in splenocytes + T cell numbers were determined by Elispot analysis using specific peptides. ***P < 0.001. P values were calculated with Prism software using two- factor ANOVA.

[0035] Figure 11 (A) to (F). Body weight of cHA-vaccinated mice challenged with a lethal dose 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), 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 after infection. Body weight changes are presented as mean ± SEM. fg or cHA mg . Body weight changes of immunized mice challenged with a lethal dose 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), 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 after infection. Body weight changes are presented as mean ± SEM. DETAILED DESCRIPTION

[0036] The practice of the present application will employ, unless otherwise indicated, conventional molecular biology, microbiology, recombinant DNA, and immunology techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Molecular Cloning A Laboratory Manual, second edition, Sambrook, Fritsch, and Maniatis eds., Cold Spring Harbor Laboratory Press, 1989; DNA Cloning, Vols. I and II, D. N. Glover ed., 1985; Culture Of Animal Cells (R. I. 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., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. 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, Harlow and Lane eds., Cold Spring Harbor Laboratory Press, 1988; and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986).

[0037] Definitions

[0038] The singular forms "a," "an," and "the" as used in the specification and claims include plural referents unless the context clearly dictates otherwise. For example, the term "a chimeric transmembrane receptor" includes multiple chimeric transmembrane receptors.

[0039] The terms "hemagglutinin" and "HA" as used herein refer to any hemagglutinin known to the skilled artisan. In certain embodiments, the hemagglutinin is an influenza hemagglutinin such as an influenza A hemagglutinin, an influenza B hemagglutinin, or an influenza C hemagglutinin. A typical hemagglutinin includes domains known to the skilled artisan including a signal peptide, a stem domain, a globular head domain, a luminal domain, a transmembrane domain, and a cytoplasmic domain.

[0040] The terms "stem domain polypeptide," "HA stem domain," "influenza virus hemagglutinin stem domain polypeptide," and "HA stalk domain" as used herein refer to a polypeptide comprising or consisting of one or more polypeptide chains that make up the stem domain of an influenza hemagglutinin. The stem domain polypeptide can be a single polypeptide chain, two polypeptide chains, or more polypeptide chains.

[0041] The terms "influenza virus hemagglutinin head domain polypeptide," "influenza virus hemagglutinin head domain," "HA globular head domain," and "HA head domain" as used herein refer to the globular head domain of an influenza hemagglutinin polypeptide.

[0042] The term "antigen" as used herein is defined as any substance capable of eliciting an immune response.

[0043] The term "immunogenicity" as used herein refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.

[0044] The term "antigenic determinant" as used herein is defined as the portion of an antigen molecule that contacts the antigen binding site of an antibody or T cell receptor.

[0045] The term "vaccine" as used herein refers to a preparation containing an antigen consisting of either the whole causative organism (killed or attenuated) or components of such organisms such as proteins, glycoproteins, peptides, glycopeptides, glycolipids, polysaccharides, or any combination thereof, which is used to confer immunity against disease caused by these organisms. The vaccine preparation can be natural, synthetic, or obtained by recombinant DNA technology.

[0046] The term "antigen specificity" as used herein refers to a property of a population of cells that causes the provision of a particular antigen or antigenic fragment to result in proliferation of specific cells.

[0047] "Effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0048] A "therapeutically effective amount" of a substance / molecule of the present application can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance / molecule are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired prophylactic result. Typically (but not necessarily), a prophylactically effective amount will be lower than a therapeutically effective amount since a prophylactic dose is used before or at an early stage of disease in an individual.

[0049] The 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, M.W. 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)). The present 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, the chimeric HA (cHA) vaccine with consensus H5 as the globular head and consensus H1 as the stem was the best and showed elicitation of strong CD4 + and CD8 + T cell immune responses. Strikingly, the monoglycosylated cHA (cHAmg) vaccine with only GlcNAc at each glycosite induced more stem-specific antibodies with higher antibody-dependent cellular cytotoxicity (ADCC), better neutralization activity, and stronger cross-protection activity against H1, H3, H5, and H7 strains and subtypes. Furthermore, the cHAmg vaccine combined with a glycolipid adjuvant designed for class switching further improved vaccine efficacy, generating more IFN-γ, IL-4, and CD8 + memory T cells.

[0050] Chimeric influenza virus hemagglutinin (HA) polypeptides

[0051] The present disclosure provides chimeric influenza virus hemagglutinin (HA) polypeptides for use as immunogens or vaccines to elicit CD4 + and CD8 + T cell immune responses. Thus, the chimeric influenza virus HA polypeptides can prevent influenza virus disease in an individual.

[0052] The chimeric influenza hemagglutinin (HA) polypeptides of the present invention comprise one or more stem domain sequences each having at least 60% homology to a stem domain consensus sequence of H1 subtype HA (H1 HA) and / or H5 subtype HA (H5 HA) fused to one or more globular head domain sequences each having at least 60% homology to a globular head consensus sequence of H1 subtype HA (H1 HA) or H5 subtype HA (H5 HA).

[0053] The term "homology" as used herein refers to the overall relatedness between polymeric molecules, such as between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Polymeric molecules (e.g., nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules) that share a similarity or identity above a threshold level determined by alignment of matching residues are said to be homologous. Homology is a qualitative term describing the relatedness between molecules and can be based on a quantitative measure of similarity or identity. Similarity and identity are quantitative terms defining the extent of sequence match between two sequences being compared. In some embodiments, polymeric 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.

[0054] In some embodiments, the polypeptides of the present invention can comprise one or more sequences having at least 60% homology to a consensus sequence of H1 HA or H5 HA of known human and avian influenza 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 H1 HA; an N-terminal stem segment of H1 HA or a C-terminal stem segment of H1 + H5 HA sequence; or an N-terminal stem segment of H5 HA or a C-terminal stem segment of H1 + H5 HA sequence.

[0055] 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.

[0056] SEQ ID NO: 1 (H1 stem)

[0057]

[0058] SEQ ID NO: 2 (H1 stem)

[0059]

[0060] SEQ ID NO: 5 (H1 stem)

[0061]

[0062] SEQ ID NO: 6 (H1+H5 stem)

[0063]

[0064] SEQ ID NO: 9 (H5 stem)

[0065]

[0066] SEQ ID NO: 10 (H5+H1 stem)

[0067]

[0068] In one embodiment, 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.

[0069] SEQ ID NO: 3 (H5 globular head)

[0070]

[0071] SEQ ID NO: 7 (H1 globular head)

[0072]

[0073] SEQ ID NO: 11 (H5 globular head)

[0074]

[0075] 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.

[0076] SEQ ID NO: 4 (chimeric H5 / 1)

[0077]

[0078] SEQ ID NO: 8 (swap H1 / 5)

[0079]

[0080] SEQ ID NO: 12 (exchange H5 / 1)

[0081]

[0082] In some embodiments, to enhance immunogenicity, one or more glycosylation sites on the HA are monoglycosylated. Preferably, the monoglycosylated HA has only N-acetylglucosamine (GlcNAc) on each glycosylation site.

