Chimeric influenza vaccines

Abstract

The invention relates to chimeric influenza vaccines. 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 which has at least 60% homology with a stem domain common sequence of HA (H1 HA) subtype and / or H5 HA (H5 HA) subtype, the one or more globular head domain sequences each have at least 60% homology with a globular head domain common sequence of H1 subtype HA (H1 HA) or H5 subtype HA (H5 HA).

Description

[0001] This application is a divisional application of the invention patent application filed on May 7, 2021, with application number "202180033945.1" and title "Chimeric Influenza Vaccine". Related applications

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

[0003] sequence list The original application for this divisional application submitted a sequence list text file named G4590-08600PCT_SeqListing.txt, which was 28 kilobytes in size. This divisional application submits a sequence list in ST.26 format. Technical Field

[0004] This invention relates to chimeric influenza virus hemagglutinin (HA) polypeptides, immunogenic / vaccine compositions containing the same, and their applications. Background Technology

[0005] Traditional methods for producing influenza vaccines involve culturing the virus in specific pathogen-free (SPF) chicken embryos, a process that often requires more than six months for large-scale production. However, some vaccine virus strains do not grow well in eggs, and safety concerns may arise for individuals with egg allergies. Novel virus-based cell culture methods have been developed to replace egg-based methods; however, cell culture methods still carry the risk of producing potentially harmful viruses. To overcome these issues, research into alternative strategies has shown that vaccines based on recombinant HA can induce neutralizing antibodies against influenza virus infection. However, antibodies induced by specific influenza virus subtypes may not be effective in neutralizing other influenza subtypes. Furthermore, due to the continuous mutation of the virus, vaccines must be updated annually.

[0006] Therefore, it is still necessary to develop a universal vaccine that can resist broad-spectrum influenza virus strains. Summary of the Invention

[0007] In one embodiment, the present invention provides a chimeric influenza virus hemagglutinin (HA) polypeptide comprising one or more stem domain sequences fused with one or more globular head domain sequences, each of the one or more stem domain sequences having at least 60% homology with a stem domain common sequence of H1 subtype HA (H1 HA) and / or H5 subtype HA (H5 HA), and each of the one or more globular head domain sequences having at least 60% homology with a globular head domain common sequence of H1 subtype HA (H1 HA) or H5 subtype HA (H5 HA).

[0008] In some embodiments, HA is influenza A HA, influenza B HA, or influenza C HA.

[0009] In some embodiments, the homology is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0010] In some embodiments, the stem domain sequence is the N-terminal stem region of H1 HA or the C-terminal stem region of H1 HA; the N-terminal stem region of H1 HA or the C-terminal stem region of the H1+H5 HA sequence; or the N-terminal stem region of H5 HA or the C-terminal stem region of the H1+H5 HA sequence.

[0011] In some embodiments, the stem domain common 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.

[0012] In some embodiments, the common sequence of the globular 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.

[0013] 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.

[0014] In some embodiments, one or more glycosites on the HA are monosaccharified. In another embodiment, the monosaccharified HA has only N-acetylglucosamine (GlcNAc) at each glycosite.

[0015] In one embodiment, the chimeric influenza virus HA peptide is used as an immunogen.

[0016] In another embodiment, the present invention provides an immunogenic composition comprising a chimeric influenza virus HA peptide and an adjuvant. In one embodiment, the adjuvant is a glycolipid adjuvant.

[0017] In another embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid sequence encoding the polypeptide of the present invention 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.

[0018] In another embodiment, the present invention provides a vector comprising the recombinant polynucleotide of the present invention. A host cell comprising the vector of the present invention is also provided.

[0019] In another embodiment, the present invention provides a method for immunizing an individual against an influenza virus, the method comprising administering to the individual an effective amount of the chimeric influenza virus hemagglutinin (HA) polypeptide or immunogenic composition of the present invention.

[0020] In another embodiment, the present invention provides a method for preventing influenza virus disease in an individual, the method comprising administering to the individual an effective amount of the chimeric influenza virus hemagglutinin (HA) polypeptide or immunogenic composition of the present invention.

[0021] In one embodiment, the method described herein triggers CD4 + and / or CD8 + T-cell immune response.

[0022] In one embodiment, the method described herein induces stem-specific antibodies that exhibit high antibody-dependent cytotoxicity (ADCC), superior neutralizing activity, and strong cross-protective activity against H1, H3, H5, and H7 viral strains and subtypes.

[0023] In one embodiment, the method described herein improves vaccine efficacy by generating more IFN-γ, IL-4, and CD8+ memory T cells. Attached Figure Description

[0024] Figure 1 (A) to (I). A chimeric H5 / 1 structure (cHA) having a common H5 bulbous head and a common H1 stem, and through cHA mg Extensive cross-protective stem-specific antibodies induced by immunogen inoculation. (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) Neutralizing activity against H1N1 California / 07 / 2009 and H5N1 Vietnam / 1194 / 2004 viruses. (C) CD8+ granzyme B (GrzB)-producing cells in splenocytes of mice inoculated with PBS (control), HA+Alu, or HA+C34 for 2 days after stimulation with HA (black bar) or PBS (white bar) control. + T cell count was assessed by flow cytometry. (DI) cHA inoculated with Al(OH)3 adjuvant. fg and cHA mg Compared to cHA with C34 adjuvant fg and cHA mgThe antibody titers in mice were measured by ELISA on day 42 using the following antigens as coating antigens: 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). Endpoint antibody titer was defined as the final dilution of antiserum used to produce an absorbance 2.5 times higher than that produced by the negative control (pre-immunization serum). Data were obtained using Stulton's method. t Test (Student's) t The differences were examined using a test and a two-factor ANOVA from Prism; differences were considered statistically significant. P < 0.05; P < 0.01. Data represent mean ± SEM.

[0025] Figure 2 (A) to (C). Analysis of ADCC reporter bodies from antiserum of cHA-inoculated mice targeting cells expressing H1N1, H3N2, or H5N1 and their subtypes. cHA was used with aluminum hydroxide or C34 adjuvant. fg or cHA mg Antiserum collected from protein-immunized mice was cultured together with MDCK cells, which were then infected with (A) H1N1 virus, (B) H5N1 virus, or (C) H3N2 virus for 30 min. Subsequently, ADCC reporter body analysis was performed using Jurkat effector cells expressing mouse FcγRIII, and relative luminescent units (RLU) were measured, with values ​​being mean ± SEM. P < 0.001. Calculated using two-factor ANOVA with Prism software. P value.

