Broad-spectrum reactive immunogen for influenza H3 virus, its composition, and method of use.

Abstract

The isolated, non-natural, broadly reactive antigens derived from the H3(H3N2) influenza virus provided herein are immunogenic and, after introduction into a subject, can induce a broadly reactive immune response against the H3(H3N2) virus, such as a broadly reactive neutralizing antibody response. Non-natural, broadly reactive immunogens, vaccines, virus-like particles (VLPs), and compositions comprising immunogens and vaccines are also provided. Methods are provided for inducing an immune response in a subject by administering isolated immunogens, vaccines, VLPs, or compositions thereof. In particular, the immunogens are recombinant and contain the hemagglutinin (HA) protein of the H3(H3N2) influenza virus lineage.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority and the benefit of U.S. Provisional Application No. 63 / 488,849, filed on March 7, 2023, the content of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application is electronically filed in XML format and includes a sequence listing, the entire content of which is hereby incorporated by reference. The XML file of the sequence listing created on March 6, 2024, is named 173093 - 011701_PCT_SL.xml and has a size of 45,335 bytes.

Background Art

[0003] Background The U.S. Centers for Disease Control and Prevention determined that the 2017 seasonal influenza vaccine was only 42% effective. This limited effectiveness was due to mutations that occurred in the influenza A (H3N2) vaccine strains that cause influenza in infected individuals. Additionally, the number of influenza cases caused by the influenza B virus has been increasing from the 2017 - 2018 period to the present. Considering that in a severe influenza season, there may be on the order of 50,000 deaths in the United States alone, there is an urgent need for improved immunogens and vaccines that can provide broad protection against the virus, particularly the influenza A H3 or H3N2 virus, in current and future circulations. In particular, considering the past and current global spread of severe influenza infections and related diseases, new immunogens and vaccine products that can more effectively protect individuals from severe or critical influenza conditions and related symptoms during any given influenza season are recognized.

Summary of the Invention

[0004] Summary As described below, non-natural, broadly reactive antigens and antigenic sequences derived from influenza H3 virus (also referred to herein as "H3 influenza," "H3 influenza virus," or "H3 virus"), for example, subtype H3N2, are provided. These H3 virus antigens are typically structural proteins or peptides, for example, comprising hemagglutinin (HA) protein or the HA1 (head) or HA2 (tail or stalk) portion of the HA protein, and are potent immunogens that, in a subject after administration, induce a broadly reactive immune response to the H3 HA protein, and ultimately to current and future H3 virus lineages. As referred herein, an H3 (H3N2) virus antigen or antigenic sequence that induces an immune response in a subject is an immunogenic antigen or immunogen. These H3 immunogens are called broadly reactive and panepitopetic because they can induce the production of antibodies against various subtypes or lineages of H3 viruses that have both sequence similarity and variability and epitope (antigenic determinant) diversity in the antigen and its sequence, particularly in the HA antigen. In one embodiment, the HA antigen recognized by the H3 viral immunogens described herein is included in past and / or historical influenza virus H3 vaccines. As will be understood by those skilled in the art, "H3N2" is a subtype of influenza A virus (IAV) that often causes serious illness. In embodiments, the terms "H3" and "H3N2" are interchangeable herein.

[0005] In one aspect, the amino acid sequences of non-natural H3 virus antigens and antigens containing such sequences (e.g., structural antigens) described herein contain broadly reactive epitopes that reflect the sequence similarities and variability of past, present, and future H3 antigens. Such antigen sequences and antigens containing such sequences are therefore referred to as “non-naturally broadly reactive pan-epitope” antigens. The antigens are immunogenic and, when introduced into or administered to a subject, induce antibodies in the subject, for example, neutralizing antibodies against the H3 virus HA antigen or its antibody-binding moiety. In one aspect, such an H3 HA antigen sequence is an amino acid sequence. In another aspect, the H3 HA antigen sequence is a polynucleotide sequence, for example, a polynucleotide sequence encoding the amino acid sequence of the antigen described herein. For convenience of reference, the “non-naturally broadly reactive pan-epitope” H3 virus antigens described herein are referred to as “broadly reactive antigens.”

[0006] The broadly reactive H3 antigens described herein are immunogens because they induce a broadly reactive immune response in a subject. The immune response is particularly in the form of a neutralizing antibody response, for example, a neutralizing antibody that is specifically directed against the HA antigen of the H3 virus and neutralizes the activity of the HA protein. Accordingly, immunogens and immunogenic compositions, such as vaccines (e.g., polypeptide or polynucleotide products), containing the broadly reactive H3 HA antigens described herein, which induce an immune response against the H3 virus in a subject, for example, directed against the HA protein of the H3 virus, are also provided. For convenience of reference, “non-naturally occurring broadly reactive panepitope” H3 virus immunogens described herein are referred to as “broadly reactive immunogens.”

[0007] Methods are also provided for using immunogens described herein to induce an immune response to H3 influenza infection, disease, and / or symptoms thereof in a subject. In certain embodiments, the H3 viral antigen is the HA, HA1, or HA2 protein of the H3 influenza virus or an H3N2 subtype of the influenza virus or a viral type associated therewith, or their antibody-binding moiety. Methods are also provided for using immunogens to induce an immune response in a subject.

[0008] In one aspect, the H3 HA immunogenic antigen has an amino acid sequence that is identical to at least 85% or 85%, at least 90% or 90%, at least 91% or 91%, at least 92% or 92%, at least 93% or 93%, at least 94% or 94%, at least 95% or 95%, at least 96% or 96%, at least 97% or 97%, at least 98% or 98%, or at least 99% or 99% of the sequence of one or more H3 HA polypeptides (or HA1 or HA2 polypeptides) of the influenza H3 HA proteins described herein as NG-4, NG-5, NG-6, NG-7, and NG-8.

[0009] In one aspect, broadly reactive H3 antigen sequences capable of eliciting an immune response against current and future H3 influenza virus strains are generated by methods such as those described in U.S. Patent Application No. 2021-0225457 A1, published on July 22, 2021, the contents of which are incorporated herein by reference. Methods that may be referred to as Next Generation of Computationally Optimized Broadly Reactive ("COBRA") HA vaccine methods include considering parameters of the H3 antigen sequence, e.g., HA antigen sequence, from a length or range of time (e.g., a linear time range), e.g., one or more influenza seasons, and the geographical location from which the H3 virus was isolated, e.g., the Southern Hemisphere or the Northern Hemisphere.

[0010] In another aspect, isolated, non-natural, broadly reactive, panepitope antigens of H3 influenza virus (H3 virus) are provided that can induce an immune response against current and future H3 virus lineages. In one embodiment, the antigen is hemagglutinin (HA), HA1 or HA2, or its antibody-binding moiety. In one embodiment, the H3 virus antigen comprises an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 98%, 99%, or more identical to one or more amino acid sequences of the sNG-4(SEQ ID NO:1), sNG-5(SEQ ID NO:2), NG-6(SEQ ID NO:3), NG-7(SEQ ID NO:4), and NG-8(SEQ ID NO:5), or their soluble forms, in other words, sNG-4(SEQ ID NO:11), sNG-5(SEQ ID NO:12), sNG-6(SEQ ID NO:13), sNG-7(SEQ ID NO:14), and sNG-8(SEQ ID NO:15) H3 (H3N2) influenza virus HA antigens. In one embodiment, the isolated non-natural broad-reactive H3 influenza virus antigen is recombinant or produced by recombinant.

[0011] The non-natural, broadly reactive, panepitope immunogens provided herein may be referred to interchangeably as “non-natural immunogens,” “broadly reactive immunogens,” or “panepitope immunogens” for brevity. In one embodiment, the immunogen is isolated. In another embodiment, the immunogen is recombinant or is produced by recombinant processes.

[0012] In one aspect, an isolated non-natural hemagglutinin (HA) polypeptide antigen or antibody-binding moiety of H3 influenza virus (H3 virus) is provided, comprising an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity with (i) an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or (ii) an amino acid sequence selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15). In one embodiment, an isolated non-natural hemagglutinin (HA) polypeptide antigen of H3 influenza virus (H3 virus) includes an amino acid sequence or antibody-binding moiety having at least 98% sequence identity with (i) an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or (ii) an amino acid sequence selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15). In one embodiment, an isolated non-natural hemagglutinin (HA) polypeptide antigen of H3 influenza virus (H3 virus) comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or (ii) an amino acid sequence selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15), or an antibody-binding moiety thereof.In one embodiment, an isolated non-natural hemagglutinin (HA) polypeptide antigen of H3 influenza virus (H3 virus) essentially consists of or comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or (ii) an amino acid sequence selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15), or their antibody-binding moieties. In one embodiment, the H3 HA polypeptide antigen is a recombinant protein or is produced by recombinant means.

[0013] In another aspect, virus-like particles (VLPs) containing an H3 virus polypeptide antigen isolated according to the above aspects and / or embodiments thereof are provided. In one aspect, the VLP comprises a polynucleotide encoding the H3 virus HA polypeptide antigen. In another aspect, the polynucleotide is RNA, e.g., mRNA, or DNA.

[0014] In another aspect, an immunogen is provided comprising an isolated, non-natural hemagglutinin (HA) polypeptide antigen of an H3 influenza virus (H3 virus) of any of the above aspects and / or embodiments thereof, the immunogen capable of eliciting an immune response against current and future H3 influenza (H3) virus lineages. In this aspect, the H3 HA polypeptide antigen is a recombinant protein or is synthesized by recombinant means.

[0015] In another aspect, a virus-like particle (VLP) containing the H3 virus HA polypeptide antigen in any of the above aspects and / or embodiments thereof is provided. In one aspect, the VLP comprises a polynucleotide encoding the H3 virus HA polypeptide antigen. In another aspect, the polynucleotide is RNA, e.g., mRNA, or DNA.

[0016] In another context, an immunogen is provided comprising virus-like particles (VLPs) in the manner described above and / or those embodiments thereof, the VLPs capable of eliciting an immune response against current and future H3 influenza (H3) virus strains.

[0017] In one embodiment, the immune response induced by the above immunogen includes the production of an antibody having hemagglutinin inhibitory activity. In one embodiment, the immune response further includes the production of T lymphocytes.

[0018] In one aspect, a pharmaceutical composition is provided comprising an isolated non-natural hemagglutinin (HA) polypeptide antigen from any one of the above aspects and / or embodiments thereof, as well as a pharmaceutically acceptable carrier, diluent, or excipient. In this aspect, the H3 HA polypeptide antigen is a recombinant protein or is produced by recombinant means.

[0019] In one aspect, a pharmaceutical composition is provided comprising virus-like particles (VLPs) in the above aspects and / or embodiments thereof, as well as a pharmaceutically acceptable carrier, diluent, or excipient.

[0020] In one aspect, a pharmaceutical composition is provided comprising an immunogen in any one of the above aspects and / or embodiments thereof, as well as a pharmaceutically acceptable carrier, diluent, or excipient.

[0021] In one embodiment, the above-mentioned pharmaceutical composition further comprises an adjuvant. In this embodiment, the adjuvant comprises a squalene oil-in-water emulsion adjuvant or a cationic lipid nanoparticle adjuvant.

[0022] In another aspect, an immunogenic composition or vaccine is provided comprising an H3 virus antigen or virus-like particle (VLP) or immunogen in any of the above aspects and / or embodiments thereof.

[0023] In another aspect, a pharmaceutically acceptable composition is provided comprising an immunogenic composition or vaccine in any of the above aspects and / or embodiments thereof, as well as a pharmaceutically acceptable carrier, diluent, or excipient. In one aspect, the pharmaceutically acceptable composition further comprises an adjuvant. In one aspect, the pharmaceutically acceptable composition comprises a squalene oil-in-water emulsion adjuvant or a cationic lipid nanoparticle adjuvant. In one aspect, the adjuvant is ADDAVAX® or R-DOTAP.

[0024] In another aspect, a method is provided for inducing an immune response in a subject, the method comprising administering an effective amount of an isolated H3 virus polypeptide antigen, virus-like particle (VLP), immunogen, pharmaceutically or pharmaceutically acceptable composition, or immunogenic composition to the subject, as described in the above aspects and / or any of those embodiments. In one aspect of the method, the immune response produced or induced in the subject includes the production of an antibody active against the hemagglutinin protein of a historical influenza vaccine, e.g., the H3N2 HA protein, in a hemagglutinin inhibition assay. In one aspect of the method, an immune response to current and future H3 influenza (H3) virus lineages is produced in the subject. In one aspect of the method, the immune response produced or induced in the subject includes the production of an antibody having hemagglutinin (HA) inhibitory activity. In one aspect of the method, the immune response in the subject is produced against a disease or condition and / or symptoms thereof resulting from infection with the H3 influenza virus or its subtypes. In one aspect of the method, an adjuvant is administered concurrently to the subject. In one embodiment of the method, a squalene oil-in-water emulsion adjuvant or a cationic lipid nanoparticle adjuvant is administered to the subject. In one embodiment, the adjuvant is ADDAVAX®. In one embodiment, the adjuvant is R-DOTAP. In one embodiment, the adjuvant is formulated together with an H3 HA polypeptide, H3 HA polypeptide antigen, VLP, immunogen, immunogenic composition, vaccine, or pharmaceutical composition as described herein. In one embodiment of the method, the immune response induced in the subject includes the production of neutralizing antibodies and / or T-lymphocytes. In one embodiment, the H3 HA polypeptide antigen is a recombinant protein or is synthesized recombinantly.

[0025] In one aspect, the H3 HA polypeptide antigen (or immunogen) described herein is isolated and / or purified. In another aspect, the H3 HA polypeptide antigen (or immunogen) described herein is formulated, for example in a pharmaceutical composition, for administration to a subject as needed. In one embodiment, the method provides prophylactic or therapeutic treatment of a disease or condition induced by H3 influenza virus infection.

[0026] In another aspect, a polynucleotide encoding the H3 virus HA polypeptide antigen of any of the above aspects and / or their embodiments is provided. In one embodiment, the polynucleotide comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with a polynucleotide sequence selected from the group consisting of SEQ ID NO:6 - 10 or SEQ ID NO:16 - 20. In one embodiment, the polynucleotide comprises, consists essentially of, or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO:6 - 10 or SEQ ID NO:16 - 20. In an embodiment, the polynucleotide is RNA, such as mRNA, or DNA.

[0027] In another aspect, an immunogenic composition or vaccine comprising a polynucleotide encoding the H3 virus HA polypeptide antigen of any of the above aspects and / or their embodiments is provided. In one embodiment, the polynucleotide is RNA, mRNA, or DNA. In one embodiment, the polynucleotide is mRNA. In one embodiment, the H3 HA polypeptide antigen is a recombinant protein and / or is recombinantly produced.

[0028] In another aspect, a pharmaceutical composition comprising the immunogenic composition or vaccine of any of the above aspects and / or their embodiments is provided.

[0029] In another aspect, a method is provided for inducing an immune response in a subject to the H3N2 influenza virus and / or its hemagglutinin (HA) protein antigen, the method comprising the step of administering an effective amount of a pharmaceutically acceptable composition of the above aspects and / or embodiments thereof to the subject. In one aspect of the method, the immune response in the subject is induced in response to a disease or condition and / or symptoms thereof resulting from infection with the H3 influenza virus or its subtype. In one aspect of the method, the immune response includes the production of antibodies active against current and future H3 influenza (H3) virus lineages and / or their HA polypeptides. In one aspect of the method, the immune response includes the production of antibodies having hemagglutinin (HA) inhibitory activity. In one aspect of the method, an adjuvant is administered concurrently to the subject. In one aspect of the method, the adjuvant is formulated together with the pharmaceutical composition. In one aspect, the adjuvant includes a squalene oil-in-water emulsion adjuvant or a cationic lipid nanoparticle adjuvant. In one aspect, the adjuvant is ADDAVAX®. In one embodiment, the adjuvant is R-DOTAP.

[0030] In one aspect, a composition is provided comprising a polynucleotide in the above aspects and / or embodiments thereof, as well as a pharmaceutically acceptable carrier, diluent, or excipient.

[0031] In another aspect, a composition comprising a combination or mixture of two of isolated non-natural hemagglutinin (HA) polypeptide antigens of any one of the above aspects and / or embodiments thereof. In one aspect of the composition, the isolated non-natural H3 influenza virus HA polypeptide antigens comprise NG-7 with SEQ ID NO:4 or SEQ ID NO:14 and NG-8 with SEQ ID NO:5 or SEQ ID NO:15. In one aspect of the composition, the isolated non-natural H3 influenza virus HA polypeptide antigens are recombinant and / or produced by recombinant. In one aspect, the composition further comprises a pharmaceutically or physiologically acceptable carrier, diluent, or excipient. In one aspect, the composition further comprises an adjuvant, which may be a squalene oil-in-water emulsion adjuvant or a cationic lipid nanoparticle adjuvant.