[0083] Chimeric influenza virus HA polypeptides can be produced by any suitable method, many of which are known to those of skill in the art. For example, proteins can be synthesized chemically, or produced using recombinant DNA technology (e.g., in bacterial cells, in cell culture (mammalian, yeast, or insect cells), in plants or plant cells, or by free prokaryotic or eukaryotic-based expression systems, by other in vitro systems, etc.). Accordingly, the present application provides recombinant polynucleotides comprising nucleic acid sequences encoding the polypeptides of the present application, and optionally, nucleic acid sequences encoding signal peptides. The present application provides vectors comprising the recombinant polynucleotides of the present application. Embodiments of the polypeptides of the present application 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 the vectors of the present application are also provided.

[0084] Immunogenic compositions

[0085] The immunogenic compositions preferably comprise 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.

[0086] The chimeric influenza virus HA polypeptides of the present application can be formulated or administered in combination with one or more pharmaceutically acceptable excipients. The immunogenic / vaccine compositions can be sterile, pyrogen-free, or sterile and pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents, such as vaccine compositions, can be found in, for example, Remington: The Science and Practice of Pharmacy 21st Ed., Lippincott Williams & Wilkins, 2005 (herein incorporated by reference in its entirety).

[0087] The 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 quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize antibodies and, if necessary, to produce cell-mediated immunity. The precise amount of active ingredient required is that which is therapeutically effective, as determined by the practitioner. However, a suitable dosage range is readily determined by one skilled in the art. The regimen utilized to achieve the desired therapeutic effect will vary depending on the particular active ingredient selected. Suitable dosages are readily determined by one skilled in the art.

[0088] The formulations of the vaccine compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing into association the active ingredients with the excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, bringing the product to a form suitable for dosing, such as a powder, a tablet or a capsule.

[0089] Applications

[0090] It has been known for some time that cytotoxic T lymphocytes (CTLs) can provide an immune response against influenza strains. Recent studies have shown that CTL responses in humans can be directed against multiple epitopes.

[0091] Provided herein are methods for preventing influenza virus disease in humans and other mammals. Also provided are methods of eliciting an immune response in an individual against influenza virus. The methods involve administering to the individual an effective amount of a chimeric influenza virus HA polypeptide or immunogenic composition / vaccine of the present application, which induces an immune response in the individual that is specific for influenza strains, such as H1, H3, H5, and H7 strains and subtypes. Preferably, these methods elicit CD4 + and CD8 + T cell immune responses. More preferably, these methods induce stem-specific antibodies that have higher antibody-dependent cellular cytotoxicity (ADCC), better neutralization activity, and stronger cross-protection activity against H1, H3, H5, and H7 strains and subtypes. These methods also improve vaccine efficacy, producing more IFN-γ, IL-4, and CD8+ memory T cells.

[0092] Antibody titers in individuals are increased following vaccination. In exemplary aspects, the immunogenic compositions or vaccines of the present application are used to provide prophylactic protection from influenza. Prophylactic protection from influenza can be achieved following administration of a vaccine or combination vaccine of the present application. The vaccine, including combination vaccine, can be administered once, twice, three times, four times, or more, but administration of the vaccine once, followed if necessary by a single booster, can be sufficient. Thus, dosing can need to be adjusted.

[0093] A prophylactically effective dose is a therapeutically effective dose of a vaccine that prevents influenza virus to a clinically acceptable degree. In some embodiments, a therapeutically effective dose is a dose listed in the package insert of the vaccine.

[0094] The chimeric influenza virus HA polypeptides or immunogenic compositions / vaccines of the present application can be administered by any route that produces a therapeutically effective result. These routes include, but are 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 application can be administered intramuscularly or intradermally, similar to the administration of inactivated vaccines known in the art.

[0095] The application of the present application is not limited to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present application is capable of other embodiments and of being practiced or being carried out in various ways.

[0096] Examples

[0097] Methods

[0098] Vaccine and plasmid construction. All 102 full-length HA sequences from H1N1 viruses available from 2009 to early 2013 were downloaded from NCBI database and aligned by ClustalW algorithm by BioEdit program. The most conserved amino acids at each position were selected to form 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 pcDNA expression vector, and the resulting plasmids were used as templates for swapping and chimeric HA construction. Swap H1 / 5 is composed of H1 (amino acids 1-327 of SEQ ID NO: 8) as HA1 and H5 (amino acids 328-503 of SEQ ID NO: 8) as HA2, resulting in H1 as the globular head and H1+H5 (HA2) stem. Swap H5 / 1 is composed of H5 (amino acids 1-330 of SEQ ID NO: 12) as HA1 and H1 (amino acids 331-506 of SEQ ID NO: 12) as HA2, resulting in H5 as the globular head and H5+H1 (HA2) stem. For the chimeric H5 / 1 construct, the globular head domain is composed of the amino acid sequence between residues C42 and C274 (H3 numbering) of SEQ ID NO: 4, and the stem region is composed of parts 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 at the C-terminus of HA with additional residues from the bacteriophage T4 fibritin foldon trimerization sequence, a thrombin cleavage site, and a (His)6-tag. The two DNA sequences of consensus HA were optimized for expression by using human-biased codons, and the various regions were amplified by PCR and then cloned into pcDNA vectors for expression. In addition, HA genes from influenza virus 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. Sequences were confirmed by DNA sequencing and prepared in high quality for protein expression and purification.

[0099] Recombinant secreted HA from expressed 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 with Mirus TransIT ® -LT1 (Mirus Bio) transfection reagent using a 3: 1 ratio of reagent to plasmid DNA. TransIT ® -LT1 reagent was diluted with Opti-MEM (Gibco) and the mixture was incubated for 5-20 minutes at room temperature. Plasmid DNA was added to the solution and mixed thoroughly, followed by incubation for 15-30 minutes. Prior to transfection, cells were replaced with fresh DMEM (Gibco) media supplemented with 10% fetal bovine serum. TransIT ® -LT1 reagent / DNA complex was added to the cells and incubated for 48 h at 37°C. Expression of hemagglutinin was confirmed with immunoblots using anti-(his)6 antibody (Qiagen) or specific anti-hemagglutinin antibody and secondary horseradish peroxidase (HRP)-conjugated antibody (PerkinElmer).

[0100] Purification of recombinant secreted hemagglutinin. For expression in human 293T cells, pcDNA harboring the gene of interest was prepared in high quality and transfected with Mirus TransIT ®- LT1 (Mirus Bio) was transfected into cells. After 48 h of transfection, the culture medium was collected and the cells were clarified by centrifugation at 1,000 x g for 10 min. The supernatant was purified by 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 out with 20 mM Tris-HCl pH 8.0 and 300 mM NaCl (buffer A) containing a gradient of 25 mM to 50 mM imidazole. Then, the HA proteins were eluted with buffer A containing a gradient of 100 mM to 300 mM imidazole. The purified HA proteins were concentrated in PBS pH 7.4 by Amicon ultrafiltration units (MW 30K cutoff) (Millipore). The purity was monitored by using SDS-PAGE and the proteins were confirmed using Western blot with anti-(his)6 antibody (Qiagen) or specific anti-hemagglutinin antibody and secondary antibody conjugated to horseradish peroxidase (PerkinElmer). Finally, the trimeric form of the HA proteins was obtained by using a size exclusion column, Superdex 200 Increase 10 / 300 GL gel filtration column (GE Healthcare).