[0026] Figure 3 (A) through (E). More CD4s + and CD8 + T-cell responses and broadly neutralizing antibodies via C34-adjuvanted cHA mg This triggers a broader cross-protection process. cHA with Al(OH)3 or C34 adjuvants... fg and cHA mgBALB / c mice were immunized; cells from the spleen of immunized mice were obtained after three immunizations, and the number of cells secreting IFN-γ (A), IL-4 (B), and GzB (C) was determined by ELISpot analysis using specific peptides. The number of speckled cells (SFCs) is expressed as mean ± SEM. Analysis of cells from cHA-inoculated mice... fg and cHA mg The neutralizing activity of mouse antiserum against (D)H1N1 and (E)H5N1 viruses was determined. Data are presented as mean ± SEM. Stuttgart assay was performed using Prism software. t Results of the test and two-factor ANOVA calculation; significant differences were marked as... P < 0.05; P < 0.01; P < 0.001.

[0027] Figure 4 (A) to (F). Cross-protective efficacy in mice challenged with lethal doses of H1N1 and H5N1 viruses. Three doses of cHA with Al(OH)3 or C34 adjuvants administered at 2-week intervals. fg and cHA mg BALB / c mice were immunized. The 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 post-infection survival for 14 days. P < 0.01. Significant differences in survival were analyzed using the logarithmic rank (Mantel-Cox) test.

[0028] Figure 5(A) to (E). Design and preparation of chimeric HA proteins. (A) The designed influenza HA sequence was constructed using a common H1N1 sequence and a common H5N1 sequence pCHA5-II to generate chimeric HA. The globular head domain consists of the amino acid sequence between residues C52 and C277 (H3 number). The stem region consists of portions of the HA1 and HA2 subunits. The protein structure was downloaded from the protein database IDs 2IBX (VN1194 H5 HA) and 3LZG (A / California / 04 / 2009). The final image was generated using PyMol. Since the structure of the common HA was not yet publicly available, 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. (BD) Purification and gel filtration chromatography analysis of the chimeric HA protein. (B) Analysis of the purified HA protein by SDS / PAGE. M: Molecular weight marker. Left: cHA fg That is, fully glycosylated cHA directly purified from HEK293T cells; (C) cHA mg That is, monoglycated cHA purified from HEK293S cells and digested with endoglucosidase H. (D) Gel filtration analysis of purified secreted HA protein. Fully glycated and monoglycated cHA from HEK293T cells were present as trimers (>200 kDa) as shown in the chromatography diagram. This figure shows the superposition dissolution profile of cHA protein expressed by HEK293T cells overlaid with calibration standards (dashed lines). (E) Labeled cHA as determined by LC-MS / MS fg and cHA mg A schematic diagram of the major polysaccharides at the glycosidic sites. This is followed by the symbols for general polysaccharides.

[0029] Figure 6 (A) and (B). Construction and purification of HA secretion. (A) The sequence encoding the extracellular domain of HA was prepared in the expression vector pcDNA and transfected into HEK293T cells. The protein was engineered to contain a stable / trimeric signal, a folding group, and a C-terminal (His)6 tag for purification. (B) Analysis of purified HA protein 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.

[0030] Figure 7(A) to (F). From cHA vaccination fg and cHA mg The HA-binding activity of mouse antiserum was assessed. cHA with Al(OH)3 or C34 adjuvants was administered at two-week intervals. fg or cHA mg BALB / c mice (n=10 mice / group) were immunized. They were then inoculated with cHA containing Al(OH)3 adjuvant. fg and cHA mg Compared to cHA with C34 adjuvant fg and cHA mg The antibody titers in mice were measured by ELISA on day 28 using the following antigens as coating antigens: 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). Endpoint antibody titer was defined as the highest dilution of serum that produced an absorbance 2.5 times higher than that produced by the negative control (pre-immunization serum). Data were examined using a two-factor ANOVA from Prism; differences were considered statistically significant. P < 0.01; P < 0.001. Data represent mean ± SEM.

[0031] Figure 8 (A) and (B). Binding of stem-reactive antibody (F10 IgG) to recombinant H1, H5, and cHA. (A) Purified F10 was analyzed by SDS / PAGE. M: Molecular weight marker. Lane 1: F10 antibody. (B) Binding affinity of F10 IgG and various HAs was measured by ELISA. The x-axis shows the concentration of various HA proteins and the y-axis shows the absorbance at OD405 nm.

[0032] Figure 9(A) to (D). Dose-dependent effect of C34 on antibody titer. BALB / c mice (n=10 / group) were injected with 20 μg cHA containing 0.5 μg, 2 μg, or 10 μg C34 adjuvant at two-week intervals. Serum was collected two weeks after the second (D28) and third (D42) immunizations. Antibody titer was measured by ELISA using HA proteins (A and C) from H1N1 A / California / 07 / 2009 and HA proteins (B and D) from H5N1 Vietnam / 1194 / 2004. P-values ​​for antibody titer were calculated using a two-factor ANOVA from Prism; differences were considered statistically significant. P < 0.05; P < 0.01. Data represent mean ± SEM.

[0033] Figure 10 (A) to (C). Dose-dependent effects of C34 on antigen-specific cytokine-secreting cells. BALB / c mice (n=5 mice / group) were injected with 20 μg of purified cHA containing three different doses of C34 adjuvant (0.5 μg, 2 μg, and 10 μg) at two-week intervals. Spleen cells from cHA-immunized mice were obtained after a second (D28) and third (D42) immunization. (A) IFN-γ and (B) IL4-secreting cells were assessed using Elispot analysis. (C) CD8+ granzyme B-producing cells in spleen cells. + T cell counts were determined using specific peptides via Elispot assays. P < 0.001. The p-value was calculated using two-factor ANOVA with Prism software.

[0034] Figure 11 (A) to (F). Inoculation with cHA after being challenged with lethal doses of H1N1 and H5N1 viruses. fg or cHA mg Body weight of mice was monitored for 14 days post-infection 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). Body weight changes are presented as mean ± SEM. Detailed Implementation

[0035] Unless otherwise indicated, the practice of this invention will employ well-known molecular biology, microbiology, recombinant DNA, and immunology techniques within the skill of the art. Such techniques are well described in the literature. See, for example, *Molecular Cloning: A Laboratory Manual*, 2nd edition, edited by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press, 1989); *DNA Cloning*, Volumes I and II (edited by DNGlover, 1985); *Culture of Animal Cells* (RI Freshney, Alan R. Liss, 1987); *Immobilized Cells and Enzymes* (IRL Press, 1986); B. Perbal, *A Practical Guide to Molecular Cloning* (1984); *The Treatise*, *Methods in Enzymology* (Academic Press, NY); *Gene Transfer Vectors For Mammalian Cells* (edited by JH Miller and MP Calos, 1987, Cold Spring Harbor Laboratory); *Methods in Enzymology*, Volumes 154 and 155 (edited by Wu et al.); *Immunochemical Methods in Cell and Molecular Biology*. (Mayer and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, in Harlow and Lanes (Cold Spring Harbor Laboratory Press, 1988); and Handbook of Experimental Immunology, Volumes I-IV (DM Weir and C.C. Blackwell, eds., 1986).