[0032] In another aspect, a method is provided for inducing an immune response in a subject to the hemagglutinin (HA) protein of the H3N2 influenza virus, the method comprising administering to the subject an effective amount of the composition comprising a combination or mixture of two isolated non-natural hemagglutinin (HA) polypeptide antigens of H3 influenza virus (H3 virus) of any of the above aspects and / or embodiments thereof. In aspects of the method, the resulting immune response includes (i) the production of antibodies active against current or future H3 influenza (H3) virus lineages; (ii) the production of antibodies having hemagglutinin inhibitory activity; (iii) the production of one or both virus-neutralizing antibodies and T-lymphocytes; and / or (iv) the production of antibodies active against the hemagglutinin (HA) protein of historical influenza vaccines in a hemagglutinin inhibitory assay.

[0033] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art of the field to which this disclosure belongs or relates. The following references provide general definitions of many terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Robert A. Meyers (ed.), published by VCH Publishers, Inc., 1995. (ISBN 1-56081-569-8); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings given below unless otherwise specified.

[0034] The terms “H3 virus HA antigen (or immunogen),” “H3 virus HA polypeptide (or protein) antigen (or immunogen),” “H3 HA antigen (or immunogen),” “H3 HA polypeptide (or protein) antigen (or immunogen),” “H3 HA polypeptide or protein,” and “H3 virus HA polypeptide or protein” are used interchangeably herein. In one embodiment, the H3 virus constitutes the H3N2 virus subtype. In another embodiment, the H3 HA polypeptide antigen is a recombinant protein or is synthesized by recombinant means.

[0035] An "adjuvant" refers to a substance or vehicle that nonspecifically enhances the immune response to an antigen. Examples of adjuvants include suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphate) on which the antigen is adsorbed; or, in some cases, water-in-oil emulsions in which an antigen solution is emulsified in mineral oil (e.g., an incomplete Freund's adjuvant), which may contain dead mycobacteria to further enhance antigenicity (Freund's complete adjuvant). Immunostimulating oligonucleotides (such as those containing CpG motifs) can also be used as adjuvants (see, for example, U.S. Patents 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). In some embodiments, adjuvants include squalene oil-in-water emulsions (e.g., ADDAVAX®; InvivoGen, San Diego, CA); surfactants, e.g., Quil A; plant saponins; or cationic lipid nanoparticles (e.g., R-DOTAP; PDS Biotechnology Corporation, Florham Park, NJ). In particular, ADDAVAX® is a squalene-based oil-in-water nanoemulsion used in adjuvant-added influenza vaccines, without limitation (Ott G. et al., 2000, Methods in Molecular Medicine, Vol 42, 211-228; Calabro, S. et al., 2013, Vaccine, 31:3363-9; Ott G. et al., 1995, Pharm Biotechnol, 6: 277-96). Squalene is an oil that is more readily metabolized than paraffin oil used in Freund's adjuvants. Squalene oil-in-water emulsions, such as ADDAVAX®, or another squalene oil-in-water adjuvant, MF59®, conveniently induce both cellular (Th1) and humoral (Th2) immune responses.In one embodiment, the adjuvant is a cationic lipid, such as 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP), which can induce memory T cell responses and pluripotent antigen-specific T cells, particularly in response to recombinant proteins (Gandhapudi, SK et al., 2023, Viruses, 15(2):432). Other adjuvants include biological molecules, such as costimulatory molecules. Exemplary biological adjuvants include, but are not limited to, interleukin-1 (IL-2), the protein memory T cell attractant "Regulated on Activation, Normal T Expressed and Secreted" (RANTES), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), granulocyte colony-stimulating factor (G-CSF), lymphocyte function-associated antigen 3 (LFA-3, also known as CD58), differentiation antigen cluster 72 (CD72) (a negative regulator of B cell responsiveness), superficial membrane protein B7-1 (B7-1, also known as CD80), superficial membrane protein B7-2 (B7-2, also known as CD86), TNF ligand superfamily member 4 ligand (OX40L), or type 2 transmembrane glycoprotein receptor (4-1BBL) belonging to the TNF superfamily.

[0036] "Administer" means to give, supply, distribute, or apply or bring into contact with a subject a composition, agent, therapeutic agent, etc. Administration or administration can be achieved by any of several routes, for example, not limited to, local, oral, subcutaneous, intramuscular, intraperitoneal, intravenous (IV) (injection or immunization), intrathecal, intramuscular, percutaneous, intradermal, intracranial, inhalation, rectal, vaginal, or intraocular.

[0037] "Agent" means any small molecule chemical compound, antibody, nucleic acid molecule, peptide, polypeptide, or fragment thereof.

[0038] "Alteration" means a change (increase or decrease) in the expression level or activity of a gene or polypeptide, as detected by standard methods known in the art, such as those described herein. As used herein, alteration includes changes of 5%, 10%, preferably 25%, more preferably 40%, and most preferably 50% or more in expression level.

[0039] "To improve" means to reduce, lessen, decrease, suppress, weaken, prevent, or stabilize the onset or progression of a disease or pathological condition.

[0040] An "analog" refers to a molecule that is not identical but possesses similar functional or structural characteristics. For example, a polypeptide analog may have certain biochemical modifications that enhance the functionality of the analog compared to the native polypeptide, while retaining the biological activity of the corresponding native polypeptide. Such biochemical modifications can, for example, increase the protease resistance, membrane permeability, or half-life of the analog without altering ligand binding. Analogs may contain non-native amino acids.

[0041] An "antibody" refers to an immunoglobulin (Ig) molecule produced by B lymphocyte cells that has a specific amino acid sequence. Antibodies are induced or triggered in a subject (human or other animal or mammal) after exposure to a specific antigen (immunogen). A subject capable of producing antibodies / immunoglobulins (i.e., an immune response) directed to a specific antigen / immunogen is said to be immunocompetent. Antibodies are characterized by their specific reaction with an antigen or immunogen in some demonstrable way (e.g., binding to the antigen or immunogen), and antibodies and antigens / immunogens are defined in relation to each other.

[0042] "To induce an antibody response" refers to the ability of an antigen, immunogen, or other molecule to induce the production of antibodies. Antibodies are of different classes, e.g., IgM, IgG, IgA, IgE, IgD, and subtypes or subclasses, e.g., IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4. An antibody / immunoglobulin response induced in a subject can neutralize pathogenic (e.g., infectious or disease-causing) activators by binding to epitopes (antigenic determinants) on the activator and blocking or inhibiting the activator's activity, and / or by forming a binding complex with the activator, which is then removed from the target system, for example, via the liver.

[0043] As used herein, “broad reactivity” means that in a subject, an immune response is induced against a viral protein (e.g., a viral antigen, antigenic sequence, protein, or protein sequence) that is sufficient to block, inhibit, interfere with, neutralize, or prevent infection by a wide range of related influenza viruses (e.g., most or all influenza viruses within a particular subtype, e.g., viruses associated with H3 influenza virus).

[0044] "Antigen" means a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal, including compositions injected or absorbed into the animal. Antigens react with products of specific humoral or cellular immunity, including those induced by heterologous immunogens. In some embodiments of the compositions and methods disclosed, the antigen is the influenza hemagglutinin (HA) protein. Often, an antigen that elicits or stimulates an immune response in a subject is called an "immunogen."

[0045] The term "antigen drift" refers to a mechanism of variation in organisms or microorganisms, such as viruses, that involves the accumulation of mutations within the genes encoding antibody-binding sites (also called antigenic determinants or epitopes). This process results in a new lineage of virus / viral particles that is not effectively inhibited or blocked by antibodies initially produced against the antigen of the pre-mutation viral lineage, thus allowing the virus to spread more easily across a partially immune population. Antigen drift occurs in both influenza A and influenza B viruses, for example.

[0046] In the context of living viruses, the term “attenuated” refers to a virus that is attenuated when its ability to infect cells or targets and / or cause disease is reduced (e.g., diminished, suppressed, or eliminated) compared to the ability of the wild-type virus to cause disease in a target. Typically, an attenuated virus retains at least some ability to elicit an immune response after administration to an immunocompetent target. In some cases, an attenuated virus can elicit a protective immune response without causing any signs or symptoms of infection. In some embodiments, the ability of the attenuated virus to cause disease or a condition in a subject is reduced by at least about 5% or 5%, or at least about 10% or 10%, or at least about 25% or 25%, at least about 50% or 50%, at least about 75% or 75%, or at least about 80% or 80%, or at least about 85% or 85%, or at least about 90% or 90%, or at least about 95% or 95%, or more, compared to the ability of the wild-type virus to cause disease or a condition in a subject.

[0047] The term "clade" refers to different categorizations (often called subtypes) of known influenza viruses, such as the influenza A H3N2 virus. Viruses within the H3N2 clade are genetically related but do not share the exact same viral genome. As will be recognized by those skilled in the art, there are many clades and subclades of H3N2 virus subtypes designated in the art. For example, one clade is 3C.2a; its subclades include 3C.2a.1, 3C.2a.2, 3C.2a.3, and 3C.2a.4. Furthermore, there are at least 10 different clades of H5N1 virus subtypes designated in the art: clade 0, clade 1, clade 2, clade 3, clade 4, clade 5, clade 6, clade 7, clade 8, and clade 9 (Abdel-Ghafar et al., N Engl J Med 358:261-273, 2008). Clade 2 is further divided into subclades (including clades 2.1, 2.2, 2.3, 2.4, and 2.5).

[0048] "Codon-optimized" nucleic acids (polynucleotides) refer to nucleic acid sequences that have been modified so that the codons are optimal for expression in a particular system (e.g., a specific species within a group of species). For example, nucleic acid sequences can be optimized for expression in mammalian cells. Codon optimization does not alter the amino acid sequence of the encoded protein.

[0049] In this disclosure, “comprises,” “comprising,” “containing,” and “having,” etc., may have the meanings given to them in U.S. patent law, and may mean “includes,” “including,” etc.; “essentially from” or “essentially from” also has the meanings given to them in U.S. patent law, and the terms are open-ended, allowing for more existences than those enumerated, as long as the basic or novel features of those enumerated are not altered by more existences than those enumerated, but excluding aspects of the prior art.

[0050] "Detecting" refers to identifying the presence, absence, or quantity of an analyte, compound, active agent, or substance to be detected. "Detectable label" means a composition that, when linked to a molecule of interest, makes the molecule of interest detectable, for example, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Non-limiting examples of useful detectable labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, high electron-density reagents, enzymes (such as those commonly used in ELISA), biotin, digoxigenin, or haptens.

[0051] "Disease" means any condition, disorder, or pathology that impairs or interferes with the normal functioning of a cell, tissue, or organ. Examples of diseases include those caused by H3 virus infection, and the symptoms and adverse effects caused by infection of the body with an H3 virus. The influenza virus causes influenza and its symptoms in an infected individual.

[0052] "Effective dose" refers to the amount of active therapeutic agent, composition, compound, or biologic (e.g., vaccine or therapeutic peptide, polypeptide, or polynucleotide) required to improve, reduce, improve, suppress, decrease, or eliminate the symptoms and / or effects of a disease, condition, or pathology compared to an untreated patient. The effective dose of an immunogen or composition containing an immunogen used to carry out a method of therapeutic treatment for a disease, condition, or pathology caused by an H3 virus will vary depending on the mode of administration, the age, weight, and overall health of the subject. Ultimately, it is considered that the attending physician or veterinarian will determine the appropriate dose and dosage regimen. Such a dose is referred to as the "effective" dose.

[0053] The “therapeutic effective dose” refers to the amount of a particular agent that is sufficient to achieve the desired effect in a subject being treated with that agent. For example, this could be the amount of H3 influenza virus immunogen or vaccine that is useful in inducing an immune response in a subject and / or preventing infection by the H3 influenza virus. Ideally, in the context of this disclosure, the therapeutic effective dose of an influenza vaccine or anti-influenza immunogenic composition is an amount sufficient to enhance resistance to infection caused by the influenza virus in a subject, prevent, improve, reduce, and / or treat such infection without causing substantial cytotoxic effects in the subject. The effective dose of an influenza vaccine of an immunogenic composition useful in enhancing resistance to infection, preventing, improving, reducing, and / or treating infection in a subject depends, as stated above, for example, on the subject being treated, the mode of administration of the therapeutic composition, and other factors.

[0054] A “fragment” refers to a portion of a polypeptide or nucleic acid molecule. This portion preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. A polypeptide portion or fragment may be a peptide. In the case of an antibody or immunoglobulin fragment, the fragment typically binds to a target antigen.

[0055] A "fusion protein" refers to a protein produced by the expression of a nucleic acid (polynucleotide) sequence manipulated from nucleic acid sequences encoding at least parts of two different (heterogeneous) proteins or peptides. To create a fusion protein, the nucleic acid sequences must be in the same reading frame and must not contain internal stop codons. For example, a fusion protein might include an H3 influenza HA protein fused to a heterogeneous protein.

[0056] "Genetic vaccine" means an immunogenic composition containing a polynucleotide that codes for an antigen. In some embodiments, the polynucleotide is RNA, such as mRNA, or DNA.

[0057] The term “geographic location or geographical area” refers to a pre-selected division of the Earth’s geographical region, such as a continent or other pre-selected territory or division (e.g., the Middle East, which spans more than one continent). Examples of different geographical areas include countries (e.g., Turkey, Egypt, Iraq, Azerbaijan, China, the United States); continents (e.g., Asia, Europe, North America, South America, Oceania, Africa); recognized geopolitical divisions (e.g., the Middle East); or hemispheres of the world (e.g., the North, South, East, or Western Hemispheres).

[0058] "H3 virus polypeptide" means an amino acid sequence that is at least 85% identical to the amino acid sequence of the HA antigen described herein, or a fragment thereof that can induce an immune response in an immunized subject. In one embodiment, the H3 virus polypeptide includes or comprises the NG-4, NG-5, NG-6, NG-7, or NG-8 HA amino acid sequences described herein (i.e., SEQ ID NO: 1-5), or their antibody-binding portions or fragments.

[0059] "H3 viral polynucleotide" refers to a nucleic acid molecule that codes for an H3 viral polypeptide (antigen or protein antigen).

[0060] The term “hemagglutinin (HA)” refers to a surface glycoprotein expressed by the influenza virus. HA mediates the binding of viral particles to host cells and subsequent transfer of the virus to host cells. Nucleotide and amino acid sequences of numerous influenza HA proteins are publicly known in the art and are publicly available, including those deposited in GenBank (see, for example, U.S. Patent Application Publication US 2015 / 0030628, Table 1). HA (together with neuraminidase (NA)) is one of two main influenza virus protein antigens that have an antigenic determinant (epitope) that is recognized and bound by antibodies / immunoglobulins. In some embodiments, HA antigens of the H3 influenza virus are provided herein as immunogens that induce a broad-spectrum reactive immune response in a subject after administration to that subject. In some embodiments, the HA protein antigen or its functional fragment may have at least about 85% or 85% amino acid sequence identity with the amino acid sequence of a representative influenza A virus HA protein or its functional fragment, for example, the influenza virus HA proteins (NG-4 to NG-8) described herein, or at least about 90%, 95%, 98%, 99%, or 90%, 95%, 98%, 99%, or higher. In one embodiment, the HA protein antigen is recombinant or is a recombinantly produced HA protein antigen.

[0061] For example, the hemagglutinin (HA) protein of the influenza H3N2 virus is a polypeptide or fragment thereof that has at least approximately 85% or 85% amino acid sequence identity with the complete sequence of influenza A virus (A / Hong Kong / 1-4 / 1968(H3N2)) segment 4, accession number CY033017, or at least approximately 90%, 95%, 98%, 99%, or 90%, 95%, 98%, 99%, or greater amino acid sequence identity: TIFF2026509251000001.tif49135

[0062] Furthermore, the hemagglutinin (HA) protein of the influenza H3N2 virus is encoded by a polynucleotide or fragment having at least approximately 85% or 85% sequence identity with the following polynucleotide sequences, or at least approximately 90%, 95%, 98%, 99%, or 90%, 95%, 98%, 99%, or greater sequence identity: TIFF2026509251000002.tif144136

[0063] "Hybridization" refers to hydrogen bonding between complementary nucleic acid bases, which can be Watson-Crick, Hoogsteen, or reverse Hoogsteen type hydrogen bonds. For example, in DNA, adenine and thymine, and cytosine and guanine are complementary nucleic acid bases that pair up through the formation of hydrogen bonds.

[0064] "Immunogen" means a compound, composition, or substance, including compositions injected or absorbed into animals, that can induce or promote an immune response, such as antibody production and / or T-cell response, in animals under appropriate conditions. As used herein, "immunogenic composition" is a composition containing an immunogen (e.g., an H3 HA polypeptide) or a vaccine containing an H3 HA polypeptide). As recognized by those skilled in the art, when administered to a subject in need before the subject contracts the disease or experiences a full-blown illness, an immunogenic composition may be prophylactic, and the subject may be able to be protected from the disease or prevent a more severe disease or condition and / or its symptoms by inducing an immune response, such as a neutralizing antibody and / or cellular immune response. When administered to a subject in need after the subject has contracted the disease, an immunogenic composition may be therapeutic, and the subject may be able to treat the disease by inducing an immune response, such as a neutralizing antibody and / or cellular immune response, for example, by reducing, decreasing, suppressing, improving, or eliminating the disease and / or its symptoms. In one embodiment, the immune response is a B-cell response, which results in the production of antibodies, such as neutralizing antibodies, against an immunogenic composition containing an immunogen or antigen or antigenic sequence. As previously stated, in some embodiments, the immunogenic composition or vaccine may be prophylactic. In some embodiments, the immunogenic composition or vaccine may be therapeutic. In one embodiment, the disease is influenza (flu).