[0101] Monoglycosylated HA proteins were prepared. HA with high-mannose glycans 31 was produced using HEK293S cells lacking N-acetylglucosaminyltransferase I. Purified HA proteins from HEK293S cells were treated with Endo H (NEB) at 20 °C overnight to produce monoglycosylated HA mg . The ratio of protein to Endo H was 3 to 1 (w / v) for HA. Then, Endo H was separated from the monoglycosylated HA proteins by a Superdex 200 Increase 10 / 300 GL gel filtration column (GE Healthcare). The HA proteins were concentrated in PBS pH 7.4 by Amicon ultrafiltration units (MW 30K cutoff) (Millipore) and these proteins were confirmed by SDS-PAGE and LC-MS / MS analysis. mg

[0102] ​N-linked glycosylation on HA proteins was identified. Ten micrograms of protein were run on SDS-PAGE and prepared for in-gel digestion. The desired protein band was excised with a sharp razor blade, cut into 1 mm pieces and placed into 1.3 ml eppendorf tubes. After washing twice with 500 μΐ of 50% ACN (acetonitrile) containing 25 mM ammonium bicarbonate for 3 min, the gel pieces were dried using a SpeedVac evaporator (Thermo). The dried samples were reduced by adding 100 μΐ of 25 mM ammonium bicarbonate (pH 8.5) containing 50 mM dithiothreitol (DTT) at 37°C for 1 h followed by centrifugation at 10,000 g for 1 min. The solution was removed and the gel samples were subjected to an alkylation step by adding 100 μΐ of 25 mM ammonium bicarbonate (pH 8.5) containing 100 mM iodoacetamide (IAA) and incubated at room temperature for 1 h in the dark. After washing with 500 μΐ of 25 mM ammonium bicarbonate (pH 8.5) containing 50% acetonitrile and 500 μΐ of 100% acetonitrile, the samples were centrifuged at 10,000 g for 1 min and the supernatant was completely removed. The gel samples were dried at a SpeedVac evaporator and re-dissolved with 200 μΐ of 25 mM ammonium bicarbonate (pH 8.5). Subsequently, the gel samples were treated with 0.5 μg of trypsin (Promega, Madison, WI, USA) and 1 μg of chymotrypsin (Promega, Madison, WI, USA) overnight. After overnight digestion, 100 μΐ of 5% TFA containing 50% acetonitrile was added to the samples. The samples were sonicated for 10 seconds and then stopped for 10 seconds. These processes were repeated 10 times. The supernatant containing the peptide mixture was removed from the sample tubes and transferred to new tubes. The procedure was repeated twice. The combined supernatants were dried in a SpeedVac concentrator and processed for LC-MS / MS analysis.

[0103] Endotoxin measurement. Pierce ®LAL chromogenic endotoxin quantitation kit (Thermo Scientific) was used to determine endotoxin content. Protein samples were diluted 10, 20, 100 and 1000 times, 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 microtiter plate in the heating block for 10 min at 37°C, the protein samples or standards were mixed with Limulus amebocyte lysate (LAL) Pyrochrome reagent (100 μl final volume) (1:1) in endotoxin-free wells for 10 min at 37°C. One hundred microliters of substrate solution was added to each well and the plate was incubated for 6 min at 37°C. 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 against the corresponding standard concentration. The endotoxin concentration of the samples was determined using the standard curve. All purified proteins had endotoxin values < 0.5 ng / ml.

[0104] Mouse vaccination. 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 immunized intramuscularly with 20 μg of purified chimeric HA fg or HA mg protein mixed with 50 μg aluminum hydroxide (alum; Sigma) or 2 μg C34. Control mice were injected with phosphate-buffered saline (PBS). Three vaccinations were given at two-week intervals. Blood was collected 14 days after the second or third immunization. Blood was incubated at 37°C for 30 min and centrifuged at 1,2000 rpm for 10 min to collect serum. Serum collected from vaccinated mice was assessed for HA-specific antibodies by enzyme-linked immunosorbent assay (ELISA) and neutralization assay.

[0105] HA-specific antibodies were determined 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 targets. Ninety-six well ELISA plates (Greiner bio-one, Frickenhausen, Germany) were coated with 100 μΐ of protein diluted in ELISA coating buffer, i.e. 100 mM NaHC03(pH 8.8), at a concentration of 5 μg / ml / well and covered with plastic sealer at 4°C overnight. After blocking the plates 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 and washing them 3 times with TBST, the plates were incubated with 200 μΐ of 2-fold serial dilutions of mouse sera for 2 h at 37°C. After sera were moved and plates were washed 6 times, HA-specific IgG was monitored by using 200 μΐ of secondary HRP-labeled anti-mouse antibody (1 :8000) (PerkinElmer, Waltham, MA, USA). After 1 h incubation at 37°C, plates were washed 6 times with TBST and developed with 100 μΐ of Super Aquablue ELISA substrate (eBioscience, San Diego, CA, USA) for 1 min. The reaction was stopped by adding 100 μΐ of 0.625 M oxalic acid. The absorbance of each well was measured at 405 nm using a SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA). The endpoint antibody titer was defined as the highest dilution of serum that produced an absorbance 2.5-fold higher than the optical density (OD) produced by the negative control (pre-immune serum). The background endpoint antibody titer was assigned as less than 1 :50.

[0106] Bone marrow-derived dendritic cells were harvested. GM-CSF-cultured bone marrow-derived dendritic cells (BMDCs) were prepared as previously described. Briefly, bone marrow single cell suspension was subjected 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 murine GM-CSF (eBioscience), 10% FBS (BenchMark), 50 μΜ 2-ME, 100 units / mL penicillin, and 100 μg / mL streptomycin. Cells were plated into each petri dish to achieve 2 x 10 6final cell density of 1 cell / Petri dish. Cultures were replenished on day 3 by adding 10 ml fresh medium containing 20 ng / mL murine GM-CSF and refreshed on day 6 with half volume of complete medium as described above. On day 8, immature BMDCs were harvested by collecting non-adherent cells using gentle pipetting and re-plated at a density of 10 6 For CD8+ T cell analysis, immature BMDCs were co-cultured with CD8+ T cells and chimeric HA protein (0.1 mg / well in 100 μΙ_) for 48 h. After washing, the number of granzyme B-producing CD8+ T cells was determined by flow cytometry.