[0036] definition

[0037] Unless the context clearly indicates otherwise, the singular forms “a / an” and “the” as used in the specification and claims include multiple references. For example, the term “a chimeric transmembrane receptor” includes multiple chimeric transmembrane receptors.

[0038] As used herein, the terms "hemagglutinin" and "HA" refer to any hemagglutinin known to those skilled in the art. In some embodiments, the hemagglutinin is an influenza hemagglutinin such as influenza A hemagglutinin, influenza B hemagglutinin, or influenza C hemagglutinin. Typical hemagglutinins contain domains known to those skilled in the art, including signal peptides, stem domains, globular head domains, luminal domains, transmembrane domains, and cytoplasmic domains.

[0039] As used herein, the terms “stem domain polypeptide,” “HA stem domain,” “influenza virus hemagglutinin stem domain polypeptide,” and “HA stalk domain” refer to polypeptide chains containing one or more stem domains that constitute influenza hemagglutinin, or polypeptides composed of such stem domains. Stem domain polypeptides may be a single polypeptide chain, two polypeptide chains, or more polypeptide chains.

[0040] As used in this article, the terms “influenza virus hemagglutinin head domain polypeptide”, “influenza virus hemagglutinin head domain”, “HA globular head domain”, and “HA head domain” refer to the globular head domain of the influenza hemagglutinin polypeptide.

[0041] As used in this article, the term "antigen" is defined as any substance that can trigger an immune response.

[0042] As used in this article, the term "immunogenicity" refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.

[0043] As used in this article, the term "antigen determinant" is defined as the portion of an antigen molecule that contacts the antigen-binding site of an antibody or T-cell receptor.

[0044] As used herein, the term "vaccine" refers to a preparation containing an antigen composed of an intact pathogenic organism (killed or attenuated) or a component of such an organism, such as a protein, glycoprotein, peptide, glycopeptide, glycolipid, polysaccharide, or any combination thereof, for the purpose of priming immunity against a disease caused by these organisms. Vaccine preparations may be natural, synthetic, or obtained through recombinant DNA technology.

[0045] As used herein, the term “antigen specificity” refers to a characteristic of a cell population that causes specific cell proliferation resulting from the supply of a particular antigen or antigen fragment.

[0046] "Effective dose" refers to the amount that can effectively achieve the desired therapeutic or preventive outcome at the necessary dosage and time.

[0047] The "therapeutic effective amount" of the substance / molecule of this invention can vary depending on factors such as the individual's disease state, age, sex, weight, and the substance / molecule's ability to elicit the desired response in the individual. Therapeutic effective amount is also the amount by which the therapeutic benefit of the substance / molecule outweighs any toxic or harmful effects. "Preventive effective amount" refers to the amount at which the desired preventive or therapeutic outcome can be effectively achieved at the necessary dose and time. Generally (but not always), because preventive doses are administered to the individual before or in the early stages of disease, preventive effective amounts will be lower than therapeutic effective amounts.

[0048] The common DNA sequence of avian influenza H5 (pCHA5-II) was used as a vaccine administered to mice, and the results showed that it provided 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 invention 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 a common H5 as the globular head and a common H1 as the stem is optimal and shows strong CD4 elicitation. + and CD8 + T-cell immune responses. Of particular interest, monosaccharidated cHA (cHAmg) vaccines containing only GlcNAc at each glycosite induce more stem-specific antibodies. These stem-specific antibodies exhibit higher antibody-dependent cytotoxicity (ADCC), better neutralizing activity, and stronger cross-protective activity against H1, H3, H5, and H7 viral strains and subtypes. Furthermore, cHAmg vaccines combined with glycolipid adjuvants designed for class switching further enhance vaccine efficacy, generating more IFN-γ, IL-4, and CD8+. + Memory T cells.

[0049] Chimeric influenza virus hemagglutinin (HA) peptide

[0050] This invention provides a method for use as an immunogen or vaccine to induce CD4. + and CD8 + Chimeric influenza virus hemagglutinin (HA) peptides involved in T-cell immune responses. Therefore, chimeric influenza virus HA peptides can prevent influenza virus disease in individuals.

[0051] The chimeric influenza virus hemagglutinin (HA) polypeptide of the present invention comprises one or more stem domain sequences fused with one or more globular head domain sequences, wherein each of the one or more stem domain sequences has at least 60% homology with the stem domain common sequence of H1 subtype HA (H1 HA) and / or H5 subtype HA (H5 HA), and each of the one or more globular head domain sequences has at least 60% homology with the globular head common sequence of H1 subtype HA (H1 HA) or H5 subtype HA (H5 HA).

[0052] As used herein, the term "homology" refers to the overall relevance between aggregate molecules, such as nucleic acid molecules (e.g., DNA and / or RNA molecules) and / or polypeptide molecules, based on the similarity or concordance of critical criterions determined by the alignment of matching residues. Homology is a qualitative term describing the relationship between molecules and can be based on quantitative similarity or concordance. Similarity and concordance are quantitative terms defining the degree of sequence matching between two compared sequences. In some embodiments, aggregate molecules are considered "homological" to each other if their sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar.

[0053] In some embodiments, the polypeptide of the present invention may comprise one or more sequences having at least 60% homology with common sequences of H1 HA or H5 HA on known human and avian influenza virus strains. In some embodiments, the homology is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the stem domain sequence is the N-terminal stem region of H1 HA or the C-terminal stem region of H1 HA; the N-terminal stem region of H1 HA or the C-terminal stem region of the H1+H5 HA sequence; or the N-terminal stem region of H5 HA or the C-terminal stem region of the H1+H5 HA sequence.

[0054] In some embodiments, the stem domain common 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.

[0055] SEQ ID NO: 1 (H1 stem)

[0056]

[0057] SEQ ID NO: 2 (H1 stem)

[0058]

[0059] SEQ ID NO: 5 (H1 stem)

[0060]

[0061] SEQ ID NO: 6 (H1+H5 stem)

[0062]

[0063] SEQ ID NO: 9 (H5 stem)

[0064]

[0065] SEQ ID NO: 10 (H5+H1 stem)

[0066]

[0067] In one embodiment, the common sequence of the globular 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.

[0068] SEQ ID NO: 3 (H5 spherical head)

[0069]

[0070] SEQ ID NO: 7 (H1 bulbous head)

[0071]

[0072] SEQ ID NO: 11 (H5 spherical head)

[0073]

[0074] 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.

[0075] SEQ ID NO: 4 (Chimera H5 / 1)

[0076]

[0077] SEQ ID NO: 8 (H1 / 5 swapped)

[0078]

[0079] SEQ ID NO: 12 (Swap H5 / 1)

[0080]

[0081] In some embodiments, to enhance immunogenicity, one or more glycosites on the HA are monosaccharified. Preferably, the monosaccharified HA has only N-acetylglucosamine (GlcNAc) at each glycosite.