[0065] The term "immune response" refers to any response mediated by immune response cells. An example of an immune response is the recruitment of white blood cells to perform various different specific functions in response to exposure to an antigen (e.g., an exogenous entity). The immune response is a multifactorial process that differs depending on the type of cells involved. Immune responses include cellular responses (e.g., T-cell responses), humoral responses (B-cell / antibody responses), innate responses, and combinations thereof.

[0066] "Immunogenic composition" means a composition containing an antigen, an antigen sequence, or an immunogen that induces an immune response in an immunized subject.

[0067] The term "to immunize" (or "to grant immunity") refers to protecting an individual from a disease, infectious disease, condition, or symptoms caused by an H3 virus, for example, through vaccination.

[0068] The term "influenza virus" refers to segmented negative-strand RNA viruses belonging to the Orthomyxoviridae family. There are three types of influenza viruses: A, B, and C. Influenza A viruses infect a wide variety of birds and mammals, such as humans, horses, marine mammals, pigs, ferrets, and chickens. In animals, most influenza A viruses cause mild, localized infections of the respiratory and intestinal tracts. However, highly pathogenic influenza A strains, such as H3N2, can cause systemic infections in poultry, with mortality rates reaching 100%.

[0069] "Inhibitory nucleic acid" means a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimic thereof, that, when administered to mammalian cells, results in a reduction in the expression of a target gene (e.g., 5%, 10%, 25%, 50%, 75%, or even 90–100%). Typically, a nucleic acid inhibitor contains at least a portion of the target nucleic acid molecule or its ortholog, or at least a portion of the complementary strand of the target nucleic acid molecule. For example, an inhibitory nucleic acid molecule contains at least a portion of any or all of the nucleic acids described herein.

[0070] The terms “isolated,” “purified,” or “biologically pure” refer to substances that contain, to varying degrees, the components normally associated with them, as they would be found in their natural state. “Isolating” indicates a degree of separation from the original source or surrounding environment. “Purifying” indicates a higher degree of separation than isolation. A “purified” or “biologically pure” protein contains so little of other substances that, as a result, no impurities substantially affect the protein’s biological properties or cause other adverse events. That is, a nucleic acid, protein, or peptide is purified if, when produced by recombinant DNA, it substantially contains no cellular material, residue, unrelated viral material, or culture medium, or, when chemically synthesized, substantially contains no chemical precursors or other chemicals. Purity and homogeneity are typically determined using standard purification methods and analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” can indicate that a nucleic acid or protein produces essentially a single band in an electrophoretic gel. In the case of proteins that can be modified, for example, phosphorylation or glycosylation, different modifications can result in different isolated proteins, which can be purified separately. The term “isolated” also includes recombinant nucleic acids, proteins, or viruses, as well as chemically synthesized nucleic acids or peptides.

[0071] "Isolated polynucleotide" means a nucleic acid (e.g., a DNA molecule) that does not contain genes adjacent to its gene in the natural genome of the organism from which the nucleic acid molecule described herein originates. Therefore, this term may include recombinant DNA that exists as an isolated molecule (e.g., cDNA or genomic fragment or cDNA fragment produced by PCR or restriction enzyme digestion), for example, in a vector; in an autonomously replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryotic or eukaryotic organism; or independently of other sequences. Furthermore, this term includes recombinant DNA that is part of an RNA molecule transcribed from a DNA molecule, and a hybrid gene encoding a further polypeptide sequence.

[0072] "Isolated polypeptide" means a polypeptide described herein that has been isolated from its naturally associated components. Typically, a polypeptide is isolated if it does not contain at least 40%, at least 50%, or at least 60% by weight of the naturally associated proteins and naturally occurring organic molecules. Preferably, the isolated polypeptide preparation does not contain at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the naturally associated proteins and naturally occurring organic molecules. Isolated polypeptides can be obtained, for example, by extraction from a natural source; by expression of recombinant nucleic acids encoding such polypeptides; or by chemical synthesis of proteins. Purity can be measured by any standard suitable method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Isolated polypeptide may refer to a broadly active viral immunogen polypeptide produced by the methods described herein.

[0073] A "linker" refers to one or more amino acids that act as spacers between two polypeptides or peptides in a fusion protein.

[0074] A "marker" refers to any protein or polynucleotide whose expression level or activity is altered in relation to a disease, condition, pathology, or disorder.

[0075] The "matrix (M1) protein" refers to the influenza virus structural protein found in the viral coat. M1 is thought to function in the assembly and budding of the virus after infection of cells.

[0076] The term "neuraminidase (NA)" refers to a membrane glycoprotein of the influenza virus. NA is involved in the destruction of the cell receptor for the virus's HA by cleaving terminal sialic acid residues from the carbohydrate portion on the surface of infected cells. NA also cleaves sialic acid residues from viral proteins, preventing viral aggregation. NA (along with HA) is one of the two main influenza virus polypeptides that possess antigenic determinants.

[0077] As used herein, "obtaining" in the phrase "obtaining the active substance" includes synthesizing, isolating, purchasing, or acquiring the active substance.

[0078] The term “functionally linked” as used herein refers to nucleic acid sequences. For example, a first nucleic acid sequence is functionally linked to a second nucleic acid sequence if the first nucleic acid sequence is positioned in a functional relationship with the second nucleic acid sequence. For example, a promoter is functionally linked to a coding sequence if the promoter influences (enables) the transcription or expression of the coding sequence. Generally, functionally linked DNA sequences are close together and, if it is necessary to link two protein-coding regions, they reside within the same reading frame. Nucleic acid sequences are typically functionally linked in plasmids, expression vectors, and / or delivery vectors. In aspects, a nucleic acid sequence is, non-limitingly, DNA, RNA, or mRNA.

[0079] A "computationally optimized" influenza HA protein generally reflects an HA protein sequence resulting from a comparison of the sequences (amino acid sequences) of two or more viruses, such as the sequence of an H3 influenza virus clade, as described in, for example, U.S. Patent Application Publication No. US 2015 / 0030628. The nucleotide sequence encoding the H3 HA protein produced by the described method can be optimized for expression in mammalian cells through codon optimization and RNA optimization (e.g., aimed at increasing RNA stability) using the procedures and techniques practiced in the Art.

[0080] A broadly reactive, pan-epitope immunogen for inducing an immune response in a subject, such as the hemagglutinin (HA) protein of H3 influenza, has a collective set of potently immunogenic epitopes (also called antigenic determinants). The H3 virus HA proteins described herein are "pan-epitope" H3 immunogens suitable for use as vaccines that, when introduced into a host subject, particularly a human subject infected with an H3 virus, induce a broadly reactive immune response, such as a neutralizing antibody response, against multiple H3 virus types that express the HA protein on the surface of the virus. Immunogenic antigens (or vaccines) are advantageous in providing anti-H3 virus immunogens (or vaccines) that induce a broadly active immune response against H3 influenza virus HA antigens with antigenic variability and similarity, and that treat or protect against infections and diseases caused by one or more H3 influenza virus subtypes.

[0081] An "open reading frame (ORF)" refers to a series of nucleoti triplets (codons) that code for an amino acid without any stop codons. These sequences are typically translatable into peptides or polypeptides.

[0082] As used herein, an “outbreak” of influenza virus refers to a collection of virus isolates from a geographical location (e.g., a single country) over a given period of time (e.g., one year).

[0083] The term “pharmaceutically acceptable vehicle” refers to conventional carriers (vehicles) and excipients that are physiologically and pharmaceutically acceptable, particularly for use in mammalian subjects, such as human subjects. Such pharmaceutically acceptable vehicles are known to those skilled in the art and can be readily found in Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975) and its updated editions, which describe compositions and formulations suitable for the pharmaceutically acceptable delivery of one or more therapeutic compositions, such as one or more influenza vaccines, and further pharmaceutical agents. Generally, the properties of a pharmaceutically acceptable carrier depend on the specific mode of administration used. For example, parenteral formulations typically include injectable fluids / liquids as vehicles, such as pharmaceutically and physiologically acceptable fluids, e.g., water, saline, equilibrium salt solutions, aqueous dextrose, glycerol, etc. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers may include, for example, typically pharmaceutically-grade mannitol, lactose, starch, or magnesium stearate, which stabilize the composition or drug and / or extend its half-life. In addition to a biologically neutral carrier, the administered pharmaceutical composition may include small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0084] A "plasmid" refers to a cyclic nucleic acid molecule that can autonomously replicate within a host cell.

[0085] A "polypeptide" (or "protein") means a polymer composed of monomers that are linked together by amide bonds, consisting of amino acid residues. If the amino acid is an alpha-amino acid, either an L-optical isomer or a D-optical isomer may be used. As used herein, the terms "polypeptide" or "protein" are intended to encompass any amino acid sequence and to include modified sequences such as glycoproteins. The term "polypeptide" is particularly intended to cover natural proteins and proteins produced recombinantly or synthetically. The terms "residue" or "amino acid residue" also refer to amino acids incorporated into proteins, polypeptides, or peptides. In embodiments, the terms polypeptide and protein are used interchangeably.

[0086] Conservative amino acid substitutions are those that, when performed, do little to alter the properties of the original protein; that is, the protein's structure and, in particular, its function are preserved and not significantly altered by such substitution. Examples of conservative amino acid substitutions are known in the art, for example, as shown in U.S. Patent Application Publication 2015 / 0030628. Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the substituted region, for example, as a sheet or helix conformation; (b) the molecular charge or hydrophobicity at the target site; and / or (c) the majority of the side chain.

[0087] Substitutions that are generally expected to alter the properties of a protein most significantly are non-conservative and conservative, for example: (a) hydrophilic residues, e.g., ceryl or threonyl, are substituted for (or by) hydrophobic residues, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) cysteine ​​or proline are substituted for (or by) any other residue; (c) residues with positively charged side chains, e.g., lysyl, arginyl, or histadyl, are substituted for (or by) negatively charged residues, e.g., glutamyl or aspartyl; or (d) residues with bulky side chains, e.g., phenylalanine, are substituted for (or by) those without side chains, e.g., glycine.

[0088] A "primer set" refers to a set of oligonucleotides that can be used, for example, in PCR. A primer set is thought to consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers.

[0089] A "promoter" refers to a set of nucleic acid regulatory sequences that direct the transcription of a nucleic acid. A promoter contains the necessary nucleic acid sequences near the transcription start site. Promoters may also optionally contain enhancer or repressor sequence elements. A "constitutive promoter" is constantly active and not regulated by external signals or molecules. In contrast, the activity of an "inducible promoter" is regulated by external signals or molecules (e.g., transcription factors). For example, a promoter could be a CMV promoter.

[0090] As is recognized by those skilled in the art, the term “purified” does not require absolute purity; rather, it is intended as a relative term. Therefore, for example, a purified peptide, protein, virus, or other active compound is one that has been isolated, in whole or in part, from the naturally occurring proteins and other impurities. In certain embodiments, the term “substantially purified” refers to a peptide, protein, virus, or other active compound that has been isolated from cells, cell culture media, or other crude preparations and subjected to conventional methods such as fractionation, chromatography, or electrophoresis to remove various components of the original preparation, such as proteins, cell debris, and other components.

[0091] "Recombinant" nucleic acids, proteins, or viruses are those that have a non-natural sequence or a sequence created by the artificial combination of two otherwise isolated sequence segments. Such artificial combinations are often achieved by chemical synthesis or by artificial manipulation of isolated nucleic acid segments, for example, by genetic engineering techniques. "Non-natural" nucleic acids, proteins, polypeptides, or viruses can be created through recombinant techniques, artificial manipulation, or procedures and techniques of genetic engineering or molecular biological engineering, for example, those commonly practiced in the art.

[0092] "To reduce" means a negative change of at least 5%, 10%, 25%, 30%, 40%, 50%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%.

[0093] "Reference" means a standard or control condition. In a non-limiting example, reference cells may be wild-type or healthy cells. Reference may be untreated cells that have not been subjected to test conditions or to placebo or normal saline, culture medium, buffer, and / or a control vector that does not contain the polynucleotide of interest. Reference may be a healthy, normal, or non-pathological subject, for comparison with a subject having a disease, disorder, or condition caused by, for example, H3 influenza virus infection.

[0094] A “reference sequence” is a defined sequence used as a criterion for sequence comparison. A reference sequence can be a subset or the whole of a particular sequence; for example, it may be a full-length cDNA or a segment of a gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is generally considered to be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is generally considered to be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides, or any integer around or between those numbers. In some embodiments, the reference sequence is the wild-type sequence of the protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence encoding a wild-type protein.

[0095] "Specifically binding" means a compound or antibody that recognizes and binds to polypeptides, such as viral polypeptides, peptides, or vaccine products, but substantially does not recognize or bind to other molecules in a sample naturally containing polypeptides, such as viral polypeptides or peptides, such as a biological sample.

[0096] Nucleic acid molecules useful in the methods described herein include any nucleic acid molecule or fragment thereof that encodes the polypeptide described. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but are typically considered to exhibit substantial identity. Polynucleotides having “substantial identity” with the endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. “Hybridize” means to pair complementary polynucleotide sequences (e.g., genes) or parts thereof to form a double-stranded molecule under various stringency conditions (see, e.g., Wahl, GM and SL Berger, (1987), Methods Enzymol., 152:399; Kimmel, AR, (1987), Methods Enzymol. 152:507).

[0097] For example, stringent salt concentrations are typically considered to be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low-stringency hybridization can be obtained in the absence of organic solvents, such as formamide, while high-stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions are typically considered to include temperatures of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Further parameters, such as hybridization time, surfactant concentration, such as sodium dodecyl sulfate (SDS), and modifications to the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In a preferred embodiment, hybridization is considered to be carried out at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization is considered to be carried out at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In the most preferred embodiment, hybridization is considered to be carried out at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful modifications to these conditions are expected to be readily apparent to those skilled in the art.

[0098] In most applications, the stringency of the washing step after hybridization is also expected to vary. The conditions for washing stringency are defined by the salt concentration and temperature. As described above, washing stringency can be increased by decreasing the salt concentration or increasing the temperature. For example, the stringent salt concentration for the washing step is preferably about 30 mM NaCl and less than 3 mM trisodium citrate, most preferably about 15 mM NaCl and less than 1.5 mM trisodium citrate. The stringent temperature conditions for the washing step are usually considered to include temperatures of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, the washing step is considered to be carried out at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step is to be carried out at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step is to be carried out at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further modifications to these conditions are to be readily apparent to those skilled in the art.Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0099] "Substantially identical" means a polypeptide or nucleic acid molecule that exhibits at least 50% identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such a sequence is identical to the sequence used for comparison by at least 60%, or at least 80% or 85%, or at least 90%, 95%, or even 99%, or 90%, 95%, or even 99%, at the amino acid level or nucleic acid level.

[0100] Sequence identity refers to the similarity between amino acid sequences or nucleic acid sequences, expressed in terms of the similarity between sequences. Sequence identity is often measured in terms of identity (or similarity or homology) percentages; a higher percentage indicates greater sequence similarity. Homologous or variant sequences of a given gene or protein are considered to have a relatively high degree of sequence identity when aligned using standard methods. Sequence identity is typically measured using sequence analysis software (e.g., the sequence analysis software packages of Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, such as BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. An exemplary approach to determining the degree of identity is to show closely related sequences e -3 from e -100The BLAST program can be used with the probability scores between these values. Furthermore, other programs and alignment algorithms are cited, for example, in Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Needleman and Wunsch, 1970, J. Mol. Biol. 48:443; Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444; Higgins and Sharp, 1988, Gene 73:237-244; Higgins and Sharp, 1989, CABIOS 5:151-153; Corpet et al., 1988, Nucleic Acids Research 16:10881-10890; Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444; and Altschul et al., 1994, Nature. It is described in Genet. 6:119-129. The NCBI Basic Local Alignment Search Tool (BLAST®) (Altschul et al. 1990, J. Mol. Biol. 215:403-410) is readily available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.), and via the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0101] "Subject" means an animal, for example, a mammal, for example, a human, a non-human primate, or a non-human mammal, for example, a mammal of the genus Bos, family Equidae, family Canidae, family Ash-like, or family Felidae, or a goat, llama, camel, or a rodent (rat, mouse), gerbil, or hamster. In non-limiting cases, subject is an individual infected with the H3 virus, or at risk of infection by such virus, or susceptible to such infection. In certain contexts described herein, subject is a human subject, for example, a patient.

[0102] The ranges provided herein are understood to be abbreviated notations for all values ​​within the range, including the first and last values ​​listed. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange of the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more, consecutively, for example, up to 100.

[0103] As used herein, the terms “to treat,” “to treat,” and “treatment” refer to reducing, decreasing, diminishing, suppressing, improving, or eliminating a disease, condition, disorder, or pathology, and / or symptoms associated therewith. “To treat” typically refers to therapeutic interventions performed to reduce the severity of a disease, condition, disorder, or pathology, and / or symptoms associated therewith, after the onset of the disease, condition, disorder, or pathology, and / or symptoms associated therewith. It will be recognized, but not excluded, that treating a disorder or condition does not require the complete elimination of the disease, condition, disorder, pathology, or symptoms associated therewith.