[0107] 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 min. To detect IFN-γ, IL-4 and granzyme B-secreting cells from chimeras-immunized mice, splenocytes were collected and cultured at 5 x 10 5 50 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 min. To detect IFN-γ, IL-4 and granzyme B-secreting cells from chimeras-immunized mice, splenocytes were collected and cultured at 5 x 10

[0108] Neutralization assay. Culture supernatant containing 100 TCID 50 virus was mixed with an equal volume of two-fold serially diluted serum and incubated for 1 h at 37°C. Then, the mixture was added to MDCK cells in each well of a 96-well plate and incubated for 3 days at 37°C. 30 μΙ CellTiter-Glo (Promega) was added to the cells to determine the number of viable cells based on the quantification of ATP present. The neutralizing activity of the serum was determined as the maximum dilution factor that significantly protected the cells from virus-induced death.

[0109] Micro-neutralization assay. Culture supernatant containing 100 TCID 50Virus infection medium (DMEM supplemented with 0.3% BSA and 2 μg / ml TPCK-trypsin) was mixed with an equal volume of serum at a serial dilution and incubated at 37°C for 1 h. Subsequently, the mixture was added to each well of a 96-well plate containing MDCK cells (1.5 × 10⁶ cells / well). 4 Cells were incubated in wells at 37°C for 16–20 h. Cells were washed with PBS, fixed in acetone / methanol solution (1:1 vol / vol), and blocked with 5% skim milk. After incubation at 37°C for 1 h, each well was washed 6 times with PBST, and viral titer was monitored using 100 μl of anti-influenza A NP mAb (1:2500). After incubation at 37°C for 1 h, each well was 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 h, each well was washed 6 times with PBST again and developed with 50 μl of 1-Step Ultra TMB receptor (Thermo) for 1 min. The reaction was stopped by adding 50 μl of 1 M H2SO4. The absorbance of each well was measured at 450 nm using a SpectraMax M5 (MolecularDevices, Sunnyvale, CA, USA).

[0110] Antibody-dependent cell-mediated cytotoxicity reporter assay. MDCK cells (1 × 10⁶) were collected from each well of a 96-well flat-bottomed dish. 4 Cells / well were cultured at 37°C for 24 h. The next day, 1 × 10⁻⁶ cells were cultured at an infection rate (MOI) of 1. 4 MDCK cells were infected with influenza virus for 24 h. The medium was then replaced with Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 4% low-IgG serum, followed by the addition of serially diluted antiserum from mice inoculated with chimeric HA protein, and incubated at 37°C for 30 min. Jurkat effector cells (Promega) expressing mouse FcγRIII were suspended in RPMI 1640 medium containing 4% low-IgG FBS, and target cells:effector cells were added to the infected MDCK cells at a 1:5 ratio. After incubation at 37°C for 6 h, the analysis tray was removed from the 37°C incubator and equilibrated at ambient temperature for 15 min, followed by the addition of Bio-Glo™ luciferase assay buffer (Promega) at a 1:1 ratio. Luminescence was measured using a CLARIOstar tray reader.

[0111] Virus challenge experiments. Two weeks after three vaccinations at two-week intervals, immunized mice were challenged intranasally with 10 LD50 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 H5N1 A / Turkey / 1 / 2005 / NIBRG23. After infection, mice were observed daily for 14 days and survival and body weight were recorded. Body weight percent for each individual animal in each group was calculated by comparing daily body weight to pre-challenge body weight, and mice that lost more than 25% of their initial body weight were euthanized and scored as dead. All animal experiments were performed under Biosafety Level 3 enhanced conditions. 50 (cause 50% of mice to die) 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 H5N1 A / Turkey / 1 / 2005 / NIBRG23. After infection, mice were observed daily for 14 days and survival and body weight were recorded. Body weight percent for each individual animal in each group was calculated by comparing daily body weight to pre-challenge body weight, and mice that lost more than 25% of their initial body weight were euthanized and scored as dead. All animal experiments were performed under Biosafety Level 3 enhanced conditions.

[0112] Expression and purification of recombinant F10 antibody. Plasmids encoding F10 antibody were transfected into serum-free adapted FreeStyle™ 293F cells using polyethylenimine and cultured in FreeStyle™ 293 expression medium (Gibco) in 125 ml sterile Erlenmeyer flasks, rotating on a roller platform at 135 rpm. Supernatant was collected 72 h post-transfection and cells were clarified by centrifugation at 1,000 x g for 10 min. Supernatant was loaded onto a Protein-A column (GE Healthcare) pre-equilibrated in 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 eluate was collected into tubes containing 0.5 mL 1 M Tris-HCl pH 9.0 for neutralization. Purity was monitored by SDS-PAGE.

[0113] Statistical analysis. Animal experiments for assessing immune responses were repeated at least three times (n = 5 / group) and virus challenge studies were performed at least twice (n = 10 / group). Responses of individual mice were counted as individual data points for statistical analysis. Data obtained from animal studies were examined by two- factor ANOVA from Prism; data are presented as mean ± SEM and differences were considered significant, *P < 0.05; **P < 0.01; ***P < 0.001.

[0114] Example 1 Preparation and characterization of monoglycosylated chimeric HA.

[0115] To design a universal vaccine, we first aimed to have a vaccine with broad protection against Group 1 influenza A viruses (H1 and H5 are major subtypes, while H2, H6, and H9 are minor subtypes). Thus, a consensus H1 sequence was generated using the HA sequences of H1N1 viruses available from early 2009 to 2013. Subsequently, consensus H5 and consensus H1 were used as templates for vaccine design. In influenza virus replication, the HA precursor (HA0) is proteolytically cleaved into two subunits, HA1 and HA2; the HA1 subunit carries 5-N-acetylneuraminic acid (sialic acid) binding sites, and the HA2 subunit is responsible for fusion of the virus with the host cell membrane Figure 5 A). On the other hand, HA can be divided into two domains, i.e., the globular head and the stem, based on the three-dimensional (3D) structure. The stem region contains the HA2 domain, the N-terminal 36~50 residues, and a short extension of the C-terminus of the HA1 domain. Thus, we designed vaccines based on various combinations of domains from H1 and H5. We first generated a swap H1 / 5 (H1 globular head and [H1+H5 (HA2) stem], a swap H5 / 1 (H5 globular head and [H5+H1 (HA2) stem], and a chimeric H5 / 1 (H5 globular head and H1 stem) for comparison Figure 1 A and Figure 5 A). The results indicated that vaccination with consensus 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 Figure 1 B). We next investigated whether this cross-protection was contributed by CD8 + T cell responses, and found that granzyme B was more secreted in mice immunized with chimeric H5 / 1, indicating that the chimeric H5 / 1 vaccine induced stronger CD8 + T cell responses than the swap H5 / 1 vaccine Figure 1 C).