[0082] Chimeric influenza virus HA peptides can be produced by any suitable method, many of which are known to those skilled in the art. For example, the protein can be synthesized chemically 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 through free prokaryotic or eukaryotic expression systems, through other in vitro systems, etc.). Therefore, the present invention provides recombinant polynucleotides comprising a nucleic acid sequence encoding the peptide of the present invention and, where appropriate, a nucleic acid sequence encoding a signal peptide. The present invention provides vectors comprising the recombinant polynucleotides of the present invention. Examples of the peptides of the present invention 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 invention are also provided.

[0083] Immunogenic compositions

[0084] 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.

[0085] The chimeric influenza virus HA peptide of the present invention can be formulated or administered in combination with one or more pharmaceutically acceptable excipients. The immunogenic / vaccine composition can be sterile, pyrogen-free, or sterile and pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical preparations such as vaccine compositions can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams and Wilkins, 2005 (incorporated herein by reference in its entirety).

[0086] Immunogenic compositions are administered in a dosage form compatible with therapeutic efficacy, protection, and immunogenicity. The amount to be administered depends on the individual being treated, including, for example, the individual's immune system's ability to synthesize antibodies and, if necessary, generate a cell-mediated immune response. The precise amount of active ingredient to be administered is determined by the physician's judgment. However, those skilled in the art can easily determine the appropriate dosage range. The regimen for initial and booster doses is also variable, but may include an initial dose followed by subsequent doses. Vaccine dosage may also vary depending on the route of administration and host body size.

[0087] The formulations of the vaccine compositions described herein can be prepared by any method known in or subsequently developed in the field of pharmacological techniques. Generally, such preparation methods include the following steps: associating the active ingredient with excipients and / or one or more other adjuncts, and subsequently, if necessary and / or required, pulverizing, shaping and / or encapsulating the product into the desired single-dose or multi-dose units.

[0088] application

[0089] It has been known for some time that cytotoxic T lymphocytes (CTLs) can provide an immune response against influenza virus strains. Recent studies have shown that CTL responses in humans can target multiple antigenic determinants.

[0090] This document provides methods for preventing influenza virus disease in humans and other mammals. Methods for inducing an immune response against an influenza virus in an individual are also provided. These methods involve administering to an individual an effective amount of the chimeric influenza virus HA peptide or immunogenic composition / vaccine of the present invention, thereby inducing a specific immune response against influenza virus strains (such as H1, H3, H5, and H7 strains and subtypes). Preferably, these methods induce CD4+. + and CD8 + T-cell immune responses. More preferably, these methods induce stem-specific antibodies with higher antibody-dependent cytotoxicity (ADCC), better neutralizing activity, and stronger cross-protective activity against H1, H3, H5, and H7 viral strains and subtypes. These methods also enhance vaccine efficacy by generating more IFN-γ, IL-4, and CD8+ memory T cells.

[0091] Antibody titers in individuals increase after vaccination. In exemplary cases, the immune composition or vaccine of the present invention is used to provide protective protection against influenza. Protective protection against influenza can be achieved after administration of the vaccine or combination vaccine of the present invention. Vaccines (including combination vaccines) may be administered once, twice, three times, four times, or more, but a single administration (followed by a single booster, if applicable) may be sufficient. Therefore, dosage adjustments may be necessary.

[0092] The effective dose for prevention and treatment is a clinically acceptable dose that is effective in preventing influenza virus infection. In some embodiments, the effective dose is the dose listed in the product information leaflet of the vaccine.

[0093] The chimeric influenza virus HA peptide or immunogenic composition / vaccine of the present invention can be administered via any route that produces a therapeutically effective outcome. These routes include, but are not limited to, intradermal, intramuscular, and / or subcutaneous administration. In some embodiments, similar to the administration of inactivated vaccines known in this art, the chimeric influenza virus HA peptide or immunogenic composition / vaccine of the present invention can be administered intramuscularly or intradermally.

[0094] The application of this invention is not limited to the details of the construction and configuration of the components described below or illustrated in the figures. This invention can have other embodiments and can be practiced or implemented in various ways.

[0095] Example

[0096] method

[0097] Vaccine and plasmid construction. All 102 full-length HA sequences from the H1N1 virus available from 2009 to early 2013 were downloaded from the NCBI database and aligned using the BioEdit program via the ClustalW algorithm. Most conserved amino acids at each position were selected to form a common H1 sequence. The common hemagglutinin H5 (pCHA5-II) sequence was generated as previously described. The nucleotide sequences of common hemagglutinin H5 (pCHA5-II) and common H1 were selected and colonized into a pcDNA expression vector, and the resulting plasmid was used as a template for the exchange and chimeric HA construction. The exchange H1 / 5 was constructed from 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. The H5 / 1 crossover is constructed from 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 an H5 head and an H5+H1(HA2) stem. For the chimeric H5 / 1 construct, the head domain is composed of the amino acid sequence between residues C42 and C274 (H3 number) of SEQ ID NO: 4, and the stem region is composed of portions of the HA1 and HA2 subunits (amino acids 1-41 of SEQ ID NO: 4 and 275-511 of SEQ ID NO: 4). The transmembrane domain at the C-terminus of HA is replaced by additional residues from the phage T4 fibrin folding trimer sequence, a thrombin cleavage site, and a (His)6-tag. The two DNA sequences of the common HA are optimized for expression using human-preferred codons, and the various regions are amplified by PCR and subsequently selected into pcDNA vectors for expression. In addition, HA genes from seasonal influenza virus H1N1 Brisbane / 59 / 2007, pandemic H1N1 California / 07 / 2009, H3N2 Brisbane / 10 / 2007, H7N9A Shanghai / 2 / 2013, and avian influenza H5N1 Vietnam / 1194 / 2004 were optimized, synthesized, and selected into pcDNA expression vectors. The sequences were confirmed by DNA sequencing and prepared with high quality for protein expression and purification.

[0098] Recombinant secretory HA from the expressed cells was expressed. 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 plates (Nunc, Roskilde, Denmark) and all procedures were performed according to the manufacturer's protocol. Briefly, 293T or 293S cells at 80% confluence were transfected using Mirus TransIT. ® -LT1 (Mirus Bio) transfection reagent, using a 3:1 reagent-to-plasmid DNA ratio for transfection. TransIT... ® -LT1 reagent was diluted with Opti-MEM (Gibco) and the mixture was incubated at room temperature for 5–20 minutes. Plasmid DNA was added to the solution and mixed thoroughly, followed by incubation for 15–30 minutes. Before transfection, cells were replaced with fresh DMEM (Gibco) medium supplemented with 10% fetal bovine serum. TransIT... ® -LT1 reagent / DNA complex was added to cells and incubated at 37°C for 48 h. Hemagglutinin expression was confirmed by immunoblotting using anti-(his)6 antibody (Qiagen) or specific anti-hemagglutinin antibody and a secondary antibody binding to horseradish peroxidase (HRP) (PerkinElmer).