[0104] As used herein, the terms “prevent,” “prevention,” “prevention,” and “preventive measures” refer to inhibiting or blocking a disease state or the complete onset of a disease in an object, or reducing the probability of developing a disease, disability, or condition in an object that does not have a disease, disability, or condition but is at risk of developing one, or is susceptible to developing one.

[0105] As used herein, “transformed” cells are cells into which nucleic acid molecules or polynucleotide sequences have been introduced by molecular biological techniques. As used herein, the term “transformed” encompasses all techniques that can introduce nucleic acid molecules or polynucleotides into such cells, including transfection using viral vectors, transformation using plasmid vectors, and introduction of naked nucleic acids (DNA or RNA) by electroporation, lipofection, and particle gun acceleration.

[0106] "Vaccine" means a preparation of an immunogenic substance (e.g., protein or nucleic acid; vaccine) that can promote (induce) an immune response, administered to a subject to treat a disease, condition, or pathological state, or to prevent a disease, condition, or pathological state, such as an infectious disease (e.g., caused by H3 virus infection). Immunogenic substances may include, for example, attenuated or killed microorganisms (e.g., attenuated viruses), or antigenic proteins, peptides, or DNA derived from such microorganisms. A vaccine can induce a prophylactic (preventive) immune response in a subject; it can also induce a therapeutic immune response in a subject. As mentioned above, the method of vaccine administration varies depending on the vaccine and may include routes or means, such as inoculation (intravenous or subcutaneous injection), ingestion, inhalation, or other forms of administration. Inoculation may be delivered parenterally, by any of several routes, including intravenous, subcutaneous, or intramuscular. Vaccines may also be administered with adjuvants to boost the immune response.

[0107] As used herein, “vector” refers to a nucleic acid (polynucleotide) molecule into which an exogenous nucleic acid can be inserted without disrupting the vector’s ability to replicate in and / or be incorporated into a host cell. A vector may include nucleic acid sequences, e.g., replication origins, that enable the vector to replicate in a host cell. An insertion vector may insert itself into a host nucleic acid. A vector may also include one or more select marker genes and other genetic elements. An expression vector is a vector that includes regulatory sequences necessary to enable the transcription and translation of one or more inserted genes in a host cell. In some aspects of this disclosure, a vector encodes an influenza HA, NA, or M1 protein. In some aspects, a vector is an RNA (e.g., mRNA) or DNA vector. In some embodiments, the vector is a pTR600 expression vector (U.S. Patent Application Publication No. 2002 / 0106798; Ross et al., 2000, Nat Immunol. 1(2):102-103; and Green et al., 2001, Vaccine 20:242-248).

[0108] "Virus-like particles (VLPs)" refer to viral particles that are composed of one of many viral structural proteins but lack a viral genome. Because VLPs lack a viral genome, they are non-infectious and result in safer and potentially more economical vaccines and vaccine products. Furthermore, VLPs are often produced by heterologous expression and can be easily purified. Most VLPs contain at least a viral core protein that causes them to budding from host cells and release particles. An example of such a core protein is influenza M1. In some aspects of this specification, H3 influenza VLPs contain the HA protein. In some aspects of this specification, H3 influenza VLPs contain the HA, NA, and M1 proteins. As described herein, H3 influenza VLPs can be produced by transfection of host cells with plasmids encoding the H3 HA, NA, and M1 proteins. In some aspects, VLPs contain polynucleotides encoding viral proteins. In some aspects, the polynucleotides are RNA (e.g., mRNA) or DNA. After incubating transfected cells for a suitable time to allow protein expression (e.g., approximately 72 hours), VLPs can be isolated from the cell culture supernatant. For example, a protocol for purifying or isolating influenza VLPs from cell supernatant includes slow centrifugation (to remove cell debris), suction filtration, and ultracentrifugation of the VLPs via 20% glycerol.

[0109] Unless otherwise stated or evident from the context, the term “or” is understood to be inclusive when used herein. Unless otherwise stated or evident from the context, the terms “a,” “an,” and “the” are understood to be singular or plural when used herein. Similarly, the word “or” is intended to include “and” unless otherwise explicitly stated in the context. Thus, “including A or B” means including A, or B, or A and B. It should be further understood that all base size or amino acid size and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximations and are provided for illustrative purposes only.

[0110] Unless otherwise stated or made clear from the context, the term “about” as used herein is understood to mean within the normal tolerance of the art, for example, within two standard deviations of the mean. “About” may be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise made clear from the context, all numerical values ​​provided herein are modified by the term “about.”

[0111] Any description of a list of chemical groups in any definition of a variable in this specification includes the definition of that variable as any single group or combination of the enumerated groups. Any description of an aspect of a variable or aspect in this specification includes that aspect as any single aspect or in combination with any other aspects or parts thereof.

[0112] Any composition or method provided herein may be combined with one or more of the other compositions and methods provided herein. [Brief explanation of the drawing]

[0113] [Figure 1A]Figures 1A and 1B show tables describing the hemagglutinin (HA) (H3N2 HA) protein antigens of the NG-4–NG-8 H3 influenza viruses described herein. In particular, five H3 HA sequences were designed using a next-generation computationally optimized broad-reactivity ("COBRA") method (next-generation computation method) with wild-type H3 HA sequences that circulated during the 2018–2021 influenza seasons. The COBRA HA antigen constructs were named NG-4–NG-8. The design timeframes for each of these antigens are shown in the table in Figure 1A. These sequences were compared with other H3N2 vaccine lineage viruses from 2017–2021, and the number of amino acid differences between them is shown in the table in Figure 1B. [Figure 1B] Refer to the explanation in Figure 1A. [Figure 2] This specification provides a diagram illustrating a phylogenetic tree comparing the NG-4 to NG-8 sequences described herein with those of the wild-type historical H3N2 influenza vaccine lineage. [Figure 3] Schematic diagrams (Examples 1 and 2) show the design of animal studies in naive mice to evaluate the development of an antibody immune response to the H3 hemagglutinin polypeptide described herein, which is used as an immunogen. [Figure 4-1] Figures 4A to 4F show graphs of hemagglutination inhibition (HAI) titers at day 70 of serum antibodies produced in research animals immunized with NG-4 (Figure 4A), NG-5 (Figure 4B), NG-6 (Figure 4C), NG-7 (Figure 4D), NG-8 (Figure 4E), and NG-7 / NG-8 (Figure 4F) H3 influenza rHA polypeptide immunogens (vaccines) as described herein (Example 3). In this study, animals were immunized with rHA immunogens containing ADDAVAX® (a squalene-based oil-in-water nanoemulsion) adjuvant. Antibody titers against historical H3N2 HA vaccine strains were evaluated by hemagglutination inhibition assays (HAI). [Figure 4-2] Refer to the explanation in Figure 4-1. [Figure 4-3] Refer to the explanation in Figure 4-1. [Figure 4-4]Refer to the explanation in Figure 4-1. [Figure 5-1] Figures 5A to 5F show graphs of hemagglutination inhibition (HAI) titers at day 70 of serum antibodies produced in research animals immunized with NG-4 (Figure 5A), NG-5 (Figure 5B), NG-6 (Figure 5C), NG-7 (Figure 5D), NG-8 (Figure 5E), and NG-7 / NG-8 (Figure 5F) H3 influenza rHA polypeptide immunogens (vaccines) as described herein (Example 4). In this study, animals were immunized with rHA immunogens containing R-DOTAP (cationic lipid) adjuvant. Antibody titers against historical H3N2 HA vaccine strains were evaluated by hemagglutination inhibition assays (HAI). [Figure 5-2] See the explanation in Figure 5-1. [Figure 5-3] See the explanation in Figure 5-1. [Figure 5-4] See the explanation in Figure 5-1. [Figure 6] The table shows the 50% neutralizing titer of serum antibodies at day 70. This table shows data indicating the reciprocal Log2 serum antibody titer at day 70 that prevented 50% of viral infections in Maidin-Derby canine kidney (MDCK)-SIAT cells across a panel of H3N2 influenza viruses from 2013 to 2021. The MDCK-SIAT cell line was obtained by stably transfecting MDCK cells with human 2,6-sialtransferase (SIAT1) cDNA. The cells express twice as much hexalinked sialic acid and twice as little trialinked sialic acid as the parental MDCK cells. In general, MDCK-SIAT1 cells, through overexpression of the sialyl-alpha-2,6-galactose moiety, provide a suitable system for testing the susceptibility of human influenza virus to neuraminidase inhibitors (NAIs) (Matrosovich M. et al., 2003, J. Virol., 77:8418-8425). In the table, the lowest neutralizing titer is shown in white, and the highest neutralizing titer is shown in dark gray shading. [Modes for carrying out the invention]

[0114] Detailed explanation H3 influenza viruses are routinely transmitted in humans and cause seasonal influenza epidemics. H3 viruses typically cause severe influenza illness and adapt to avoid eradication by constantly altering their surface proteins, such as the HA protein. H3 influenza A virus was found to be the dominant strain in the United States and globally during the 2017-2018 influenza season, for example, in Australia and the United Kingdom. The H3 strain has been particularly problematic to treat due to its unusually high mutation rate and the inability to produce vaccines effective against the relatively rapid changes that occur in its HA surface protein during vaccine development.

[0115] This specification addresses isolated, synthetic (non-natural) immunogenic antigens derived from the H3(H3N2) lineage of influenza A virus ("influenza") hemagglutinin (HA) protein, such as protein antigens and glycoprotein antigens, that induce a potent, broad-spectrum, and long-lasting immune response in subjects, particularly in human subjects. Such immunogenic antigens are also referred to herein as "immunogens."

[0116] Immunogens are provided that protect against diseases caused by influenza H3 strains or seasonal influenza H3 strains over several years, including drift strains that do not yet exist. In one embodiment, isolated, fully synthetic protein antigens, such as influenza H3 virus HA protein antigens, are addressed. Such H3 HA antigens are synthetic proteins not found in nature, and furthermore, they retain all the functions of the natural H3 HA virus protein and are immunogenic, that is, they can induce an immune response, in particular a broad-spectrum active immune response in the form of neutralizing antibodies and / or reactive T lymphocytes, after administration, delivery, or introduction to a subject. Immunogenic compositions, such as vaccines, containing synthetic H3 virus protein antigens or nucleic acids encoding said antigens are also provided.

[0117] H3 HA amino acid sequences and protein antigens having such sequences are intended for use as immunogens or in immunogenic compositions, such as vaccines, to induce a broad-spectrum reactive immune response in subjects to whom a composition or vaccine is administered, particularly in human subjects. H3 viral immunogens contain antigenic determinants representing different “antigenic spaces” derived from sequences of many H3 viral lineages analyzed based on seasonal periods (either overlapping or non-overlapping seasonal periods). Such overlapping or non-overlapping seasonal periods can encompass different time intervals, for example, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 10 years or more, including time intervals between the following:

[0118] The H3 virus antigens described herein encompass seasonal, panepitope, broadly reactive antigens of H3 influenza viruses and their subtypes, particularly antigens containing sequences based on H3 drift variants, which are designed to evoke a broadly active immune response in subjects, especially human subjects, particularly in the form of neutralizing antibodies. Such antigens are beneficial as immunogens that induce an immune response (e.g., production of neutralizing antibodies) against H3 viruses where multiple H3 strains are circulating simultaneously. Broadly reactive H3 immunogenic antigens may originate from H3 viruses that frequently mutate parts of their genome to evade immunosuppression, and as a result evade immune surveillance in subjects that have not been primed or stimulated to produce antibodies against antigenic epitopes (determinants) on the H3 antigen after infection. Therefore, synthetic H3 antigens, e.g., H3 HA antigens, contain amino acid (or polynucleotide) sequences that are thought to induce a greater number of neutralizing antibodies against potential H3 drift variants within and across multiple seasons compared to wild-type antigen sequences.

[0119] H3 HA immunogenic proteins or immunogens can be used in immunogenic compositions or as vaccines, as described herein, to provide protection from many H3 virus lineages over several years. The broad-reactive H3 influenza immunogens and vaccines described herein are advantageous in that they are designed to provide broader and longer-lasting protection from several seasonal H3 influenza lineages (or clades) prevalent in different geographical locations. The immunogens and sequences described herein provide a versatile and broad-reactive H3 influenza vaccine that can reduce the need for seasonal influenza vaccines (immunogenic compositions) against H3 lineages and subtypes of influenza viruses administered annually.

[0120] The immunogenic H3 virus HA antigens described herein can be used in immunogenic compositions (e.g., influenza vaccines) that can provide protective immunity against H3 influenza infection and disease in a subject. Protective immunity is induced in the subject through the induction of a potent, broadly reactive anti-H3 HA-specific antibody response that protects the subject from drifted seasonal H3 influenza virus strains and pandemic H3 influenza virus strains. The immunogenic compositions and vaccines offer advantages over earlier and conventional immunogenic compositions and vaccines against H3 viruses, which typically rely on the selection of candidate vaccine viruses by public health authorities after analysis of data collected through proactive surveillance of annually circulating influenza viruses.

[0121] Influenza virus Influenza viruses are segmented negative-strand RNA viruses belonging to the Orthomyxoviridae family. There are three types of influenza viruses: A, B, and C. Influenza A viruses infect a wide variety of birds and mammals, such as humans, horses, marine mammals, pigs, ferrets, and chickens. In animals, most influenza A viruses cause mild, localized infections of the respiratory and intestinal tracts. However, highly pathogenic influenza A strains, such as H3, can cause systemic infections in poultry, with mortality rates reaching 100%. Animals infected with influenza A often act as reproductive hosts for the influenza virus, and certain subtypes have been shown to cross the species barrier to humans, where they can cause severe illness and devastating influenza outbreaks that can lead to death in infected human subjects.

[0122] Influenza A viruses can be classified into subtypes based on allele mutations in the antigenic regions of two genes that encode surface glycoproteins necessary for viral attachment and cell release: hemagglutinin (HA) and neuraminidase (NA). Currently, 16 HA subtypes (H1-H16) and 9 NA subtypes (N1-N9) of influenza A viruses are known antigenic variants. Previously, only three subtypes were known to cause outbreaks in humans (H1N1, H1N2, and H3N2). However, in recent years, for example, the pathogenic H5N1 subtype of avian influenza A has been reported to cross species barriers, infect humans, and lead to the deaths of several patients, as recorded in Hong Kong in 1997 and 2003.

[0123] In humans, avian influenza viruses infect cells in the respiratory tract as well as the intestines, liver, spleen, kidneys, and other organs. Symptoms of avian influenza infection include difficulty breathing, including fever, shortness of breath and cough, lymphopenia, diarrhea, and difficulty regulating blood glucose levels. In contrast to seasonal influenza, the group most at risk is healthy adults, who constitute the majority of the population. Due to the high pathogenicity of certain avian influenza A subtypes, particularly H3, and their demonstrated ability to transspecies transmission to humans, there are considerable economic and public health risks associated with these virus lineages, including the threat of real epidemics and pandemics.

[0124] The influenza A virus genome encodes nine structural proteins and one non-structural (NS1) protein with regulatory functions. The segmented genome of the influenza virus contains eight minus-strand RNA (nsRNA) gene segments (PB2, PB1, PA, NP, M, NS, HA, and NA) encoding at least 10 polypeptides, e.g., RNA-dependent RNA polymerase proteins (PB2, PB1, and PA), nucleoproteins (NP), neuraminidase (NA), hemagglutinin (HA), e.g., subunit HA1, often referred to as the “head” subunit; and HA2, often referred to as the “tail” or “stem” subunit; matrix proteins (M1 and M2); and non-structural proteins (NS1 and NS2) (see, e.g., Krug et al., 1989, In: The Influenza Viruses, RM Krug, ed., Plenum Press, NY, pp. 89-152).

[0125] The ability of influenza viruses, such as H3, to cause widespread disease is due to their ability to evade the immune system by undergoing antigenic changes, which is thought to occur when a host is simultaneously infected with both animal and human influenza viruses. During mutation and reassortment in the host, the virus can incorporate HA and / or NA surface protein genes from another virus into its genome, thereby resulting in a new influenza subtype that evades the immune system.

[0126] The effectiveness of vaccines against H3 influenza viruses has often been below optimal and below standard, particularly due to antigenic drift in the HA and NA proteins of the virus in strains during H3 influenza virus outbreaks. The methods described herein provide a broadly reactive pan-epitope HA antigen of H3 (H3N2) influenza virus that, in particular, induces a broadly reactive immune response in the form of a neutralizing antibody that binds to the H3 viral antigen and neutralizes the activity of the virus (e.g., its ability to infect cells), for more effectively treating H3 influenza and its symptoms.

[0127] Hemagglutinin (HA) and neuraminidase (NA) proteins of the influenza virus HA is a viral surface glycoprotein that generally consists of approximately 560 amino acids (e.g., 566 amino acids) and accounts for 25% of the total viral protein. As described herein, HA is a very useful protein antigen as an immunogen against H3 viruses because it contains a diverse repertoire of epitopes that produce antibodies in targets or hosts that encounter the H3 HA antigen during infection.