[0116] Example 2 Effect of glycosylation on immune responses to chimeric H5 / 1 (cHA)

[0117] 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 known to be specific for high-mannose but not complex-type poly-N-acetylglucosamine. HA glycoprotein expressed in HEK293S cells, which lack N-acetylglucosamine transferase I and produce glycoproteins with high-mannose type N-polyglycosylation, was treated with Endo-H to cleave the N-polyglycosylation into a single GlcNAc residue. To generate cHA mgcHA 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-glycan to produce HA with only one N-acetylglucosamine (GlcNAc) attached to each glycosylation site. After Endo-H treatment, the mixture was passed through gel filtration to separate Endo-H from trimeric cHA mg . After concentration, cHA mg was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS / PAGE) and liquid chromatography-mass spectrometry (LC-MS / MS) analysis to ensure purity and glycan composition Figure 5 C). Since influenza HA exists as a trimer on the virus surface, gel filtration was performed to confirm that cHA fg and cHA mg existed as trimers (>200 kDa) Figure 5 D). We also produced another fully glycosylated cHA fg from human cells (HEK293T) for comparison Figure 5 B), and cell culture produced ~6 mg / L of cHA fg .

[0118] The N-linked glycosylation sites and glycan profile of recombinant cHA fg and cHA mg were analyzed by LC-MS / MS, showing seven glycosylation sites (N28, N40, N171, N182, N292, N303, and N497); the N-glycans of cHA fg were mostly complex, and ~99% of cHA mg could be obtained in the form of a single glycoform with only GlcNAc at each of its N-glycosylation sites Figure 5 E and Table 1).

[0119] Table 1. N-linked glycan structures of cHA in fully glycosylated and monoglycosylated proteins analyzed by LC-MS / MS.

[0120] Example 3. Cross-reactivity of antisera from mice immunized with fully glycosylated chimeric H5 / 1 (cHA fg ) and monoglycosylated chimeric H5 / 1 (cHA mg )

[0121] To assess the binding activity of antibodies elicited by cHA constructs, BALB / c mice were immunized intramuscularly with 20 μg of cHAfgor cHAmgprotein adjuvanted with Al(OH)3or C34 (an alpha-galactosylceramide (a-GalCer) analog). Mice were immunized at weeks 0, 2, and 4, and HA-induced sera were obtained at days 28 and 42 and measured using enzyme-linked immunosorbent assay (ELISA) with various recombinant HA Figure 6 ). Compared to the maximum dilution of antisera after two immunizations, three immunizations actually produced antisera with higher titers of HA-specific antibodies Figure 1 D-I and Figure 7 ), and cHA fg vaccination induced better antibody responses mg than cHA Figure 1 D, E, and G). In addition, antisera from cHA mg showed slightly better binding to H3 and H7 HA proteins Figure 1 F and H), and no significant difference was observed between Al(OH)3and C34 adjuvants. These data indicate that cHA vaccines can elicit cross-reactive antibodies that recognize HA from H1N1, H3N2, H5N1, and H7N9 viral strains.

[0122] F10 is a broadly neutralizing IgG antibody known to target the stem region of HA, which is highly conserved among various subtypes of influenza viruses. To compare the binding of F10 to recombinant H1, H5, and cHA, the binding affinity of F10 to various HA was measured, and the results showed that F10 can bind to H1, H5, and cHA proteins Figure 8 . To investigate whether F10-like antibodies are elicited by cHA vaccination, the binding of cHA-induced sera to HA stem no. 4900 was measured by using ELISA. The results showed that cHA mg vaccines can induce higher titers of stem-specific antibodies fg than cHA Figure 1 I and Figure 7 F), and better results were observed with cHA vaccines adjuvanted with C34 Figure 1 I.

[0123] Example 4 cHA mg and adjuvant C34 elicit strong CD4 + and CD8 + T cell responses and antibody-dependent effector functions and neutralizing activity against H1, H3, H5 viruses and their subtypes

[0124] Besides antibody-mediated neutralization, Fc-mediated effector function also plays an important role in influenza infection prevention. Therefore, we examined whether antibodies induce Fc receptor-mediated immune responses. Mouse adaptive ADCC analysis was performed using Jurkat effector cells expressing FcγRIII to assess ADCC activity in serum from mice immunized with cHAfg and cHAmg. Figure 2 As expected, from cHA vaccination fg or cHA mg Serum from 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, in cHA with Al(OH)3 adjuvant... mg Better ADCC activity was observed in the group. Figure 2 B), and in response to H1N1 A / California / 07 / 2009, A / Brisbane / 59 / 2007, A / Solomon Islands / 3 / 2006, A / New Caledonia / 20 / 1999 ( Figure 2 A), H3N2 A / Wisconsin / 67 / 2005 and A / Victoria / 361 / 2011 viruses ( Figure 2 Similar results were observed in experiment C).

[0125] To assess the role of antigen-specific cytokine-secreting cells in cHA-immunized mice, spleen cells were collected after two and three immunizations, and IFN-γ, IL-4, and granzyme B (GzB) secretory cells were estimated using enzyme-linked immunosorbent assay (ELISpot) analysis with specific peptides of HA used for stimulation. Figure 3 As shown, cHA with Al(OH)3 adjuvant fg and cHA mg The vaccine produces similar levels of interferon-secreting cells. However, this is different from cHA with Al(OH)3 adjuvant. mg Compared to vaccination, cHA with C34 adjuvant is more effective. mg Vaccination triggers more CD4 + / IFN-γ + Th1 cells ( Figure 3 A) CD4 + / IL-4 + Th2 ( Figure 3 B) and CD8 + GzB secretory cells ( Figure 3 C). These results confirm that, with cHA fgcHA with C34 adjuvant mg stimulated more CD4 + T helper cell responses and stronger CD8 + cytotoxic effects.

[0126] To assess the dose dependency of C34 on antibody titers and cell-mediated immunity, cHA fg was adjuvanted with three different doses of C34, 0.5 μg, 2 μg and 10 μg fg The results indicated that after two or three immunizations, cHA fg adjuvanted with 0.5 μg and 10 μg C34 fg induced higher titers of antibodies fg than cHA fg adjuvanted with 2 μg C34 fg induced more IFN-γ fg than cHA + adjuvanted with 0.5 μg C34 mg There was no difference in the increase of CD8 mg GzB secreting cells when cHA mg was adjuvanted with 0.5 μg, 2 μg or 10 μg C34 after two and three immunizations. fg Based on these observations, 2 μg C34 was used throughout the experiments.

[0127] The neutralizing activity of cHA induced antisera was further investigated. Antisera from cHA mg vaccinated mice were shown to have better neutralizing activity against homologous virus H1N1 A / California / 07 / 2009 Figure 3 (D) and heterologous H5N1 NIBRG14 (A / Vietnam / 1194 / 2004), NIBRG23 (A / Turkey / 1 / 2005), RG5 (A / Anhui / 1 / 2005) or RG2 (A / Indonesia / 5 / 2005) Figure 3 (E). In addition, antisera from mice vaccinated with cHA mg exhibited significant neutralizing activity against heterologous viruses H1N1 A / Brisbane / 59 / 2007, A / New Caledonia / 20 / 1999 and A / Solomon Islands / 3 / 2006 Figure 3D). Antiserum from mice immunized with cHA significantly blocked H1N1 and H5N1 virus infection, and cHA mg Its neutralizing activity is generally higher than that of cHA. fg Even better, especially against foreign viruses.