[0099] Purification of recombinant secreted hemagglutinin. For expression in human 293T cells, the pcDNA carrying the gene of interest was prepared with high quality and controlled by Mirus TransIT. ®-LT1 (Mirus Bio) was transfected into cells. 48 h after transfection, the culture medium was collected and the cells were clarified by centrifugation at 1,000 × g for 10 min. The supernatant was purified using a Ni-NTA (nickel-nitrotriacetic acid) affinity column (GE Healthcare). The supernatant was loaded onto a Ni-NTA affinity column pre-equilibrated with 20 mM Tris-HCl pH 8.0 and 300 mM NaCl. Unbound protein was washed away with 20 mM Tris-HCl pH 8.0 and 300 mM NaCl (buffer A) containing a 25 mM to 50 mM imidazole gradient. Subsequently, the HA protein was dissociated with buffer A containing a 100 mM to 300 mM imidazole gradient. The purified HA protein was concentrated in PBS pH 7.4 using an Amicon ultrafiltration unit (MW30K cutoff) (Millipore). Purity was monitored using SDS-PAGE, and protein was confirmed using Western Ink spot assay with anti-(his)6 antibody (Qiagen) or specific anti-hemagglutinin antibody and a secondary antibody binding to horseradish peroxidase (PerkinElmer). Finally, the trimer form of the HA protein was obtained using a particle size exclusion column and a Superdex 200 Increase 10 / 300 GL gel filtration column (GE Healthcare).

[0100] Preparation of monosaccharidated HA protein. High-mannose polysaccharide protein was generated using HEK293S cells lacking N-acetylglucosamine transferase I. 31 HA was produced by treating purified HA protein from HEK293S cells overnight with Endo H (NEB) at 20°C. mg For HA, the protein to Endo H ratio was 3:1 (w / v). Endo H and monosaccharidated HA protein were then separated using a Superdex 200 Increase 10 / 300 GL gel filtration column (GE Healthcare). HA was then concentrated in PBS at pH 7.4 using an Amicon ultrafiltration unit (MW30K cutoff) (Millipore). mg The proteins were identified and confirmed by SDS-PAGE and LC-MS / MS analysis.

[0101] N-linked glycosylation on HA proteins was identified. Ten micrograms of protein were processed on SDS-PAGE and prepared for in-gel digestion. The desired protein band was excised with a sharp scraper, cut into 1 mm pieces, and placed in 1.3 ml eppendorf tubes. After washing twice with 500 µl of 50% ACN (acetonitrile) containing 25 mM ammonium bicarbonate for 3 min each time, the gel pieces were dried using a SpeedVac evaporator (Thermo). The dried sample was regenerated by adding 100 μl of 25 mM ammonium bicarbonate 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 sample was subjected to a further alkylation step by adding 100 μl of 25 mM ammonium bicarbonate containing 100 mM iodoacetamide (IAA) (pH 8.5) and incubated in the dark at room temperature for 1 h. After washing with 500 µl of 25 mM ammonium bicarbonate (pH 8.5) containing 50% acetonitrile and 500 µl of 100% acetonitrile, the sample was centrifuged at 10,000 g for 1 min and the supernatant was completely removed. The gel sample was dried in a SpeedVac evaporator and redissolved with 200 µl of 25 mM ammonium bicarbonate (pH 8.5). Subsequently, the gel sample was treated overnight with 0.5 μg of trypsin (Promega, Madison, WI, USA) and 1 μg of chymotrypsin (Promega, Madison, WI, USA). After overnight digestion, 100 µl of 5% TFA containing 50% acetonitrile was added to the sample. The sample was sonicated for 10 seconds, followed by a 10-second pause. These steps were 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 twice. The supernatant of the mixture was dried in a SpeedVac concentrator and processed for LC-MS / MS analysis.

[0102] Endotoxin measurement. Pierce was used. ®Endotoxin concentration was determined using a LAL (Thermo Scientific) chromogenic endotoxin assay kit. Protein samples were diluted 10, 20, 100, and 1000 times, while endotoxin standards were prepared at concentrations of 10, 5, 2.5, 1.25, 0.63, 0.31, 0.15, and 0 ng / ml. After equilibrating the micro-quantitative disk in a heated assembly at 37°C for 10 min, the protein sample or standard was mixed with LAL (Lactobacillus amoeba-like cell lysate) Pyrochrome reagent (100 μl final volume) (1:1) in endotoxin-free wells at 37°C for 10 min. One hundred μl of the absorbance solution was added to each well, and the disk was incubated at 37°C for 6 min. 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 absorbance relative to the concentration of the corresponding standard. The endotoxin concentration of the samples was determined using the standard curve. The endotoxin values ​​of all purified proteins were < 0.5 ng / ml.

[0103] Mouse vaccination. Adjuvant C34 was chemically synthesized as described and dissolved in DMSO. The vaccine was administered using a mixture containing 20 μg of purified chimeric HA. fg or HA mg Female 6- to 8-week-old BALB / c mice (n=10 / group) were intramuscularly immunized with the protein in PBS pH 7.4, mixed with 50 μg aluminum hydroxide (alum; Sigma) or 2 μg C34. Control mice were injected with phosphate-buffered saline (PBS). Three vaccinations were administered at two-week intervals. Blood was collected 14 days after the second or third immunization. Serum was collected by incubating the blood at 37°C for 30 minutes and centrifuging at 12,000 rpm for 10 minutes. HA-specific antibodies in the serum collected from vaccinated mice were assessed by enzyme-linked immunosorbent assay (ELISA) and neutralization assay.

[0104] HA-specific antibodies were determined by ELISA. The titers of HA-specific antibodies were detected using HA proteins from the following strains: H1N1 A / Brisbane / 59 / 2007, H1N1A / California / 07 / 2009, H3N2 / Brisbane / 10 / 2007, H7N9 A / Shanghai / 2 / 2013, and H5N1 / Vietnam / 1194 / 2004. 100 μl of protein diluted in ELISA spread buffer (100 mM sodium bicarbonate, pH 8.8) was spread into 96-well ELISA pans (Greiner bio-one, Frickenhausen, Germany) at a concentration of 5 μg / ml / well, and the pans were sealed overnight at 4°C in a plastic container. Dishes were blocked at 37°C for 1 h with TBST containing 1% BSA (137 mM NaCl, 20 mM Tris base, 0.05% Tween 20, pH 7.4) and washed three times with TBST. The dishes were then incubated at 37°C for 2 h with a 2-fold serial dilution containing 200 μl of mouse serum. After serum transfer and six washes, HA-specific IgG was detected using 200 μl of secondary HRP-labeled anti-mouse antibody (1:8000) (PerkinElmer, Waltham, MA, USA). After 1 h of incubation at 37°C, the dishes were washed six times with TBST and developed with 100 μl of Super Aquablue ELISA substrate (eBioscience, San Diego, CA, USA) for 1 min. The reaction was stopped by adding 100 μl of 0.625 M oxalic acid. The absorbance of each well was measured at 405 nm using a SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA). Endpoint antibody titer was defined as the highest dilution of serum required to produce an absorbance 2.5 times higher than that produced by the negative control (pre-immunization serum). Background endpoint antibody titer was specified as less than 1:50.