[0128] HA is involved in the initial stages of infection, allowing viral particles to adhere to and invade host cells, particularly the respiratory epithelium. The cleavage of the viral HA (HA0) precursor into HA1 and HA2 subfragments is a necessary step for the virus to infect cells. Therefore, cleavage is required in host cells to convert new viral particles into virions capable of infecting new cells. It is known that cleavage occurs during the transport of the complete HA0 membrane protein from the endoplasmic reticulum to the plasma membrane of infected cells. During transport, HA undergoes a series of co-translational and post-translational modifications, including proteolytic cleavage of the precursor HA into the amino-terminal fragment HA1 ("head") and carboxy-terminal HA2 ("tail" or "stem"). One of the main difficulties in the proliferation of H3 influenza strains in primary tissue cultures or established cell lines is the requirement for activation of proteolytic cleavage of influenza hemagglutinin in host cells.

[0129] Uncleaved HA is known to mediate viral attachment to neuraminic acid-containing receptors on the cell surface, but this prevents the next step in the infection cycle, namely fusion. It has been reported that cleavage, which exposes the hydrophobic amino terminus of HA2, is necessary for HA2 to be inserted into the target cell, thereby forming a crosslink between the virus and the target cell membrane. This process is followed by the fusion of the two membranes and the transfer of the virus into the target cell.

[0130] Proteolytic activation of hemagglutinin is often performed by calcium-dependent intracellular enzymes, including cleavage at arginine residues by trypsin-like endoproteases under optimal neutral pH conditions. Since the activating protease is a cellular enzyme, whether or not HA is cleaved depends on the infected cell type. HA from mammalian influenza viruses and non-pathogenic avian influenza viruses is susceptible to proteolytic cleavage in only a limited number of cell types. There are also host range differences, stemming from differences in hemagglutinin cleavage, which correlate with viral pathogenicity.

[0131] Neuraminidase (NA) is the second membrane glycoprotein of the influenza virus. The presence of NA in viruses has been shown to be important in generating a multifaceted protective immune response against infecting viruses. In most influenza A viruses, NA is 413 amino acids long and encoded by a 1413 nucleotide gene. Nine different NA subtypes have been identified in influenza viruses (N1, N2, N3, N4, N5, N6, N7, N8, and N9), all of which are found in wild birds. NA is involved in the disruption of the cell receptor for viral HA by cleaving terminal neuraminic acid (also called sialic acid) residues from the carbohydrate moiety on the surface of infected cells. NA also cleaves sialic acid residues from viral proteins, preventing viral aggregation. Using this mechanism, it is hypothesized that NA facilitates the release of viral offspring by preventing newly formed viral particles from accumulating along the cell membrane and by promoting the transport of viruses through mucus present on mucosal surfaces. NA is an important antigenic determinant involved in antigen variation.

[0132] In addition to the surface proteins HA and NA, the H3 influenza virus contains six further internal genes that give rise to eight distinct proteins, including polymerase genes PB1, PB2, and PA, matrix proteins M1 and M2, nucleoprotein (NP), and non-structural proteins NS1 and NS2 (see, for example, Horimoto et al., 2001, Clin Microbiol Rev. 14(1):129-149).

[0133] To be packaged within progeny virions, H3 viral RNA associates with influenza virus matrix 1 (M1) protein and nuclear export protein, and is transported from the nucleus as a ribonucleoprotein (RNP) complex consisting of three influenza virus polymerase proteins, a nucleoprotein (NP), and viral RNA (Marsh et al., 2008, J Virol, 82:2295-2304). The M1 protein within the envelope is thought to function in assembly and budding. A limited number of M2 proteins are incorporated into the virions (Zebedee, 1988, J. Virol. 62:2762-2772). These M2 proteins possess H+ ion channel activity and, when activated by low pH in endosomes, form tetramers that acidify the interior of the virion, thereby facilitating its shedding of the envelope (Pinto et al., 1992, Cell 69:517-528). Amantadine is an anti-influenza drug that prevents viral infection by inhibiting M2 ion channel activity, thereby inhibiting the removal of the viral sheath.

[0134] NS1, a non-structural protein, has multiple functions, including the regulation of splicing and nuclear export of cellular mRNA, as well as the stimulation of translation. The primary function of NS1 appears to be to counteract host interferon activity, as NS1 knockout viruses were viable but did not replicate as efficiently as the parent virus in interferon-free cells (Garcia-Sastre, 1998, Virology 252:324-330).

[0135] The NS2 non-structural protein has been detected in viral particles (Richardson et al., 1991, Arch. Virol. 116:69-80; Yasuda et al., 1993, Virology 196:249-255). The average number of NS2 proteins in viral particles was estimated to be 130–200 molecules. Direct protein-protein contact between M1 and NS2 was demonstrated by in vitro binding assays. The NS2-M1 complex was also detected by immunoprecipitation in solubles of virus-infected cells. The NS2 protein is thought to play a role in the export of RNP from the nucleus through interaction with the M1 protein (Ward et al., 1995, Arch. Virol. 140:2067-2073).

[0136] Broadly reactive influenza proteins and virus-like particles (VLPs) Provided are non-natural, broadly reactive, immunogenic H3 influenza HA polypeptides (immunogens) and influenza virus-like particles (VLPs) containing H3 HA polypeptide antigens with diverse epitopes (antigenic determinants) that, after administration and delivery to susceptible or in need subjects, confer upon the HA polypeptide antigen the ability to induce a broadly reactive immune response against the influenza virus and its symptoms, either prophylactically or therapeutically. For example, a typical H3 HA polypeptide immunogen includes the amino acid sequence described in Example 1 herein. In specific cases, the broadly reactive H3 HA polypeptide immunogen or the polynucleotide encoding the polypeptide immunogen is administered as part of a VLP.

[0137] It will be understood that the H3 influenza virus immunogens and sequences described and provided herein are non-natural and broadly reactive, regardless of whether these features and characteristics are explicitly stated. It will also be recognized that the H3 HA protein antigens described herein and used as immunogens are non-natural or synthetic antigens that induce an immune response, such as neutralizing antibodies, in a subject.

[0138] In one embodiment, a broadly reactive and immunogenic H3 antigen sequence capable of eliciting an immune response against H3 influenza virus lineages, including current and future H3 viruses, can be generated by a method such as that described in WO 2020 / 014675, published on January 16, 2020, whose entire contents are incorporated herein by reference, and which includes considering parameters of H3 antigen sequences, e.g., HA antigen sequences, from a length of time or range (e.g., a linear time range), e.g., one or more influenza seasons and the geographical location from which the H3 virus was isolated, e.g., from the Southern Hemisphere or Northern Hemisphere.

[0139] In one embodiment, the VLP of H3 influenza contains the viral HA protein. In another embodiment, the VLP may contain the HA1 and / or HA2 proteins. In some cases, it will be recognized that the VLP of H3 influenza virus may contain the viral NA and M1 proteins. The generation of influenza VLPs has been described in the art and is within the scope of the skills and expertise of those skilled in the art. Briefly and as described, influenza VLPs can be generated by transfecting host cells with one or more plasmids containing polynucleotide sequences encoding the HA, NA, and M1 proteins. After incubating the transfected cells for a time appropriate to allow protein expression (e.g., approximately 72 hours), the H3 VLPs can be isolated from the cell culture supernatant. The H3 influenza VLPs can be purified from the cell supernatant using procedures performed in the art, for example, the VLPs can be isolated by slow centrifugation (to remove cell debris), suction filtration, and ultracentrifugation through 20% glycerol.

[0140] Influenza VLPs can be used as immunogenic compositions or influenza vaccines to induce an immune response against the H3 influenza virus. In particular, the components, namely the broadly reactive pan-epitope H3 influenza HA polypeptide of the immunogenic composition or vaccine (or VLP), contain an antigen (pan-epitope) determinant that is broadly reactive and works to induce an immune response in the subject that can treat the subject infected with the H3 virus (e.g., neutralize the infectious virus) and / or protect the subject from full-blown viral infection or its signs and symptoms (e.g., production of neutralizing antibodies and / or activated T cells).

[0141] In one embodiment, the antigenic sequence of a broadly reactive and immunogenic H3 influenza antigen described herein, for example, an H3 HA antigen, includes a diverse repertoire of epitope determinants that can reflect, for example, antigenic drift and sequence diversity in the H3 virus antigenic protein across seasons (time) and different geographical locations. In particular, the H3 virus HA antigen described herein may include an amino acid sequence containing antigenic determinants (epitopes) derived from influenza virus strains with sequence diversity, including drift variants, and in particular, this antigen can produce broadly reactive neutralizing antibodies against it when used as an immunogenic product (immunogen) introduced into a target, for example, an antiviral vaccine.

[0142] In one aspect, the H3 viral antigen amino acid sequence ultimately provides a complex immunogenic antigen sequence containing an epitope determinant, i.e., a “panepitope” antigen that elicits a broad-spectrum reactive immune response when used as an immunogen, vaccine, or VLP, which can be derived from both past and more recent seasons of viral infection or disease, and / or from viruses of different geographical locations, and / or from different subtypes or clades of H3 viruses. In one embodiment, the immunogenic H3 viral HA antigen sequence contains an epitope resulting from an antigenic change in the sequence of the H3 HA surface antigen, which is caused by a point mutation during viral replication that gives rise to a new H3 influenza variant. As a result, administration of the H3 immunogen described herein to a subject can elicit a broad-spectrum reactive immune response in the subject, which is directed to an epitope that reflects such an antigenic change.

[0143] The broadly reactive H3 HA antigens and sequences described herein, used as immunogens or immunogenic compositions, e.g., vaccines, induce a broadly reactive immune response, e.g., antibody production, in immunocompetent subjects. Therefore, they provide excellent vaccines that capture antigenic epitopes of many different H3 influenza isolates (subtypes or strains), resulting in a broadly active immune response (e.g., a broadly active neutralizing antibody). Note that the terms “broadly active” and “broadly reactive” are used synonymously herein.

[0144] In one embodiment, the H3 virus antigen described herein is a polypeptide or peptide antigen of the H3 virus that currently causes a disease or infection and its symptoms, such as seasonal H3 influenza, and is specific to a particular geographical location. In another embodiment, the H3 virus antigen is a polypeptide or peptide antigen that is expected to cause a disease and symptoms of H3 infection in the future. In one embodiment, the H3 virus antigen is a polynucleotide sequence. In one embodiment, the H3 virus antigen is a polynucleotide sequence encoding a polypeptide or peptide antigen described herein. As an example, representative broadly reactive H3 virus HA polypeptide immunogens (in other words, NG-4, NG-5, NG-6, NG-7, and NG-8) are shown in Example 1 herein. The full-length and soluble amino acid sequences of the NG-4, NG-5, NG-6, NG-7, and NG-8 H3 HA polypeptide immunogens, as well as the polynucleotide sequences encoding the full-length and soluble forms of these polypeptide immunogens, are shown in Example 1.

[0145] In particular, isolated immunogenic full-length H3 HA viral protein antigens (polypeptides) having the amino acid sequences shown in SEQ ID NO:1(NG-4(H3 HA)), SEQ ID NO:2(NG-5(H3 HA)), SEQ ID NO:3(NG-6(H3 HA)), SEQ ID NO:4(NG-7(H3 HA)), and SEQ ID NO:5(NG-8(H3 HA)) are provided. The isolated polynucleotide sequences encoding the isolated full-length H3 HA antigens (polypeptides) are shown in SEQ ID NO:6-10, respectively. Furthermore, soluble forms of the H3 HA protein antigens are provided. In particular, isolated immunogenic soluble H3 HA viral protein antigens (polypeptides) having amino acid sequences shown in SEQ ID NO:11 (soluble NG-4 (H3 HA)), SEQ ID NO:12 (soluble NG-5 (H3 HA)), SEQ ID NO:13 (soluble NG-6 (H3 HA)), SEQ ID NO:14 (soluble NG-7 (H3 HA)), and SEQ ID NO:15 (soluble NG-8 (H3 HA)) are provided. Isolated polynucleotide sequences encoding the isolated soluble H3 HA antigens (polypeptides) are shown in SEQ ID NO:16-20, respectively. In some embodiments, the H3 HA polypeptides are recombinant and / or produced by recombinant. In one embodiment, a delivery agent, such as a delivery vector or construct, e.g., a mammalian expression vector or construct, or nucleic acid, e.g., mRNA, contains a polynucleotide sequence encoding the full-length HA viral protein antigen. In one embodiment, the soluble form of the isolated immunogenic HA viral protein antigen is used as an immunogen and / or in a composition / vaccine or a formulation thereof.

[0146] In another embodiment, the H3 immunogen sequences described herein are expressed in cells as polypeptides, proteins, or peptides. In one embodiment, the H3 immunogen is isolated and / or purified. In one embodiment, the immunogen is formulated for administration to a target requiring it. In one embodiment, the immunogen is administered to a target requiring it in an effective amount to induce an immune response. In one embodiment, the immune response induces a neutralizing antibody. In one embodiment, the immune response is prophylactic or therapeutic.

[0147] In one embodiment, a non-natural H3 virus immunogen (immunogen sequence), such as a vaccine, is provided, which induces a broad-spectrum reactive immune response in a subject after the immunogen is introduced, administered, or delivered to the subject. The route of introduction, administration, or delivery is not limited and may include, for example, intravenous, subcutaneous, intramuscular, or oral. The vaccine may be therapeutic (e.g., administered to a subject following symptoms of an H3 virus-induced illness (influenza)) or prophylactic (protective) (e.g., administered to a subject before the subject has or develops symptoms of an H3 virus-induced illness (influenza) or full-blown illness).

[0148] In one embodiment, the final amino acid sequence of an antigen, such as HA, is backtranslated and optimized for expression in mammalian cells. As will be recognized by those skilled in the art, nucleic acid sequence optimization includes codon optimization and RNA optimization (e.g., RNA stability) for sequence expression in mammalian cells.

[0149] In one embodiment, an isolated nucleic acid molecule (polynucleotide) is provided that contains a nucleotide sequence encoding a polypeptide or peptide antigen, for example, an H3 influenza HA polypeptide (or HA1 or HA2 polypeptide). In this embodiment, the polynucleotide is RNA, for example mRNA, or DNA. In a particular embodiment, the nucleotide sequence encoding the H3 HA polypeptide is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the polynucleotide encoding the HA polypeptide (or HA1 or HA2 polypeptide) sequence of the H3 HA polypeptide described in Example 1.

[0150] In other embodiments, a nucleotide sequence encoding an H3 influenza HA polypeptide (or HA1 or HA2 polypeptide) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the polynucleotide encoding the H3 HA polypeptide sequence described in Example 1 lacks a start codon encoding an N-terminal methionine.

[0151] A vector is provided comprising a nucleotide sequence encoding a non-naturally occurring broadly reactive polypeptide or peptide antigen, such as an H3 influenza HA polypeptide (or HA1 or HA2 polypeptide). In some embodiments, the vector comprises a nucleotide sequence encoding a polypeptide or peptide antigen, such as an influenza H3 HA polypeptide antigen, which is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the polynucleotide encoding the H3 HA polypeptide (or HA1 or HA2 polypeptide) sequence described in Example 1. In some embodiments, the vector further comprises a promoter functionally linked to the nucleotide sequence encoding the H3 HA polypeptide (or HA1 or HA2 polypeptide). In certain embodiments, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the nucleotide sequence of the vector is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the polynucleotide encoding the H3 HA polypeptide (or HA1 or HA2 polypeptide) sequence described in Example 1. In certain embodiments, the nucleotide sequence of the vector includes a polynucleotide encoding the H3 HA polypeptide (or HA1 or HA2 polypeptide) sequence described in Example 1. In embodiments, the nucleotide sequence is RNA, e.g., mRNA, or DNA. In embodiments, the vector is a prokaryotic or eukaryotic vector. In one embodiment, the vector is an expression vector, e.g., a eukaryotic (e.g., mammalian) expression vector. In another embodiment, the vector is a plasmid (prokaryotic or bacterial) vector. In yet another embodiment, the vector is a viral vector.

[0152] The vectors used to express H3 viral antigens, such as H3 viral proteins, such as HA proteins, as described herein may be any suitable expression vectors known and used in the art. The vectors may be, for example, mammalian expression vectors or viral vectors. In some embodiments, the vector is a pTR600 expression vector (U.S. Patent Application Publication No. 2002 / 0106798, incorporated herein by reference; Ross et al., 2000, Nat Immunol. 1(2):102-103; and Green et al., 2001, Vaccine 20:242-248).

[0153] Provided are non-natural polypeptide antigens derived from H3 influenza virus, such as H3 influenza HA polypeptide antigens, which are produced by transfecting host cells with an expression vector, as is known and used in the art, under conditions sufficient to enable the expression of HA polypeptides in cells. Isolated cells containing the vector are also provided. The H3 HA polypeptide antigens described herein can be isolated or purified from cells expressing the polypeptide antigen.