[0128] Example 5: Inoculation of mice with cHA in an attack study mg / C34 provides cross protection against H1N1 and H5N1 and their subtypes.

[0129] In order to assess cHA mg Whether vaccination provides broad cross-protective immunity against various H1N1 and H5N1 viruses was assessed by challenging vaccinated mice with lethal doses of multiple H1N1 and H5N1 viruses via intranasal injection, and by recording survival rate and weight changes for 14 days. Figure 4 and Figure 11 All cHA vaccines provided 100% protection in mice challenged with the H1N1 A / California / 07 / 2009 virus. Figure 4 A). Additionally, with cHA fg In comparison, cHA with C34 adjuvant mg Immunized mice showed the least amount of weight loss. Figure 11 A) cHA with C34 adjuvant fg Immunized mice received only 30% protection against A / New Caledonia / 1999 challenge; however, cHA with C34 adjuvant... mg The vaccine provides 90% protection against cross-strain A / New Caledonia / 1999 virus, and similar results have been observed in cHA vaccines with Al(OH)3 adjuvant. Figure 4 B). In mice challenged with the cross-linked virus strain A / WSN / 1933, all mice immunized with cHA adjuvanted with Al(OH)3 survived; however, mice immunized with cHA adjuvanted with C34 survived. fg The immunized mice received only 80% protection. Figure 4 C). Lethal challenge was also performed using A / Solomon Islands / 03 / 2006. 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 Immunized mice showed better protection against cross-linked strain A / Solomon Islands / 03 / 2006 virus. Figure 4 D). Of the mice challenged with H5N1 NIBRG14 (A / Vietnam / 1194 / 2004) and NIBRG23 (A / Turkey / 1 / 2005), all immunized mice survived. Figure 4 E and F). Weight changes following viral attack were also assessed.Figure 11 ). The data show that cHA effectively elicited significant protective immunity against various H1N1 and H5N1 viruses, and cHA mg provided broader cross-protection ability than cHA fg .

[0130] The development of a universal influenza vaccine to provide protection against multiple strains and subtypes of influenza viruses is currently of interest, and antigenic determinants for universal vaccine development include the highly conserved ectodomain of M2 containing 24 non-glycosylated amino acids, the nucleoprotein NP, and various HA constructs, which have been shown to induce higher titered broadly neutralizing antibodies targeting the HA-stem region or blocking virus entry. For example, soluble trimeric HA (microHA) vaccines with realigned stem subunits showed complete protection of mice from lethal challenge with heterologous and heterosubtypic viruses, and vaccination with chimeric HA to the same stem region with DNA prime-protein boost and exposure of divergent ectodomains showed elicitation of broadly protective stem-specific antibodies. However, the results showed that CD8 + T cells do not play a key role in cross-protection activity. Although DNA vaccines are promising, they are still in early development. In this study, cHA constructs expressing the common H5 and common H1 stem regions of the globular head were designed to mimic the actual state of influenza viruses that spread from avian viruses to human viruses. Fully glycosylated cHA fg and mono-glycosylated cHA mg were prepared for comparison, and the results showed that cHA mg vaccines elicited higher titers of cross-reactive antibodies against H1, H3, H5, and H7 subtypes + and CD8 + T cell responses Figure 3 A-C). cHA Figure 1 D-H) elicited higher titers of cross-reactive antibodies against H1, H3, H5, and H7 subtypes

[0131] HA glycosylation plays an important role in protein folding and stability and in modulating its biological activities, including masking of antigenic sites from neutralizing antibodies to reduce immunogenicity. In addition, hyperglycosylated HA evolved to mask antigenic sites in the highly variable head domain, and thus the immune response is redirected to the conserved stem region. In our results, cHA mg induced antisera significantly outperformed cHA fg induced antisera, particularly against heterologous H1N1 A / Brisbane / 59 / 2007, A / Solomon Islands / 03 / 2006, and A / New Caledonia / 20 / 1999 Figure 3 D). cHA mgThe broader neutralizing activity of the vaccine can be attributed to its induction of more antibody variants as previously reported. IgG is the predominant antibody present in mice and is the major subtype of HA-specific antibodies on immune cells with high avidity for the FcyRIII receptor, inducing ADCC. We show that vaccination with cHA mg Immunization induced higher ADCC and more stem-specific antibodies with better protective activity Figure 1 I and 2), consistent with studies showing that ADCC is necessary for in vivo influenza protection. Aluminum hydroxide (alum) is known to stimulate Th2 responses and is FDA-approved for use as a vaccine adjuvant; however, its mode of action has not been fully studied. Glycolipid C34 is a ligand for CD1d on dendritic cells and is presented by it to interact with receptors on invariant natural killer T (iNKT) cells, causing stimulation of iNKT cells to produce Th1 cytokines (e.g., IFN-g) with adjuvant effects and Th2 cytokines (e.g., IL-4) with class switching activity. In our results, IFN-g (Th1 cytokine), IL4 (Th2 cytokine) secreting cells, and CD8 + T cells were significantly increased in mice immunized with cHA mg Immunization with cHA mg Immunization with cHA Figure 3 A-C).