[0105] Bone marrow-derived dendritic cells were harvested. Bone marrow-derived dendritic cells (BMDCs) cultured in GM-CSF were prepared as previously described. In short, red blood cells (RBCs) were removed by lysing the bone marrow single-cell suspension with RBCs. The remaining cells were cultured in 10 ml RPMI 1640 supplemented with 20 ng / mL mouse GM-CSF (eBioscience), 10% FBS (BenchMark), 50 μM 2-ME, 100 units / mL penicillin, and 100 μg / mL streptomycin. The cells were plated into Piper dishes to achieve a 2 × 10⁻⁶ cell culture ratio. 6The final cell density was determined by adding 10 ml of fresh medium containing 20 ng / mL mouse GM-CSF on day 3, and replenished on day 6 with half a volume of complete medium as described above. Immature BMDCs were harvested on day 8 by gently pipetting non-adherent cells, and the culture was then divided into 10 cells / Pietrochlear culture dish. 6 Cells were re-spread at a density of cells / ml. For CD8+ T cell analysis, immature BMDCs were co-cultured with CD8+ T cells and chimeric HA protein (0.1 mg / well in 100 μL) for 48 h. After washing, the number of CD8+ T cells producing granzyme B was determined by flow cytometry.

[0106] Enzyme-linked immunospot (ELISpot) analysis. ELISpot discs 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. The discs were washed four times and incubated for 30 min with RPMI-1640 supplemented with 10% fetal bovine serum (Gibco). To detect IFN-γ, IL-4, and granzyme B-secreting cells from chimeric-immunized mice, spleen cells were collected and incubated at 37°C in 5% CO2 at 5 × 10⁻⁶ ppm. 5 Cells / well were cultured for 24 h with a specific peptide for HA used for restimulation. Cells were removed and incubated with biotin-labeled anti-mouse IFN-γ, IL-4 (Mabtech AB) or granzyme B (R&D Systems) specific antibodies. The discs were washed five times, then an antibiotic streptavidin-ALP conjugate was added, and the discs were developed with ready-to-use BCIP / NPT receptors. After drying, the number of spots was analyzed using an immunospot reader (Cellular Technology Ltd.). Data were obtained from three replicate wells.

[0107] Neutralization analysis. To obtain a solution containing 100 TCID. 50 Viral culture supernatant was mixed with an equal volume of serially diluted serum (2-fold) and incubated at 37°C for 1 h. The mixture was then added to MDCK cells in each well of a 96-well plate and incubated at 37°C for 3 days. 30 μl of CellTiter-Glo (Promega) was added to the cells to determine the number of viable cells based on the quantification of ATP present. The neutralizing activity of the serum was determined to be the maximum dilution that significantly protected cells from virus-induced death.

[0108] Trace neutralization analysis. 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).

[0109] 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.

[0110] Viral challenge experiment. Two weeks after three vaccinations administered at two-week intervals, 10 LD was used. 50 (Viral doses resulting in 50% mouse mortality) Intranasal challenge with H1N1 California / 07 / 2009, H1N1 A / New Caledonia / 1999, H1N1 A / WSN / 1933, H1N1 A / Solomon Islands / 03 / 2006, and reassortant H5N1 virus A / Vietnam / 1194 / 2004 / NIBRG14 and H5N1 A / Turkey / 1 / 2005 / NIBRG23. Mice were observed daily for 14 days post-infection, and survival and body weight were recorded. The percentage of body weight for each animal in each group was calculated by comparing daily body weight to pre-challenge body weight. Mice that lost more than 25% of their initial body weight were euthanized and scored as dead. All animal experiments were performed under enhanced biosafety level 3 conditions.

[0111] Expression and purification of recombinant F10 antibody. The plasmid encoding the F10 antibody was transfected into serum-free adapted FreeStyle™ 293F cells using polyethyleneimine and cultured in 125 mL sterile Erlenmeyer flasks in FreeStyle™ 293 expression medium (Gibco) at 135 rpm on a rotary shaker platform. The supernatant was collected 72 h post-transfection, and the cells were clarified by centrifugation at 1,000 × g for 10 min. The supernatant was loaded onto a Protein-A column (GE Healthcare) pre-equilibrated with 5 column volumes (CV) of phosphate-buffered saline (PBS) wash buffer (pH 7.0), followed by 5 CV of wash buffer. The F10 antibody was dissociated with 0.2 M glycine buffer (pH 2.5), and the dissociated fraction was collected in a tube containing 0.5 mL of 1 M Tris-HCl (pH 9.0) for neutralization. Purity was monitored using SDS-PAGE.

[0112] Statistical analysis. Animal experiments used to assess the immune response were repeated at least three times (n = 5 mice / group), and viral challenge studies were performed at least twice (n = 10 mice / group). The responses of each mouse were counted as individual data points for statistical analysis. Data from animal studies were examined using a two-factor ANOVA from Prism; data are presented as mean ± SEM, and differences were considered statistically significant. P < 0.05; P < 0.01; P < 0.001.

[0113] Example 1: Preparation and characterization of monosaccharide chimeric HA.

[0114] To design a universal vaccine, we initially aimed for a vaccine with broad protection against group 1 influenza A viruses (H1 and H5 being the major subtypes, while H2, H6, and H9 are the minor subtypes). Therefore, a common H1 sequence was generated using the HA sequences of H1N1 viruses available from early 2009 to 2013. Subsequently, common H5 and common H1 were used as templates for vaccine design. During influenza virus replication, the HA precursor (HA0) was cleaved into two subunits, HA1 and HA2, via proteolytic hydrolysis; the HA1 subunit carries 5- N - Acetylneuramine gluconic acid (sialic acid) binding site, and the HA2 subunit is responsible for the fusion of the virus with the host cell membrane. Figure 5 A On the other hand, HA can be divided into two domains based on its three-dimensional (3D) structure: a globular head and a stem. The stem region contains the HA2 domain, N-terminal residues 36-50, and a short C-terminal extension of the HA1 domain. Therefore, we designed vaccines based on various combinations of domains from H1 and H5. We first generated 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 (H5 globular head and H1 stem) for comparison. Figure 1 A and Figure 5 A The results indicated that inoculation with co-H1N1 and exchange H1 / 5 did not induce cross-protective activity, but exchange H5 / 1 and chimeric H5 / 1 induced cross-neutralizing activity against both H1N1 and H5N1 viruses. Figure 1 B We will next investigate whether this cross-protection is caused by CD8. + T cell responses are contributed, and granzyme B was found to be secreted more in mice immunized with chimeric H5 / 1, indicating that the chimeric H5 / 1 vaccine induces stronger CD8+ responses compared to the exchange H5 / 1 vaccine. + T cell response ( Figure 1 C ).