[0154] Non-natural broadly reactive H3 polypeptide antigens described herein, such as broadly reactive H3 influenza HA polypeptide immunogens, are also provided. In certain embodiments, the amino acid sequence of the polypeptide is at least 95% to 99% (including both ends) identical to the amino acid sequence of the HA polypeptide described in Example 1. In certain embodiments, the amino acid sequence of the H3 influenza HA polypeptide, which is at least 95% to 99% (including both ends) identical to the amino acid sequence of the HA polypeptide described in Example 1, lacks an N-terminal methionine residue. In certain embodiments, the amino acid sequence of the H3 influenza HA polypeptide is at least 95% to 99% (including both ends) identical to amino acids 1 to 566 of the H3 HA polypeptide described in Example 1.

[0155] In some embodiments, fusion proteins comprising the broadly reactive H3 virus polypeptide antigen described herein are also provided. In some embodiments, the H3 influenza HA polypeptide can be fused with any heterologous amino acid sequence to form a fusion protein. For example, H3 HA1 polypeptide and HA2 polypeptide can be generated independently and then fused together to generate an H3 HA polypeptide antigen containing, for example, 566 amino acids.

[0156] Virus-like particles (VLPs), particularly VLPs of H3 influenza, are also provided, comprising panepitope broad-spectrum reactive protein antigens, for example, the H3 influenza HA proteins described herein, for example, full-length polypeptide antigens of SEQ ID NO: 1-5, their soluble forms shown in SEQ ID NO: 11-15, polynucleotides encoding full-length H3 HA polypeptide antigens shown in SEQ ID NO: 6-10, or polynucleotide polypeptide antigens encoding soluble H3 HA shown in SEQ ID NO: 16-20, respectively, or their immunogenic portions. In certain embodiments, the HA protein of the VLP is at least 94% or 94%, at least 95% or 95%, at least 96% or 96%, at least 97% or 97%, at least 98% or 98%, at least 99% or 99%, or 100% identical to the H3 HA protein described in Example 1. A viral or influenza VLP may further contain any additional viral or influenza proteins necessary to form a viral particle. In certain embodiments, a viral or influenza VLP may further contain the influenza neuraminidase (NA) protein, the influenza matrix (M1) protein, or both.

[0157] VLPs of H3 influenza containing the H3 influenza HA polypeptide described herein are also provided, which are produced by transfecting host cells with a vector containing a polynucleotide encoding the H3 HA polypeptide. In one embodiment, the polynucleotide is RNA, e.g., mRNA, or DNA. In one embodiment, VLPs of H3 influenza containing the H3 influenza HA polypeptide described herein are also provided, which are produced by transfecting host cells with a vector encoding the H3 HA polypeptide, a vector encoding the influenza NA protein, and a vector encoding the influenza M1 protein, under conditions sufficient to allow the expression of the H3 HA, NA, and M1 proteins. Such VLPs containing sequences encoding SEQ ID NO: 1-5 or SEQ ID NO: 11-15) as described in Example 1 and used as immunogens produce antibodies with high hemagglutinin inhibitory (HAI) titers against various strains of H3 influenza virus, as observed in Figures 4A-4F, 5A-5F, and 6.

[0158] Plasmid (vector or nucleic acid / polynucleotide) collections are also intended. In certain embodiments, the plasmid collection includes a plasmid encoding influenza H3 NA, a plasmid encoding H3 influenza MA, and a plasmid encoding the broadly reactive H3 HA protein described herein. In some embodiments, the nucleotide sequence encoding the H3 influenza HA protein in the HA-encoding plasmid is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the polynucleotide encoding the H3 HA amino acid sequence described in Example 1. In some embodiments, the H3 HA antigen is a full-length or soluble polypeptide containing the amino acid sequence described herein. In some embodiments, the polynucleotide sequence described herein encodes a full-length or soluble H3 HA polypeptide. In some embodiments, the nucleotide sequence encodes a codon-optimized influenza H3 HA protein. In some embodiments, the nucleotide sequence encoding the codon-optimized H3 HA protein of the HA-encoding plasmid is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the polynucleotide encoding the H3 HA amino acid sequence described in Example 1.

[0159] In the context of this disclosure, “broadly reactive” or “broadly active” means that an H3 protein (e.g., an H3 HA protein sequence) is immunogenic and contains a diverse range of epitopes (antigenic determinants; panepitopetic) that are sufficient to treat a disease or infection and / or inhibit, neutralize, or prevent an infection caused by most or all H3 influenza viruses within a particular subtype or by associated viral lineages. In some embodiments, a broadly reactive H3 virus-derived protein antigen, e.g., an HA protein, can induce a protective immune response against most or all known H3 influenza virus isolates, such as about 80%, about 85%, about 90%, about 95%, or about 96%–99% of known H3 influenza virus isolates.

[0160] Compositions for administration and pharmaceutical compositions The following compositions are provided herein, comprising a broadly reactive, panepitopeous H3 influenza HA protein, or a fusion protein or VLP containing such a broadly reactive H3 influenza HA protein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient, or vehicle. In some embodiments, an adjuvant (e.g., a pharmacological or immunological agent that modifies or boosts the immune response to produce more long-lasting antibodies) is also used. For example, non-limiting adjuvants may be inorganic compounds, e.g., alum, aluminum hydroxide, or aluminum phosphate; minerals or paraffinic oils; squalene; squalene oil-in-water emulsion (ADDAVAX®; InvivoGen, San Diego, CA); surfactants, e.g., Quil A; plant saponins; Freund's complete or incomplete adjuvants; biological adjuvants (e.g., cytokines, e.g., IL-1, IL-2, or IL-12); bacterial products, e.g., dead Bordetella pertussis or toxoids; immunostimulatory oligonucleotides (e.g., CpG oligonucleotides); or cationic lipid nanoparticles (e.g., R-DOTAP; PDS Biotechnology Corporation, Florham Park, NJ).

[0161] Compositions and preparations (e.g., physiologically or pharmaceutically acceptable compositions) for parenteral administration, comprising non-natural, broadly reactive, panepitopeous H3 influenza HA polypeptides and H3 influenza virus-like particles (VLPs), include, but are not limited to, sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Non-limited examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil and canola oil), and organic esters for injection (e.g., ethyl oleate). Aqueous carriers include water, alcohol solutions / aqueous solutions, emulsions, or suspensions, including saline and buffering media. Parenteral vehicles include, for example, sodium chloride solutions, ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or non-volatile oils. Intravenous vehicles include, for example, fluids and nutritional supplements, and electrolyte replacement agents (e.g., those based on ringer's dextrose). Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, colorants, stabilizers, and inert gases, may also be present in such compositions and preparations.

[0162] Some compositions may be administered as pharmaceutically acceptable acids or base addition salts formed by reacting them with inorganic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, as well as organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reacting them with inorganic bases, such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases, such as mono, di, trialkyl and arylamines, and substituted ethanolamines.

[0163] Pharmaceutical compositions comprising a therapeutically effective amount of a non-naturally occurring broadly reactive panepitope H3 viral protein antigen, or VLP of H3 influenza, alone or in combination with a pharmaceutically acceptable carrier, are provided herein. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition may be sterile, and the formulation may be suitable for the mode of administration. The composition may also contain small amounts of wetting or emulsifying agents or pH buffers. The composition may be a liquid or aqueous solution, suspension, emulsion, dispersion, tablet, pill, capsule, powder, or sustained-release formulation. Liquid or aqueous compositions may be lyophilized and reconstituted with a solution or buffer before use. The composition may be formulated as a suppository using conventional binders and carriers, such as triglycerides. Oral formulations may contain standard carriers, such as pharmaceutically grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Any of the generally known pharmaceutical carriers, such as sterile saline or sesame oil, can be used. The medium may also include conventional pharmaceutical adjuncts, such as pharmaceutically acceptable salts, buffers, and preservatives for adjusting osmotic pressure. Other media that can be used in the described compositions and methods of administration are typically saline and sesame oil.

[0164] Methods of treatment, administration, and delivery A method is provided for treating a disease, condition, or infection caused by the H3 influenza virus, or a symptom thereof. The method comprises administering a therapeutically effective amount of a broadly reactive panepitope immunogen, or a pharmaceutical composition containing said immunogen, or a vaccine (e.g., a VLP vaccine) described herein to a subject (e.g., a mammal, particularly a human subject). One embodiment includes a method for treating a subject that is suffering from, at risk of suffering from, or susceptible to suffering from, a disease or infection caused by the H3 influenza virus, or a symptom thereof. The method comprises administering to a subject (e.g., a mammalian subject) a sufficient or therapeutic amount of an immunogenic composition or vaccine containing a non-natural broadly reactive panepitope H3 virus polypeptide antigen, e.g., HA polypeptide, or HA polypeptide VLP, to treat a disease, infection, or symptom thereof caused by the H3 influenza virus under conditions in which the disease, infection, and / or symptoms thereof are being treated.

[0165] In one embodiment, the method herein includes the step of administering an effective amount of a non-naturally occurring broadly reactive panepitope H3 virus polypeptide antigen, e.g., H3 virus HA polypeptide, or vaccine or composition described herein, or a vaccine or composition, to a subject (including a human subject identified as needing such treatment) in order to produce such an effect. The treatment method is appropriately administered to subjects, particularly humans, who have, are susceptible to, or are at risk of having, a disease, disorder, infection, or symptoms thereof, i.e., influenza, i.e., influenza. Identification of subjects needing such treatment may be based on the judgment of the subject or a healthcare professional and may be subjective (e.g., opinion) or objective (e.g., measurable by tests or diagnostic methods). Briefly, the determination of subjects needing treatment, or being at risk of or susceptible to treatment, may be made by any objective or subjective determination, including the opinion of the subject or healthcare provider, such as diagnostic tests (e.g., genetic tests, enzyme or protein marker assays), marker analyses, or family history. Non-natural, broadly reactive, panepitope H3 immunogens, such as the H3 HA polypeptide immunogens and vaccines described herein, may also be used to treat any other disorder that may involve infection or disease caused by the H3 influenza virus. The subjects receiving treatment may be non-human mammals, such as veterinary subjects, or human subjects (also referred to as “patients”).

[0166] Furthermore, preventive methods are provided for preventing or protecting against diseases or infections caused by H3 influenza viruses, or their symptoms. Such methods include, in particular, administering a therapeutically effective amount of a pharmaceutical composition, including an H3 immunogenic composition or vaccine (e.g., an H3 VLP vaccine) described herein, to a subject (e.g., a mammal, e.g., a human) prior to infection of the subject or prior to the onset of a disease, e.g., an H3 virus-related disease.

[0167] In another embodiment, a method is provided for monitoring the progression of an H3 virus infection or disease caused by an H3 virus, or for monitoring the treatment of an H3 infection or disease. The method includes determining the level or diagnostic measurement (e.g., a screening assay or detection assay) of a diagnostic marker or biomarker (e.g., an H3 virus protein, e.g., H3 HA) in a subject who is suffering from or susceptible to an infection, disease, or symptoms associated with an H3 influenza virus, and who has been administered a sufficient amount (e.g., a therapeutic dose) of the non-naturally occurring broadly reactive panepitope H3 virus HA protein or the vaccine described herein to treat the infection, disease, or symptoms thereof. The level or amount of the marker or biomarker (e.g., protein) determined by this method can be compared to known levels of the marker or biomarker in samples from healthy normal controls; pre-infection or pre-disease samples of the subject; or other suffering / infected / sick patients to confirm the disease status of the subject being treated. For monitoring, a second level or amount of a marker or biomarker in a sample obtained from a subject can be determined at a later time than the determination of the first level or amount, and the two levels or amounts of the marker or biomarker can be compared to monitor the course of the disease or infection, or the effectiveness of the therapy / treatment. In certain embodiments, the pre-treatment level of the marker or biomarker in a subject (e.g., a sample obtained from the subject) can be determined before initiating the described treatment; and then this pre-treatment level of the marker or biomarker can be compared to the level of the marker or biomarker in the subject after initiating the treatment and / or during the course of the treatment to determine (monitor the effectiveness of the disease treatment).

[0168] Isolated non-natural H3 virus polypeptide antigens, such as the H3 virus HA polypeptides described herein, and VLPs containing H3 HA polypeptides, or compositions thereof, can be administered to a subject by any of the routes commonly used to introduce recombinant proteins, compositions containing recombinant proteins, or recombinant viruses into a subject. Routes and methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intrathecal, parenteral (e.g., intravenous (IV) or subcutaneous (SC)), vaginal, rectal, nasal, inhalation, intraocular, intracranial, or oral. Parenteral administration, such as subcutaneous, intravenous, or intramuscular administration, is generally achieved by injection (immunization). Injectable preparations can be prepared in conventional forms and formulations, either as liquid solutions or suspensions, as solid forms suitable for liquid solutions or suspensions before injection (e.g., lyophilized forms), or as emulsions. Injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets. The administration may be systemic or local.

[0169] Isolated non-natural H3 virus polypeptides, such as the H3 virus HA polypeptide and H3 HA polypeptide described herein, VLPs or compositions thereof, can be administered in any suitable manner, for example, together with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is determined to some extent by the specific immunogen or composition administered and by the specific method used to administer the composition. Thus, non-natural broadly reactive panepitope H3 virus polypeptide antigens, such as pharmaceutically compositions containing H3 virus HA polypeptide and VLPs or compositions thereof, can be prepared using a wide variety of suitable and physiologically and pharmaceutically acceptable formulations.

[0170] Administration of broadly reactive, panepitopetic H3 virus polypeptide antigens, such as H3 virus HA polypeptides and VLPs containing HA polypeptides, or compositions thereof, can be achieved by single or multiple doses. The dose administered to a subject should be sufficient to induce a beneficial therapeutic response over time in the subject, for example, to inhibit, block, reduce, improve, protect against, or prevent disease or infection caused by H3 influenza virus. The required dose is considered to vary from subject to subject depending on the species, age, weight, and general condition of the subject, the severity of the infection being treated, the specific composition used, and the mode of administration. The appropriate dose can be determined by a person skilled in the art, such as a clinician or practitioner, using only routine experiments.

[0171] Further methods are provided for inducing an immune response against the H3 influenza virus in a subject by administering to the subject a non-naturally occurring broadly reactive panepitope H3 influenza HA protein, a fusion protein containing the H3 influenza HA protein, a VLP containing the influenza HA protein, or any of the compositions thereof described herein. In some embodiments, the H3 HA protein, HA fusion protein, or VLP can be administered using any suitable route of administration, such as by intramuscular injection. In some embodiments, the H3 HA protein, fusion protein, or VLP is administered as a composition comprising a pharmaceutically acceptable carrier. In some embodiments, the composition comprises, for example, alum, a Freund's complete or incomplete adjuvant, a biological adjuvant, or an immunostimulatory oligonucleotide (e.g., CpG oligonucleotide), ADDAVAX®, or an adjuvant selected from R-DOTAP, as described herein. In other embodiments, the composition can be administered in combination with another therapeutic agent or molecule.

[0172] A method for conferring immunity to an infection or disease or symptoms thereof caused by an H3 influenza virus is also provided, comprising the steps of administering a VLP containing a non-natural panepitope broadly reactive H3 influenza HA protein as described herein to the subject, or administering an immunogenic composition thereof. In some embodiments of the method, the composition further comprises a pharmaceutically acceptable carrier and / or adjuvant. For example, the adjuvant may be alum, a Freund's complete or incomplete adjuvant, a biological adjuvant, or an immunostimulatory oligonucleotide (e.g., CpG oligonucleotide). In one embodiment, the H3 VLP (or a composition thereof) is administered intramuscularly.

[0173] In some embodiments of methods for inducing an immune response to or immunizing a subject against a viral infection or disease caused by or associated with the H3 influenza virus, the subject is administered at least 1 μg of VLP containing non-naturally occurring broadly reactive pan-epitope H3 virus HA protein, for example, at least 5 μg, at least 10 μg, at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 40 μg, or at least 50 μg of VLP containing non-naturally occurring broadly reactive pan-epitope H3 virus HA protein, for example, about 1 to about 50 μg or about 1 to about 25 μg of VLP containing H3 HA protein. In specific cases, the subject is administered about 5 to about 20 μg of VLP or about 10 to about 15 μg of VLP. In certain further non-limiting cases, the subject is administered about 15 μg of VLP. However, those skilled in the art can determine a therapeutically effective dose of VLP (e.g., a dose that provides a therapeutic effect or protection from H3 influenza virus infection) suitable for administration to subjects in need of treatment for or protection from viral infection.

[0174] Administration of VLPs containing the non-naturally occurring, broadly reactive, pan-epitope H3 HA protein described herein is expected to induce high titers of neutralizing antibodies against a diverse repertoire of epitope determinants on the H3 HA protein immunogen, and protective levels of H3 HA inhibitory (HAI) antibodies against several representative H3 isolates, as well as complete protection from lethal attacks by H3 viruses and / or associated H3 virus types. VLPs containing the non-naturally occurring, broadly reactive, pan-epitope H3 influenza HA protein described herein induce a broader immune response (e.g., inducing neutralizing antibodies against a wider range of H3 virus isolates compared to the immune response induced by a polyvalent H3 influenza virus vaccine).