[0132] In summary, the development of next-generation influenza vaccines with broadly protective immune responses is currently of interest, and some promising results have been reported, making the development of a universal vaccine an achievable goal. In an effort to this end, we have successfully demonstrated in this study the proof of principle that a single glycosylated cHA vaccine with a common H5 head and a 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. With the successful development of a broadly protective vaccine against different strains and subtypes of influenza A viruses, we aim to use the strategy developed in this study to design a broader universal vaccine against influenza A and B viruses. SEQUENCE LISTING <110> Chou, Mei-Yin <120> Chimeric influenza vaccine <130> 63 / 022,328 <160> 14 <170> PatentIn Version 3.5 <210> 1 <211> 41 <212> PRT <213> Influenza A virus <400> 1 Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val 1 5 10 15 Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn Leu 20 25 30 Leu Glu Asp Lys His Asn Gly Lys Leu 35 40 <210> 2 <211> 237 <212> PRT <213> Influenza A virus <400> 2 Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro 1 5 10 15 Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val 20 25 30 Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn Val Pro Ser 35 40 45 Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly 50 55 60 Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn 65 70 75 80 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala 85 90 95 Ile Asp Lys Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn 100 105 110 Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg 115 120 125 Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp 130 135 140 Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu 145 150 155 160 Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Asn 165 170 175 Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe 180 185 190 Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr 195 200 205 Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu 210 215 220 Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr Gln 225 230 235 <210> 3 <211> 233 <212> PRT <213> Influenza A virus <400> 3 Cys Asp Leu Asp Gly Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val 1 5 10 15 Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Ile Asn Val 20 25 30 Pro Glu Trp Ser Tyr Ile Val Glu Lys Ala Asn Pro Ala Asn Asp Leu 35 40 45 Cys Tyr Pro Gly Asn Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu 50 55 60 Ser Arg Ile Asn His Phe Glu Lys Ile Gln Ile Ile Pro Lys Ser Ser 65 70 75 80 Trp Ser Asp His Glu Ala Ser Ser Gly Val Ser Ser Ala Cys Pro Tyr 85 90 95 Gln Gly Lys Ser Ser Phe Phe Arg Asn Val Val Trp Leu Ile Lys Lys 100 105 110 Asn Ser Thr Tyr Pro Thr Ile Lys Arg Ser Tyr Asn Asn Thr Asn Gln 115 120 125 Glu Asp Leu Leu Val Leu Trp Gly Ile His His Pro Asn Asp Ala Ala 130 135 140 Glu Gln Thr Arg Leu Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly 145 150 155 160 Thr Ser Thr Leu Asn Gin Arg Leu Val Pro Lys He Ala Thr Arg Ser 165 170 175 Lys Val Asn Gly Gin Ser Gly Arg Met Glu Phe Phe Trp Thr He Leu 180 185 190 Lys Pro Asn Asp Ala He Asn Phe Glu Ser Asn Gly Asn Phe He Ala 195 200 205 Pro Gin Tyr Ala Tyr Lys He Val Lys Lys Gly Asp Ser Thr He Met 210 215 220 Lys Ser Glu Leu Glu Tyr Gly Asn Cys 225 230 <210> 4 <211> 511 <212> PRT <213> Artificial Sequence <220> <223> Chimeric Influenza Virus HA Polypeptide <400> 4 Asp Thr Leu Cys He Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val 1 5 10 15 Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn Leu 20 25 30 Leu Glu Asp Lys His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 35 40 45 Pro Leu lie Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 50 55 60 Pro Met Cys Asp Glu Phe lie Asn Val Pro Glu Trp Ser Tyr lie Val 65 70 75 80 Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 85 90 95 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg lie Asn His Phe Glu 100 105 110 Lys lie Gin lie lie Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser 115 120 125 Ser Gly Val Ser Ser Ala Cys Pro Tyr Gin Gly Lys Ser Ser Phe Phe 130 135 140 Arg Asn Val Val Trp Leu lie Lys Lys Asn Ser Thr Tyr Pro Thr lie 145 150 155 160 Lys Arg Ser Tyr Asn Asn Thr Asn Gin Glu Asp Leu Leu Val Leu Trp 165 170 175 Gly lie His His Pro Asn Asp Ala Ala Glu Gin Thr Arg Leu Tyr Gin 180 185 190 Asn Pro Thr Thr Tyr lie Ser Val Gly Thr Ser Thr Leu Asn Gin Arg 195 200 205 Leu Val Pro Lys lie Ala Thr Arg Ser Lys Val Asn Gly Gin Ser Gly 210 215 220 Arg Met Glu Phe Phe Trp Thr lie Leu Lys Pro Asn Asp Ala lie Asn 225 230 235 240 Phe Glu Ser Asn Gly Asn Phe lie Ala Pro Glu Tyr Ala Tyr Lys lie 245 250 255 Val Lys Lys Gly Asp Ser Thr lie Met Lys Ser Glu Leu Glu Tyr Gly 260 265 270 Asn Cys Asn Thr Thr Cys Gin Thr Pro Lys Gly Ala lie Asn Thr Ser 275 280 285 Leu Pro Phe Gin Asn lie His Pro lie Thr lie Gly Lys Cys Pro Lys 290 295 300 Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn Val 305 310 315 320 Pro Ser lie Gin Ser Arg Gly Leu Phe Gly Ala lie Ala Gly Phe lie 325 330 335 Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His His 340 345 350 Gln Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gin 355 360 365 Asn Ala Ile Asp Lys Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys 370 375 380 Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu Glu 385 390 395 400 Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp 405 410 415 Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg 420 425 430 Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val 435 440 445 Arg Asn Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe 450 455 460 Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys Asn 465 470 475 480 Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn Arg 485 490 495 Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr Gln 500 505 510 <210> 5 <211> 41 <212> PRT <213> Influenza virus A <400> 5 Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val 1 5 10 15 Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn Leu 20 25 30 Leu Glu Asp Lys His Asn Gly Lys Leu 35 40 <210> 6 <211> 228 <212> PRT <213> Artificial Sequence <220> <223> Common sequence of stem domain of H1 HA and H5 HA <400> 6 Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro 1 5 10 15 Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val 20 25 30 Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn Val Pro Ser 35 40 45 Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly 50 55 60 Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn 65 70 75 80 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala 85 90 95 Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn 100 105 110 Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu Arg Arg 115 120 125 Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp 130 135 140 Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu 145 150 155 160 Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Leu 165 170 175 Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe 180 185 190 Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr 195 200 205 Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys Arg Glu Glu 210 215 220 Ile Ser Gly Val 225 <210> 7 <211> 234 <212> PRT <213> Influenza virus A <400> 7 Cys Lys Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile 1 5 10 15 Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala 20 25 30 Ser Ser Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr 35 40 45 Cys Tyr Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gin Leu 50 55 60 Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser 65 70 75 80 Ser Trp Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro 85 90 95 His Ala Gly Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys 100 105 110 Lys Gly Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys 115 120 125 Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Thr 130 135 140 Ala Asp Gin Gin Ser Leu Tyr Gin Asn Ala Asp Ala Tyr Val Phe Val 145 150 155 160 Gly Thr Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu lie Ala lie Arg 165 170 175 Pro Lys Val Arg Asp Gin Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu 180 185 190 Val Glu Pro Gly Asp Lys lie Thr Phe Glu Ala Thr Gly Asn Leu Val 195 200 205 Val Pro Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly lie 210 215 220 Ile lie Ser Asp Thr Pro Val His Asp Cys 225 230 <210> 8 <211> 503 <212> PRT <213> Artificial Sequence <220> <223> Chimeric Influenza Virus HA Polypeptide <400> 8 Asp Thr Leu Cys lie Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val 1 5 10 15 Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn Leu 20 25 30 Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val Ala 35 40 45 Pro Leu His Leu Gly Lys Cys Asn lie Ala Gly Trp lie Leu Gly Asn 50 55 60 Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile Val 65 70 75 80 Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe Ile 85 90 95 Asp Tyr Glu Glu Leu Arg Glu Gin Leu Ser Ser Val Ser Ser Phe Glu 100 105 110 Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp Ser 115 120 125 Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser Phe 130 135 140 Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro Lys 145 150 155 160 Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val Leu 165 170 175 Trp Gly Ile His His Pro Ser Thr Thr Ala Asp Gin Gin Ser Leu Tyr 180 185 190 Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Thr Ser Arg Tyr Ser Lys 195 200 205 Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gin Glu 210 215 220 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys Ile 225 230 235 240 Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala 245 250 255 Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro Val 260 265 270 His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr 275 280 285 Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro 290 295 300 Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn 305 310 315 320 Val Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 325 330 335 Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His 340 345 350 His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr 355 360 365 Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp 370 375 380 Lys Met Asn Thr Gin Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu 385 390 395 400 Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu 405 410 415 Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu 420 425 430 Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys 435 440 445 Val Arg Leu Gin Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys 450 455 460 Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg 465 470 475 480 Asn Gly Thr Tyr Asp Tyr Pro Gin Tyr Ser Glu Glu Ala Arg Leu Lys 485 490 495 Arg Glu Glu Ile Ser Gly Val 500 <210> 9 <211> 41 <212> PRT <213> Influenza virus A <400> 9 Asp Gin Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gin Val 1 5 10 15 Asp Thr lie Met Glu Lys Asn Val Thr Val Thr His Ala Gin Asp lie 20 25 30 Leu Glu Lys Thr His Asn Gly Lys Leu 35 40 <210> 10 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Common sequence of stem domain of H1 HA and H5 HA <400> 10 Asn Thr Lys Cys Gin Thr Pro Met Gly Ala lie Asn Ser Ser Met Pro 1 5 10 15 Phe His Asn lie His Pro Leu Thr lie Gly Glu Cys Pro Lys Tyr Val 20 25 30 Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gin 35 40 45 Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala lie Ala Gly 50 55 60 Phe lie Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr 65 70 75 80 His His Gin Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser 85 90 95 Thr Gin Asn Ala lie Asp Lys lie Thr Asn Lys Val Asn Ser Val lie 100 105 110 Glu Lys Met Asn Thr Gin Phe Thr Ala Val Gly Lys Glu Phe Asn His 115 120 125 Leu Glu Lys Arg lie Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe 130 135 140 Leu Asp lie Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn 145 150 155 160 Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu 165 170 175 Lys Val Arg Asn Gin Leu Lys Asn Asn Ala Lys Glu lie Gly Asn Gly 180 185 190 Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val 195 200 205 Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu 210 215 220 Asn Arg Glu Glu lie Asp Gly Val 225 230 <210> 11 <211> 233 <212> PRT <213> Influenza virus A <400> 11 Cys Asp Leu Asp Gly Val Lys Pro Leu lie Leu Arg Asp Cys Ser Val 1 5 10 15 Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Ile Asn Val 20 25 30 Pro Glu Trp Ser Tyr Ile Val Glu Lys Ala Asn Pro Ala Asn Asp Leu 35 40 45 Cys Tyr Pro Gly Asn Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu 50 55 60 Ser Arg Ile Asn His Phe Glu Lys Ile Gln Ile Ile Pro Lys Ser Ser 65 70 75 80 Trp Ser Asp His Glu Ala Ser Ser Gly Val Ser Ser Ala Cys Pro Tyr 85 90 95 Gln Gly Lys Ser Ser Phe Phe Arg Asn Val Val Trp Leu Ile Lys Lys 100 105 110 Asn Ser Thr Tyr Pro Thr Ile Lys Arg Ser Tyr Asn Asn Thr Asn Gln 115 120 125 Glu Asp Leu Leu Val Leu Trp Gly Ile His His Pro Asn Asp Ala Ala 130 135 140 Glu Gln Thr Arg Leu Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly 145 150 155 160 Thr Ser Thr Leu Asn Gln Arg Leu Val Pro Lys Ile Ala Thr Arg Ser 165 170 175 Lys Val Asn Gly Gin Ser Gly Arg Met Glu Phe Phe Trp Thr Ile Leu 180 185 190 Lys Pro Asn Asp Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala 195 200 205 Pro Glu Tyr Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser Thr Ile Met 210 215 220 Lys Ser Glu Leu Glu Tyr Gly Asn Cys 225 230 <210> 12 <211> 506 <212> PRT <213> Artificial Sequence <220> <223> Chimeric Influenza Virus HA Polypeptide <400> 12 Asp Gin Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gin Val 1 5 10 15 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gin Asp Ile 20 25 30 Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 35 40 45 Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 50 55 60 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val 65 70 75 80 Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 85 90 95 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 100 105 110 Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser 115 120 125 Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser Ser Phe Phe 130 135 140 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile 145 150 155 160 Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 165 170 175 Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Arg Leu Tyr Gln 180 185 190 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg 195 200 205 Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 210 215 220 Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn 225 230 235 240 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile 245 250 255 Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly 260 265 270 Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser 275 280 285 Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 290 295 300 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 305 310 315 320 Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile 325 330 335 Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr 340 345 350 Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu 355 360 365 Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn Lys Val Asn Ser 370 375 380 Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe 385 390 395 400 Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp 405 410 415 Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu 420 425 430 Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu 435 440 445 Tyr Glu Lys Val Arg Asn Gin Leu Lys Asn Asn Ala Lys Glu Ile Gly 450 455 460 Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu 465 470 475 480 Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala 485 490 495 Lys Leu Asn Arg Glu Glu Ile Asp Gly Val 500 505 <210> 13 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Signal Peptide <400> 13 Met Glu Lys Ile Val Leu Leu Leu Ala Ile Val Ser Leu Val Lys Ser 1 5 10 15 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide <400> 14 Met Lys Ala lie Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn 1 5 10 15 Ala