[0115] Example 2: Effects of glycosylation on the immune response to chimeric H5 / 1 (cHA)

[0116] To investigate the immunogenicity of chimeric H5 / 1 (cHA) vaccines with different glycosylation states, this study compared monoglycated cHA (cHA) vaccines. mg ) and fully saccharified cHA (cHA) fg )vaccine( Figure 5Endo-H is known to be specific for high-mannose but not complex polysaccharides. It will be used in the absence of... N - Acetylglucosamine transferase I and produces a high-mannose type N - The HA glycoprotein expressed in HEK293S cells was treated with Endo-H to... N - The polysaccharide is cleaved into single GlcNAc residues. This is to generate cHA. mg cHA was produced by human cells (HEK293S), and purified cHA with high levels of mannose was removed by treatment with Endo-H. N - The outer portion of the polysaccharide is used to produce an asparagine residue with only one type of attachment to each glycoside site. N HA of acetylglucosamine (GlcNAc). After Endo-H treatment, the mixture is passed through gel filtration to separate Endo-H from trimer-cHA. mg After concentration, cHA is made mg Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS / PAGE) and liquid chromatography-mass spectrometry (LC-MS / MS) to ensure purity and glycan composition. Figure 5 C Since influenza HA exists as a trimer on the surface of the virus, gel filtration was performed to confirm cHA. fg and cHA mg It exists in the form of a trimer (>200 kDa). Figure 5 D We also generate another fully glycosylated cHA from human cells (HEK293T). fg Therefore, it can be used for comparison ( Figure 5 B Furthermore, the cell culture produced ~6 mg / L of cHA. fg .

[0117] LC-MS / MS analysis of recombinant cHA fg and cHA mg N-linked glycosylation sites and polysaccharide profile, showing seven glycosylation sites (N28, N40, N171, N182, N292, N303, and N497); cHA fg of N - Most polysaccharides are complex, and cHA mg of 99% can be found in it N - Each of the glycosylation sites is obtained by having only a single glycoform of GlcNAc. Figure 5 E (and Table 1).

[0118] Table 1. N-linked polysaccharide structures of cHA in fully glycosylated and monosaccharidated proteins analyzed by LC-MS / MS.

[0119]

[0120]

[0121] Example 3: From fully saccharified chimeric H5 / 1 (cHA) fg ) and monosaccharide chimeric H5 / 1 (cHA) mg Cross-reactivity of antiserum in immunized mice

[0122] To assess antibody binding activity induced by cHA constructs, BALB / c mice were intramuscularly immunized with 20 μg of cHAfg or cHAmg protein adjuvanted with Al(OH)3 or C34 (α-galactosylceramide (α-GalCer) analogue). Mice were immunized at weeks 0, 2, and 4, and HA-induced serum was obtained at days 28 and 42. Various recombinant HAs were measured using enzyme-linked immunosorbent assay (ELISA). Figure 6 Compared to the maximum dilution of antiserum after two immunizations, three immunizations actually produced antiserum with higher titers of HA-specific antibodies. Figure 1 D - I and Figure 7 ), and with cHA fg Compared to vaccination, cHA mg Vaccination induces a better antibody response. Figure 1 D , E and G Additionally, from cHA mg The antiserum showed slightly better binding to H3 and H7 HA proteins. Figure 1 F and H Furthermore, no significant differences were observed between Al(OH)3 and C34 adjuvant. These data indicate that cHA vaccines can induce cross-reactive antibodies that recognize HA from H1N1, H3N2, H5N1, and H7N9 viral strains.

[0123] F10 is a broadly neutralizing IgG antibody known to target the stem region of HA, and this antibody is highly conserved across various influenza virus subtypes. To compare the binding of F10 to recombinant H1, H5, and cHA, the binding affinity of F10 to various HAs was measured, and the results showed that F10 can bind to H1, H5, and cHA proteins (…). Figure 8To investigate whether F10-like antibodies are induced by cHA vaccination, the binding of cHA-induced serum to HA stem number 4900 was measured using an ELISA. Results showed that cHA... mg Vaccines can induce cHA fg Compared to higher stem-specific antibody titers ( Figure 1 I and Figure 7 F Furthermore, even better results were observed with cHA vaccines containing C34 adjuvants that induced more stem-specific antibodies. Figure 1 I ).

[0124] Example 4: Inoculation of mice with cHA mg And adjuvant C34 induces strong CD4 + and CD8 + T cell response and antibody-dependent effector function, and neutralizing activity against H1, H3, and H5 viruses and their subtypes.

[0125] 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 C Similar results were observed in the experiment.

[0126] 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 fg In comparison, cHA with C34 adjuvant mg It can stimulate more CD4 + T helper cell response and stronger CD8 + Cytotoxic effects.

[0127] To evaluate the dose-dependent effect of C34 on antibody titer and cell-mediated immunity, cHA with three different doses of C34 adjuvant (0.5 μg, 2 μg, and 10 μg) was used. fg Intramuscular immunization was induced in mice. Results indicated that after two or three immunizations, cHA with 0.5 μg and 10 μg C34 adjuvant was effective. fg In comparison, cHA with 2 μg C34 adjuvant fg Inducing higher force value ( Figure 9 Additionally, following three immunizations, cHA with 0.5 μg and 10 μg C34 adjuvants was administered. fg Compared to vaccines, cHA with 2 μg C34 adjuvant... fg Vaccines induce more IFN-γ ( Figure 10 A ), and with cHA containing 0.5 μg C34 adjuvant. fg Compared to vaccines, cHA with 2μg and 10μg C34 adjuvants... fg Vaccines induce more IL-4 ( Figure 10 BOn the other hand, when cHA fg When vaccines contain 0.5 μg, 2 μg, or 10 μg C34 adjuvant, there is no effect on CD8 levels after two or three immunizations. + Differences in the increase of GzB secretory cells ( Figure 10 C Based on these observations, 2 μg of C34 was used throughout the experiment.

[0128] Further investigation was conducted into the neutralizing activity of cHA-induced antiserum. (Source: cHA) mg The antiserum from the vaccine showed protection against the 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 The better neutralizing activity of cHA. Additionally, cHA from inoculation... mg The mouse antiserum exhibited significant neutralizing activity against heterologous viruses H1N1 A / Brisbane / 59 / 2007, A / New Caledonia / 20 / 1999, and A / Solomon Islands / 3 / 2006. Figure 3 D 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.

[0129] Example 5: Inoculating mice with cHA in an attack study mg / C34 provides cross protection against H1N1 and H5N1 and their subtypes.