[0175] Adjuvant and combination therapy H3 virus immunogens or immunogenic compositions containing H3 HA protein antigen (e.g., H3 HA antigen) or H3 virus VLPs described herein can be administered alone or in combination with other therapeutic agents to enhance antigenicity or immunogenicity in a subject, i.e., to increase the immune response, e.g., the induction of specific antibodies. For example, H3 HA protein antigen or H3 influenza VLPs can be administered or formulated with adjuvants, e.g., alum, Freund's incomplete adjuvant, biological adjuvants, immunostimulatory oligonucleotides (e.g., CpG oligonucleotides), ADDAVAX™ (squalene-based oil-in-water nanoemulsion adjuvant), or R-DOTAP (cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane or its lipid nanoparticles).

[0176] One or more cytokines, such as interleukin-1 (IL-2), interleukin-6 (IL-6), interleukin-12 (IL-12), protein memory T cell attractant (RANTES) which is regulated by activation and expressed and secreted by normal T cells, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-α), or interferon-gamma (IFN-γ); one or more growth factors, such as GM-CSF or granulocyte colony-stimulating factor (G-CSF); one or more molecules, such as TNF ligand superfamily member 4 ligand (OX40L), or type 2 transmembrane glycoprotein receptor (4-1BBL) belonging to the TNF superfamily, or combinations of these molecules, can be used as biological adjuvants if desired or approved (e.g., Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze See also: et al., 2000, Cancer J. Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can be administered systemically (or topically) to the target.

[0177] Several methods for inducing cellular responses both in vitro and in vivo are known and implemented in the art. Lipids have been identified as active agents that can assist in priming cytotoxic lymphocytes (CTLs) in vivo against various antigens. For example, palmitic acid residues can be attached to the alpha and epsilon amino groups of lysine residues and then linked to immunogenic peptides (e.g., via one or more linking residues, e.g., glycine, glycine-glycine, serine, serine-serine, etc.) (U.S. Patent No. 5,662,907). The lipid-modified peptides can then be injected directly in micelle form, incorporated into liposomes, or emulsified in an adjuvant. As another example, Escherichia coli lipoproteins, such as tripalmitoyl-S-glycerylcysteine ​​glyceryl-serine, can be used to prime tumor-specific CTLs when covalently bound to a suitable peptide (see, e.g., Deres et al., 1989, Nature 342:561). Furthermore, induction of neutralizing antibodies can also be primed with the same molecule conjugated to a peptide that presents a suitable epitope, and the two compositions can be combined to induce both humoral and cellular responses when such a combination is considered desirable.

[0178] Treatment methods may include the administration of VLPs containing the non-naturally occurring broadly reactive pan-epitope H3 HA immunogenic protein described herein, but those skilled in the art will recognize that the non-naturally occurring broadly reactive pan-epitope H3 influenza HA protein itself (in the absence of viral particles) can be administered to subjects in need to induce an immune response, either as a component of a pharmaceutically acceptable composition or as a fusion protein.

[0179] kit For example, kits are also provided that include a described non-natural, broadly reactive, pan-epitope H3 virus immunogen or vaccine, or a pharmaceutically acceptable composition comprising the immunogen and a pharmaceutically acceptable carrier, diluent, or excipient, for administration to a subject. The immunogen may be in the form of an H3 virus protein (polypeptide) or polynucleotide (H3 virus polypeptide, e.g., polynucleotide encoding the H3N2 HA protein), e.g., an H3 HA polypeptide immunogen with SEQ ID NO: 1-5 or SEQ ID NO: 11-15, or a coding polynucleotide with SEQ ID NO: 6-10 or SEQ ID NO: 16-20, as described herein. In some embodiments, the coding polynucleotide is RNA, e.g., mRNA, or DNA. Kits are also provided that include one or more plasmids or plasmid collections, as described herein. As will be recognized by those skilled in the art, such kits may optionally include one or more containers for containing the immunogen, vaccine, or composition, diluent, or excipient, and instructions for use.

[0180] Recombinant polypeptide expression The aspects and embodiments described herein, unless otherwise indicated, utilize techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the scope of those skilled in the art. Such techniques are well described in the literature, for example, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); and “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the described polypeptides and coding polynucleotides and can therefore be considered in the preparation and implementation of the described embodiments. Techniques particularly useful for specific embodiments are discussed in the following sections.

[0181] The following examples are provided to give a complete disclosure and explanation of the methods for preparing and using the polypeptides and therapeutic methods described herein, and are not intended to limit the scope of the embodiments described. [Examples]

[0182] The following examples are provided to illustrate certain features and / or embodiments. The examples should not be construed as limiting the disclosure to any particular features or embodiments described.

[0183] Example 1 H3 influenza virus HA polypeptide and coding polynucleotide In this specification, the amino acid sequences of five representative hemagglutinin (HA) polypeptide antigens (proteins) of the H3 influenza A virus lineage, referred to herein as NG-4, NG-5, NG-6, NG-7, and NG-8, are provided. These act as broadly reactive immunogens that, when administered to a target, e.g., a mammalian target, a human target, or a patient, induce an immune response, e.g., antibody production, against the H3 virus and H3 virus HA protein. The amino acid sequences of the NG-4, NG-5, NG-6, NG-7, and NG-8 polypeptides were generated by a next-generation computationally optimized broadly reactive ("COBRA") HA vaccine method and contain antigenic determinants representative of H3 influenza virus HA polypeptides present during the period of 2018–2021 (e.g., the seasonal influenza season) (Figures 1A and 1B). For example, computationally optimized broad-spectrum reactive antigen (COBRA) methodologies and next-generation COBRA technologies and methods align and analyze multiple consensus sequences from thousands of viral isolates to generate final immunogens that stimulate cross-protective immune responses against a large panel of lineages of each subtype (see, e.g., Sautto, GA et al., Virology, 2018. 15(1):1-12; Huang, Y., et al., Vaccines, 2021. 9(7):793; WO 2020 / 014675; and WO 2020 / 014673).

[0184] The wild-type HA sequences that served as starting material for the design of the NG-4, NG-5, NG-6, NG-7, and NG-8 H3 HA sequences were obtained from the influenza sequence database, i.e., the GISAID (Global Initiative on Sharing Avian Influenza Flu Data) initiative. A phylogenetic tree illustrating the relationship between the NG-4, NG-5, NG-6, NG-7, and NG-8 H3 HA sequences and previous strains included in past and present seasonal commercial influenza vaccines is shown in Figure 2.

[0185] Isolated NG-4, NG-5, NG-6, NG-7, and NG-8 H3 HA polypeptides are provided for use as immunogens administered to subjects / recipients to induce a potent immune response, e.g., antibody production, against HA polypeptide antigens of other H3 influenza viruses. In one embodiment, these immunogens administered to subjects / recipients can induce a potent immune response, e.g., antibody production, against HA polypeptide antigens of other influenza virus types or subtypes. In another embodiment, the isolated NG-4, NG-5, NG-6, NG-7, and NG-8 H3 HA polypeptides are recombinant and / or produced by recombinant processes.

[0186] The full-length HA polypeptide is 566 amino acids long. The amino acid sequences of the full-length NG-4 (SEQ ID NO: 1), NG-5 (SEQ ID NO: 2), NG-6 (SEQ ID NO: 3), NG-7 (SEQ ID NO: 4), and NG-8 (SEQ ID NO: 5) polypeptides, as well as the soluble forms of these polypeptides (i.e., SEQ ID NO: 11-15, respectively), are shown herein. In some embodiments, the HA protein antigen or a fragment thereof may have at least about 85% or 85% amino acid sequence identity with a representative influenza A virus HA protein or a functional fragment thereof, for example, the amino acid sequences of the influenza virus HA proteins NG-4 to NG-8 described herein, or at least about 90%, 95%, 98%, 99%, or 90%, 95%, 98%, 99%, or more.

[0187] The amino acid sequence (SEQ ID NO:1) of the full-length H3 HA protein (polypeptide), NG-4, is provided below: NG-4: The above NG-4 H3 HA polypeptide sequence (TIFF2026509251000003.tif43144) was designed from wild-type HA sequences obtained from the GISAID database from May 1, 2018 to September 30, 2020.

[0188] The nucleic acid sequence encoding the full-length NG-4 HA immunogenic protein antigen is provided below: Nucleic acid sequence encoding full-length NG-4: TIFF2026509251000004.tif122145

[0189] The amino acid sequence (SEQ ID NO:2) of the full-length H3 HA protein (polypeptide), NG-5, is provided below: NG-5: The above NG-5 H3 HA sequence (TIFF2026509251000005.tif43144) was designed from wild-type HA sequences obtained from the GISAID database from October 1, 2018 to April 30, 2021.

[0190] The nucleic acid sequence encoding the full-length NG-5 HA immunogenic protein antigen is provided below: Nucleic acid sequence encoding full-length NG-5: TIFF2026509251000006.tif122145

[0191] The amino acid sequence (SEQ ID NO:3) of the full-length H3 HA protein (polypeptide), NG-6, is provided below: NG-6: The above NG-6 H3 HA sequence (TIFF2026509251000007.tif43144) was designed from wild-type HA sequences obtained from the GISAID database from May 1, 2019 to September 30, 2021.

[0192] The nucleic acid sequence encoding the full-length NG-6 HA immunogenic protein antigen is provided below: Nucleic acid sequence encoding full-length NG-6: TIFF2026509251000008.tif122145

[0193] The amino acid sequence (SEQ ID NO:4) of the full-length H3 HA protein (polypeptide), NG-7, is provided below: NG-7: The above NG-7 H3 HA sequence (TIFF2026509251000009.tif43144) was designed from wild-type HA sequences obtained from the GISAID database from May 1, 2019 to April 30, 2022. Amino acid substitutions were performed at 176N to remove the glycosyl moiety.

[0194] The nucleic acid sequence encoding the full-length NG-7 HA immunogenic protein antigen is provided below: Nucleic acid sequence encoding full-length NG-7: TIFF2026509251000010.tif122145

[0195] The amino acid sequence (SEQ ID NO:5) of the full-length H3 HA protein (polypeptide), NG-8, is provided below: NG-8: The above NG-8 H3 HA sequence (TIFF2026509251000011.tif43144) was designed from wild-type HA sequences obtained from the GISAID database from May 1, 2019 to April 30, 2022. Amino acid substitutions were performed at 176T to add glycosylation sites.

[0196] The nucleic acid sequence encoding the full-length NG-8 HA immunogenic protein antigen is provided below: Nucleic acid sequence encoding full-length NG-8: TIFF2026509251000012.tif122145

[0197] The amino acid sequence (SEQ ID NO:11) of the soluble form of the NG-4 HA immunogenic protein antigen is provided below: NG-4-Soluble HA TIFF2026509251000013.tif43144

[0198] The nucleic acid sequence encoding the soluble form of the NG-4 HA immunogenic protein antigen is provided below: Nucleic acid sequences encoding soluble NG-4: TIFF2026509251000014.tif127145

[0199] The amino acid sequence (SEQ ID NO:12) of the soluble form of the NG-5 HA immunogenic protein antigen is provided below: NG-5-Soluble HA TIFF2026509251000015.tif43144

[0200] The nucleic acid sequence encoding the soluble form of the NG-5 HA immunogenic protein antigen is provided below: Nucleic acid sequences encoding soluble NG-5: TIFF2026509251000016.tif127145

[0201] The amino acid sequence (SEQ ID NO:13) of the soluble form of the NG-6 HA immunogenic protein antigen is provided below: NG-6-Soluble HA TIFF2026509251000017.tif43144

[0202] The nucleic acid sequence encoding the soluble form of the NG-6 HA immunogenic protein antigen is provided below: Nucleic acid sequences encoding soluble NG-6: TIFF2026509251000018.tif127145

[0203] The amino acid sequence (SEQ ID NO:14) of the soluble form of the NG-7 HA immunogenic protein antigen is provided below: NG-7-Soluble HA TIFF2026509251000019.tif43144

[0204] The nucleic acid sequence encoding the soluble form of the NG-7 HA immunogenic protein antigen is provided below: Nucleic acid sequences encoding soluble NG-7: TIFF2026509251000020.tif127145

[0205] The amino acid sequence (SEQ ID NO:15) of the soluble form of the NG-8 HA immunogenic protein antigen is provided below: NG-8-Soluble HA TIFF2026509251000021.tif43144

[0206] The nucleic acid sequence encoding the soluble form of the NG-8 HA immunogenic protein antigen is provided below: Nucleic acid sequences encoding soluble NG-8: TIFF2026509251000022.tif127145

[0207] The soluble H3 HA amino acid and coding polynucleotide sequences described above may include amino acid additions to the carboxyl (COOH) terminus of the HA polypeptide sequence. As an unrestricted example, and not intended to be theoretically constrained, terminal 50-55 amino acids (or the coding nucleic acid sequence in its polynucleotide sequence), which may include a His tag (e.g., six consecutive histidine (H) amino acids), may be added to or included in the carboxyl (COOH) terminus of the NG-4, NG-5, NG-6, NG-7, or NG-8 HA soluble protein. In one embodiment, such carboxyl-terminal amino acids, or their coding nucleic acid sequences, may not be present in the carboxyl terminus or its coding polynucleotide of the soluble form of the HA protein. In one embodiment, the soluble H3 HA amino acid sequences described above, without the addition of carboxyl-terminal amino acids, for example, without the carboxyl-terminal 50-55 amino acids, retain their immunogenicity and function as an immunogen. In one embodiment, the 50, 51, 52, 53, 54, or 55 amino acids at the carboxyl terminus of the above-mentioned soluble amino acid sequence may be absent without affecting the immunogenicity or immunogenic function of the HA polypeptide antigen.

[0208] In some embodiments, for example, the above amino acid sequences have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of the above HA polypeptides for use as an immunogen. Nucleic acid sequences encoding these polypeptides can be used to generate virus-like particles (VLPs) containing the H3 protein antigen, which are used as immunogens / vaccines to generate neutralizing antibodies in an immunized subject.

[0209] Example 2 Research design and outline for NG-4~NG-8 H3N2 influenza virus HA 130 DBA / 2J mice were randomly divided into 13 groups (10 mice / group) and intramuscularly immunized with 50 μL of phosphate-buffered saline (PBS) containing 3 μg of NG-4~NG-8 H3 recombinant hemagglutinin (rHA) protein, either immunized with ADDAVAX® (squalene oil-in-water emulsion) (groups 1-6) or R-DOTAP (cationic lipid nanoparticles) (groups 7-12). Each animal group was immunized with a monovalent formulation of H3 NG-4, NG-5, NG-6, NG-7, or NG-8 rHA protein, and one group was immunized with a mixture of NG-7 / NG-8 H3 rHA protein containing 3 μg of each rHA protein (groups 6 and 12). As a control, additional groups of mice were immunized with a placebo vaccine ("mock") containing only PBS without adjuvants. Table 1 below shows the experimental plan.

[0210] [Table 1]

[0211] On days 0, 28, and 56, the immunogen (vaccine) was administered in a homogeneous prime-boost-boost regimen (Figure 3). Blood was collected from each animal 14 days after each vaccination (days 14, 42, and 70) to assess antibody content against a panel of historical H3N2 vaccine strains. On day 70, all animals in the study were humanely euthanized according to the method approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia in the approved animal use protocol (No. A2021-06-016-Y2-A6).

[0212] Example 3 Antibody titers in a hemagglutination inhibition assay (HAI) at day 70 against historical H3N2 vaccine strain viruses in mice immunized with H3 recombinant HA (rHA) immunogen (vaccine) containing ADDAVAX® adjuvant. Serum collected at day 70 from 60 DBA / 2J mice that had been vaccinated three times with the H3 recombinant HA (rHA) polypeptide immunogen (vaccine) formulated with the ADDAVAX® (squalene oil-in-water emulsion) adjuvant described herein was evaluated by HAI assay against a panel of eight historical H3N2 vaccine virus strains. Animals were immunized (vaccinated) with either NG-4 (Figure 4A), NG-5 (Figure 4B), NG-6 (Figure 4C), NG-7 (Figure 4D), NG-8 (Figure 4E), or NG-7+NG-8 (Figure 4F) polypeptide immunogen and adjuvant. The H3N2 influenza viruses (X axis) in the historical vaccine lineage panel included: A / Hong Kong 4801 / 2014 (HK / 14) H3N2 clade 3c.2a, A / Singapore / IFNIMH-16-0019 / 2016 (Sing / 16) H3N2 clade 3c.2a1, A / Kansas / 14 / 2017 (Kan / 17) H3N2 clade 3c.3a, A / Switzerland / 8060 / 2017 (Switz / 1 7) H3N2 clade 3c.2a2, A / South Australia / 34 / 2019 (SA / 19) H3N2 clade 3c.2a1b.2, A / Hong Kong / 2671 / 2019 (HK / 19) H3N2 clade 3c.2a1b.1b, A / Tasmania / 503 / 2020 (Tas / 20) H3N2 clade 3c.2a1b.2a.1, and A / Darwin / 6 / 2021 (Dar / 21) H3N2 clade 3c.2a1b.2a.2. Antibody titers are shown as the standard deviation from the mean of individual Log2 HAI titers (Y axis) + / -. The dotted line below the graph represents the protective HAI titer of 1:40, and the dotted line above represents the HAI titer of 1:80. The results demonstrate that animals immunized with NG-4~NG-8 H3 rHA immunogen (and ADDAVAX® adjuvant) produced antibodies and, as evaluated by hemagglutination inhibition assay (HAI), possessed antibody titers that were reactive against historical H3N2 vaccine strains of viruses (Figures 4A~4F and 6).