Claims

1. A chimeric influenza virus hemagglutinin polypeptide, wherein the chimeric influenza virus hemagglutinin polypeptide comprises the amino acid sequence of SEQ ID NO:

4.

2. The chimeric influenza virus hemagglutinin polypeptide of claim 1, wherein one or more of the seven glycosylation sites N28, N40, N171, N182, N292, N303 and N497 on the hemagglutinin of the chimeric influenza virus hemagglutinin polypeptide are monosaccharified, wherein the monosaccharified hemagglutinin has only N-acetylglucosamine GlcNAc at each glycosylation site.

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

4. An immunogenic composition comprising a chimeric influenza virus hemagglutinin polypeptide as described in any one of claims 1 to 3 and an adjuvant.

5. The immunogenic composition of claim 4, wherein the adjuvant is a glycolipid adjuvant, and the glycolipid adjuvant is a C34 adjuvant.

6. Use of a chimeric influenza virus hemagglutinin polypeptide as described in any one of claims 1 to 3 or an immunogenic composition as described in claim 4, for the manufacture of a medicament for the prevention of H1N1 or H5N1 influenza virus disease.

7. Use of the immunogenic composition as described in claim 5 for manufacturing a medicament for preventing H1N1 or H5N1 influenza virus disease.

8. The use as claimed in claim 7, wherein the immunogenic composition triggers CD4. + and CD8 + T-cell immune response.

9. The use as claimed in claim 7, wherein the immunogenic composition induces an antibody that triggers antibody-dependent cytotoxic ADCC, neutralizing activity, and cross-protective activity against H1N1 and H5N1 viral strains and subtypes.

10. The use as claimed in claim 7, wherein the immunogenic composition induces an antibody that triggers antibody-dependent cytotoxic ADCC against H3N2 virus strains and subtypes.

11. The use as claimed in claim 7, wherein the immunogenic composition induces the production of IFN-γ, IL-4, and CD8. + Memory T cells.

12. A recombinant polynucleotide comprising a nucleic acid sequence encoding a chimeric influenza virus hemagglutinin polypeptide as described in any one of claims 1 to 3 and a nucleic acid sequence encoding a signal peptide, wherein the sequence of the signal peptide is SEQ ID NO:13 or SEQ ID NO:

14.

13. A vector comprising the recombinant polynucleotide as described in claim 12.

14. A host cell comprising the vector as described in claim 13, wherein the host cell is not a plant cell.

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