[0130] 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 adjuvantmg 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 For 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 challenges were 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 better 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 Also assess weight changes following viral attack. Figure 11 Data shows that cHA effectively induces significant protective immunity against various H1N1 and H5N1 viruses, and cHA... mg Provide with cHA fg Compared to its broader cross-protection capabilities.

[0131] The development of universal influenza vaccines to provide protection against multiple strains and subtypes of influenza viruses is currently attracting attention. Antigenic determinants used in universal vaccine development include the highly conserved extracellular domain of M2 containing 24 unglycosylated amino acids, nucleoprotein NPs, and various HA constructs, which have been shown to induce broadly neutralizing antibodies with higher valences, thereby targeting the HA-stalk region or blocking viral entry. For example, a soluble trimeric HA (micro HA) vaccine with realigned stalk subunits showed complete protection in mice from lethal challenges with heterologous and heterologous subtype viruses, and chimeric HA vaccination with DNA primary injection-protein booster injection and exposure to the same stalk region, as well as diffusing foreign head domains, showed the induction of broadly protective stalk-specific antibodies. However, results showed that CD8... + T cells do not play a key role in cross-protective activity. Although DNA vaccines are promising, they are still in the early stages of development. In this study, cHA constructs expressing the common H5 of the globular head and the common H1 of the stem region were designed to simulate the actual state of influenza viruses transmitted from avian to human viruses. Fully glycosylated cHA was prepared. fg and monosaccharified cHA mg This was used for comparison, and the results showed that cHA mg Vaccine via CD4 + and CD8 + T cell response ( Figure 3 A - C This triggered a response against subtypes H1, H3, H5, and H7. Figure 1 DH The higher potency of cross-reactive antibodies.

[0132] HA glycosylation plays a crucial role in protein folding and stability, as well as in regulating its biological activity, including masking antigenic sites to prevent neutralizing antibodies from reducing immunogenicity. Furthermore, over-glycosylated HA has evolved to mask antigenic sites in highly variable head domains, thus reorienting immune responses to conserved stem regions. In our results, cHA… mg The neutralizing activity of the antiserum was significantly superior to that of cHA. fg Induced antiserum, especially against heterologous H1N1 A / Brisbane / 59 / 2007, A / Solomon Islands / 03 / 2006 and A / New Caledonia / 20 / 1999. Figure 3 D cHA mg The broader neutralizing activity of the vaccine may be attributed to its induction of more antibody variants, as previously reported. IgG is the major antibody present in mice and is the main isotype of HA-specific antibodies on immune cells that have high affinity for the FcγRIII receptor, thereby inducing ADCC. We show that with cHA... mgImmunization induces higher ADCC and more stem-specific antibodies with better protective activity. Figure 1 I And 2), this is consistent with studies showing that ADCC is necessary for in vivo influenza protection. Aluminum hydroxide (alum) is known to stimulate Th2 responses and is approved by the FDA for use as a vaccine adjuvant; however, its mechanism of action has not been adequately studied. Glycolipid C34 is a ligand for CD1d on dendritic cells and is presented thereto interacts with receptors on invariant natural killer T (iNKT) cells, thereby stimulating iNKT cells to produce adjuvant Th1 intercytokines (e.g., IFN-γ) and class-switching Th2 intercytokines (e.g., IL-4). In our results, IFN-γ (Th1 intercytokine), IL4 (Th2 intercytokine) secreted cells and CD8 cells producing granzyme B... + The number of T cells and cHA adjuvanted with Al(OH)3 mg Immunization compared to cHA with C34 adjuvant mg A significant increase in immunity ( Figure 3 AC ).

[0133] In summary, the development of next-generation influenza vaccines with broad-spectrum protective immune responses is currently a focus, and some promising results have been reported, facilitating the development of universal vaccines. In efforts toward this goal, we have successfully demonstrated in this study that monosaccharidated cHA vaccines with a common H5 head and a common H1 stem are effective influenza vaccines exhibiting broad-spectrum protective activity against heterologous influenza viruses, including H1, H3, H5, and H7 viruses and subtypes from the neutralization study, and H1N1, H5N1, and subtypes from the challenge study. Having successfully developed broad-spectrum protective vaccines against different strains and subtypes of influenza A virus, we aim to use the strategies developed in this study to design broader universal vaccines against both influenza A and influenza B viruses.

Claims

1. 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 Hl subtype HA (Hl 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 consensus sequence of an Hl subtype HA (Hl HA) or an H5 subtype HA (H5 HA).

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

3. The chimeric influenza virus hemagglutinin polypeptide of claim 1, wherein the homology is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

4. The chimeric influenza virus hemagglutinin polypeptide of claim 1, wherein the stem domain sequence is an N-terminal stem segment of an Hl HA or a C-terminal stem segment of an Hl HA; an N-terminal stem segment of an Hl HA or a C-terminal stem segment of an Hl+H5 HA sequence; or an N-terminal stem segment of an H5 HA or a C-terminal stem segment of an Hl+H5 HA sequence.

5. The chimeric influenza virus hemagglutinin polypeptide of claim 1, wherein the stem domain consensus sequence of an Hl 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.

6. The chimeric influenza virus hemagglutinin polypeptide of claim 1, wherein the globular head consensus sequence of an H5 HA comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO:

11.

7. The chimeric influenza virus hemagglutinin polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO:

12.

8. The chimeric influenza virus hemagglutinin polypeptide of claim 1, wherein one or more glycosylation sites on the HA are monoglycosylated, wherein the monoglycosylated HA has only N-acetylglucosamine (GlcNAc) on each glycosylation site.

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

10. An immunogenic composition comprising the chimeric influenza virus HA polypeptide of any one of claims 1-9 and an adjuvant.

11. The immunogenic composition of claim 10, wherein the adjuvant is a glycolipid adjuvant.

12. Use of the chimeric influenza virus hemagglutinin polypeptide of any one of claims 1-9 or the immunogenic composition of claim 10 in the manufacture of a medicament for immunizing an individual against an influenza virus or preventing an influenza virus disease.

13. The use of claim 12, wherein the chimeric influenza virus hemagglutinin polypeptide or immunogenic composition elicits a CD4 + and CD8 + T cell immune response.

14. The use of claim 12, wherein the chimeric influenza virus hemagglutinin polypeptide or immunogenic composition induces 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.

15. The use of claim 12, wherein the chimeric influenza virus hemagglutinin polypeptide or immunogenic composition increases vaccine efficacy, produces more IFN-γ, IL-4, and CD8 + memory T cells.

16. A recombinant polynucleotide comprising a nucleic acid sequence encoding the chimeric influenza virus hemagglutinin polypeptide of any one of claims 1 to 9 and optionally a nucleic acid sequence encoding a signal peptide.

17. A vector comprising the recombinant polynucleotide of claim 16.

18. A host cell comprising the vector of claim 17.

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