[0213] Example 4 Antibody titers in a hemagglutination inhibition assay (HAI) at day 70 against historical H3N2 vaccine strain viruses in mice immunized with an H3 rHA immunogen (vaccine) containing R-DOTAP adjuvant. Serum collected at day 70 from 60 DBA / 2J mice that had been vaccinated three times with the H3 rHA polypeptide immunogen (vaccine) formulated with the R-DOTAP (cationic lipid nanoparticle) adjuvant described herein was evaluated by HAI assay against a panel of eight historical H3N2 vaccine virus strains. Animals were immunized (vaccinated) with either the NG-4 (Figure 5A), NG-5 (Figure 5B), NG-6 (Figure 5C), NG-7 (Figure 5D), NG-8 (Figure 5E), or NG-7+NG-8 (Figure 5F) polypeptide immunogen and adjuvant. The H3N2 influenza viruses (X axis) in the historical vaccine lineage panel included: A / Hong Kong 4801 / 2014 (HK / 14) H3N2 clade 3c.2a, A / Singapore / IFNIMH-16-0019 / 2016 (Sing / 16) H3N2 clade 3c.2a1, A / Kansas / 14 / 2017 (Kan / 17) H3N2 clade 3c.3a, A / Switzerland / 8060 / 2017 (Switz / 1 7) H3N2 clade 3c.2a2, A / South Australia / 34 / 2019 (SA / 19) H3N2 clade 3c.2a1b.2, A / Hong Kong / 2671 / 2019 (HK / 19) H3N2 clade 3c.2a1b.1b, A / Tasmania / 503 / 2020 (Tas / 20) H3N2 clade 3c.2a1b.2a.1, and A / Darwin / 6 / 2021 (Dar / 21) H3N2 clade 3c.2a1b.2a.2. Antibody titers are shown as the standard deviation from the mean of individual Log2 HAI titers (Y axis) + / -. The dotted line below the graph represents the protective HAI titer of 1:40, and the dotted line above represents the HAI titer of 1:80. The results demonstrate that animals immunized with NG-4~NG-8 H3 rHA immunogen (and R-DOTAP adjuvant) produced antibodies and, as evaluated by hemagglutination inhibition assay (HAI), possessed antibody titers that were reactive against historical H3N2 vaccine strains of viruses (Figures 5A~5F and 6).

[0214] Example 5 Hemagglutination inhibition (HAI) assay We evaluated functional antibodies against viral hemagglutinin (HA) that can inhibit viral agglutination to guinea pig erythrocytes (red blood cells (RBCs)). To inactivate nonspecific inhibitors, serum samples were treated with receptor-destroying enzymes (RDEs) (Denka Seiken, Co., Japan) prior to the test. Briefly, 3 parts RDE were added to 1 part serum and incubated overnight at 37°C. The RDEs were inactivated by incubation at 56°C for 30 minutes. Individual RDE-treated serum samples were then diluted in a series of 2x serial dilutions in v-bottom microtiter plates (Thermo Fisher, Waltham, PA, USA). Equivalent volumes of each A(H3N2) virus, adjusted to approximately 8 hemagglutination units (HAUs) / 50 μl in the presence of 20 nM oseltamivir carboxylate, were added to each well. The plate was covered and incubated at room temperature for 30 minutes, then 0.75% guinea pig erythrocytes (Lampire Biologicals, Pipersville, PA, USA) suspended in PBS were added. Before use, the erythrocytes (RBCs) were washed twice with PBS, stored at 4°C, and used within 24 hours of preparation. The plate was mixed by gentle agitation, covered, and the RBCs were allowed to settle at room temperature for 1 hour.

[0215] Subsequently, the HAI titer was determined by reciprocal dilution of the last well containing non-aggregated RBCs. Positive and negative serum controls were included in each plate. All mice were "seronegative" (HAI ≤ 1:10) for pre-existing antibodies against human influenza virus prior to immunization (vaccination). For the studies described in the above examples, "serological protection" is defined by the WHO and the European Committee for Medicinal Products to evaluate influenza vaccines as an HAI titer ≥ 1:40.

[0216] Example 6 Virus-like particle (vaccine) preparation Mammalian 293T cells were transfected with each of three mammalian expression plasmids expressing one of the following: influenza neuraminidase (A / Mallard / Alberta / 24 / 01, H7N3), HIV p55 Gag sequence, or broadly reactive HA expression plasmid (e.g., using the method described above, containing sequences encoding HA immunogens for NG-4, NG-5, NG-6, NG-7, and NG-8 (see, e.g., U.S. Patent Application Publication No. US 2015 / 0030628)). After incubation at 37°C for 72 hours, supernatant from transiently transfected cells was collected, centrifuged to remove cell debris, and filtered through a 0.22 μm pore membrane. Mammalian virus-like particles (VLPs) were purified and ultracentrifuged at 135,000 × g for 4 hours at 4°C on a 20% glycerol cushion. The VLPs were precipitated. The VLPs were resuspended in phosphate-buffered saline (PBS), and the total protein concentration was evaluated using a conventional bicinchoninate assay (BCA). The hemagglutination activity of each VLP preparation was determined by adding an equal volume of turkey or guinea pig erythrocytes (RBCs) to a V-bottom 96-well plate and incubating them with serially diluted VLPs at room temperature (RT) for 30 minutes. The highest dilution of VLP that resulted in complete agglutination of RBCs was considered the endpoint HA titer.

[0217] Example 7 Determination of HA content by enzyme-linked immunosorbent assay (ELISA) High affinity 96-well flat-bottom ELISA plates were coated with 5–10 μg of VLP total protein, and serial dilutions of recombinant H3 antigen (3006_H3_Vc, Protein Sciences, Meriden, CT) in ELISA carbonate (50 mM carbonate, pH 9.5) were added to the wells. The plates were incubated overnight at 4°C on a rocker. The following morning, the plates were washed in PBS (PBST) containing 0.05% Tween-20, and nonspecific epitopes were blocked by RT for 1 hour with 1% bovine serum albumin (BSA) in PBST solution. The buffer was removed, and the stem-specific group 2 antibody CR8020 (Tharakaraman, K. et al., 2014, Cell Host & Microbe, Vol. 15, pp. 644-651; Ekiert, DC et al., 2012, Science, 333(6044):843-850; Creative Biolabs, Shirley, NY) was added to the plate, followed by incubation at 37°C for 1 hour. The plate was washed, and then probed with goat anti-human IgG horseradish peroxidase conjugate secondary antibody (2040-05, Southern Biotech, Birmingham, AL) at 37°C for 1 hour.

[0218] The plates were washed. Freshly prepared o-phenylenediamine dihydrochloride (OPD) (P8287, Sigma, City, State, USA) substrate in citrate buffer (P4922, Sigma) was then added to the wells, followed by 1N H2SO4 stop reagent. The plates were read using a microplate reader (Powerwave XS, Biotek, Winooski, VT) at 492 nm absorbance. Background signals were subtracted from the negative wells. Linear regression calibration analysis was performed using recombinant standard antigens of known concentrations to estimate the HA content in the VLP lot.

[0219] Example 8 Studies in mice and ferrets Research using mice DBA / 2J mice (female mice (Mus musculus), 6-8 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME, USA), housed in a micro-isolator unit, and given free access to food and water. The animals were reared according to the University of Georgia Research Animal Resources guidelines for laboratory animals. All procedures were reviewed and approved by the Animal Experimentation Committee (IACUC). Based on HA content quantified by ELISA, mice in each treatment group were vaccinated with purified virus-like particles (VLPs) (3.0 μg / mouse), and the VLP immunogen (vaccine) was delivered to the animals via intramuscular injection at week 0. The animals were boosted at weeks 4 and 8 with the same dose of the same immunogen (vaccine). Each dose of vaccine was formulated with an adjuvant. For squalene-based adjuvants (e.g., ADDAVAX®), the final concentration after 1:1 mixing with VLP was 2.5% squalene. Blood samples were collected subchinnally via the cheek at appropriate times after each vaccination and transferred to microcentrifuge tubes. The tubes were centrifuged at 10,000 rpm for 10 minutes. Serum samples were removed and frozen at -20°C ± 5°C.

[0220] Research in ferrets Purchase influenza-naive and deodorized Fitch ferrets (ferret (Mustela putorius faro), female, 6-12 months old) from Marshall Farms (Sayre, Pa., USA). House the ferrets in pairs in stainless steel cages (Shor-line, Kansas City, Kans., USA) containing Sani-chips laboratory animal bedding (PJ Murphy Forest Products, Montville, NJ, USA). Provide the ferrets with Teklad Global Ferret Diet (Harlan Teklad, Madison, Wis., USA) and fresh water free of charge.

[0221] The purified VLP was diluted in PBS, pH 7.2 to obtain the final concentration. Based on the HA content determined by densitometry assay, ferrets were immunized (vaccinated) by intramuscular injection of 15 μg of purified VLP in the quadriceps muscle at a volume of 0.25 ml during week 0, and then boosted with the same dose at a predetermined week following. The vaccine was stored at -80°C before use and formulated immediately before use with an adjuvant, for example, non-limitingly, IMJECT® Alum Adjuvant (Pierce Biotechnology, Rockford, IL USA), ADDAVAX®, or R-DOTAP. Animals were monitored weekly during the vaccination regimen for adverse events, such as weight loss, body temperature, loss of activity, nasal discharge, sneezing, and diarrhea. Prior to vaccination, animals were confirmed to be seronegative for circulating influenza A (e.g., H3N2 or H1N1) and influenza B viruses by HAI assay. 14–21 days after each vaccination, blood was collected from anesthetized ferrets via the anterior vena cava and transferred to a microcentrifuge tube. The tube was centrifuged; serum was removed and frozen at -20±5°C.

[0222] Example 9 Design and production of immunogens / vaccines The recombinant influenza HA proteins described herein were generated using computationally optimized broad-spectrum reactive antigen (COBRA) methodologies and next-generation COBRA methodologies. The recombinant influenza HA protein antigens generated and used as described herein are also referred to as “COBRA antigens” or “recombinant COBRA antigens.” The design and generation of COBRA influenza antigens are described (Huang, Y. et al., Vaccines, 2021. 9(7): p. 793; Skarlupka, AL et al. Journal of Virology, 2021. 95(17): p. e00759-21; WO 2020 / 014675; and WO 2020 / 014673). Briefly, full-length wild-type (WT) HA sequences were downloaded for each subtype from the Global Organization for the Sharing of Avian Influenza Information (GISAID). The sequences were aligned, and based on percent similarity, primary consensus sequences were created from clusters of WT sequences. We created a secondary consensus sequence from the primary sequence and continued this multi-layered consensus-building method until we obtained the final COBRA sequence.

[0223] Other embodiments From the above description, it will be clear that the embodiments described herein can be modified and altered to suit various uses and conditions. Such embodiments are also within the scope of the following claims.

[0224] Any description of a list of elements in any definition of a variable in this specification includes the definition of that variable as any single element or as a combination (or partial combination) of the enumerated elements. Any description of an aspect in this specification includes any single aspect or as a combination of any other aspect or parts thereof.

[0225] All patents and publications referenced herein are incorporated herein by reference to the same extent as each individual patent and publication is specifically and individually indicated as being incorporated by reference.

Claims

1. (i) an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or (ii) an amino acid sequence selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15) or an isolated non-natural hemagglutinin (HA) polypeptide antigen or antibody-binding moiety of H3 influenza virus (H3 virus) having at least 95% sequence identity with that amino acid sequence.

2. (i) an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or (ii) an amino acid sequence having at least 98% sequence identity with an amino acid sequence selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15), an isolated non-natural hemagglutinin (HA) polypeptide antigen or antibody-binding moiety of H3 influenza virus (H3 virus) according to claim 1.

3. An isolated, non-natural hemagglutinin (HA) polypeptide antigen or antibody-binding moiety of the H3 influenza virus (H3 virus) according to claim 1, comprising an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:

15.

4. A virus-like particle (VLP) comprising the H3 virus HA polypeptide antigen according to any one of claims 1 to 3.

5. The VLP according to claim 4, comprising a polynucleotide encoding the H3 virus HA polypeptide antigen.

6. The VLP according to claim 5, wherein the polynucleotide is RNA, mRNA, or DNA.

7. An immunogen comprising an isolated, non-natural hemagglutinin (HA) polypeptide antigen of an H3 influenza virus (H3 virus) according to any one of claims 1 to 3, which can induce an immune response against current and future H3 influenza (H3) virus strains.

8. An immunogen comprising virus-like particles (VLPs) according to any one of claims 4 to 6, which can induce an immune response against current and future H3 influenza (H3) virus strains.

9. The immunogen according to claim 7 or 8, wherein the immune response comprises the production of an antibody having hemagglutinin inhibitory activity.

10. The immunogen according to claim 9, wherein the immune response further comprises the production of one or both a neutralizing antibody and / or T-lymphocytes.

11. A pharmaceutical composition comprising a non-natural hemagglutinin (HA) polypeptide antigen according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier, diluent, or excipient.

12. A pharmaceutical composition comprising virus-like particles (VLPs) according to any one of claims 4 to 6, and a pharmaceutically acceptable carrier, diluent, or excipient.

13. A pharmaceutical composition comprising an immunogen according to any one of claims 7 to 10 and a pharmaceutically acceptable carrier, diluent, or excipient.

14. A pharmaceutical composition according to any one of claims 11 to 13, further comprising an adjuvant.

15. The pharmaceutical composition according to claim 14, wherein the adjuvant comprises a squalene oil-in-water emulsion adjuvant or a cationic lipid nanoparticle adjuvant.

16. An immunogenic composition or vaccine comprising the H3 virus HA polypeptide antigen according to any one of claims 1 to 3.

17. An immunogenic composition or vaccine comprising a virus-like particle (VLP) according to any one of claims 4 to 6.

18. An immunogenic composition or vaccine comprising the immunogen described in any one of claims 7 to 10.

19. A pharmaceutically acceptable composition comprising an immunogenic composition or vaccine according to any one of claims 16 to 18 and a pharmaceutically acceptable carrier, diluent, or excipient.

20. A pharmaceutically acceptable composition according to claim 19, further comprising an adjuvant.

21. The pharmaceutically acceptable composition according to claim 20, wherein the adjuvant comprises a squalene oil-in-water emulsion adjuvant or a cationic lipid nanoparticle adjuvant.

22. A method for inducing an immune response in a subject, comprising the step of administering an effective amount of the H3 virus polypeptide antigen described in any one of claims 1 to 3 to the subject.

23. A method for inducing an immune response in a subject, comprising the step of administering an effective amount of virus-like particles (VLPs) according to any one of claims 4 to 6 to the subject.

24. A method for inducing an immune response in a subject, comprising the step of administering an effective amount of the immunogen described in any one of claims 7 to 10 to the subject.

25. A method for inducing an immune response in a subject, comprising the step of administering an effective amount of the pharmaceutical composition described in any one of claims 11 to 15 to the subject.

26. A method for inducing an immune response in a subject, comprising the step of administering an effective amount of an immunogenic composition or vaccine according to any one of claims 16 to 18 to the subject.

27. A method for inducing an immune response in a subject, comprising the step of administering an effective amount of a pharmaceutically acceptable composition according to any one of claims 19 to 21 to the subject.

28. The method according to any one of claims 22 to 27, wherein the immune response comprises the production of an antibody having activity against the hemagglutinin protein of a historical influenza vaccine in a hemagglutinin inhibition assay.

29. The method according to any one of claims 22 to 27, wherein an immune response to current and future H3 influenza (H3) virus strains occurs in the subject.

30. The method according to claim 29, wherein the immune response comprises the production of an antibody having hemagglutinin (HA) inhibitory activity.

31. The method according to claim 29, wherein the immune response comprises the production of one or both a neutralizing antibody and T lymphocytes.

32. The method according to any one of claims 22 to 31, wherein the immune response in the subject occurs in response to a disease or condition and / or symptoms thereof resulting from infection by the H3 influenza virus or its subtype.

33. A method according to any one of claims 22 to 32, providing a preventive or therapeutic measure for a disease or condition induced by H3 influenza virus infection.

34. The method according to any one of claims 22 to 33, wherein the adjuvant is administered simultaneously to the subject.

35. The method according to claim 34, wherein the adjuvant is formulated together with the H3 virus polypeptide immunogen, immunogenic composition, pharmaceutical composition, or vaccine.

36. The method according to claim 34 or 35, wherein the adjuvant comprises a squalene oil-in-water emulsion adjuvant or a cationic lipid nanoparticle adjuvant.

37. A polynucleotide encoding the H3 virus HA polypeptide antigen according to any one of claims 1 to 3.

38. The polynucleotide according to claim 37, comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with a polynucleotide sequence selected from the group consisting of SEQ ID NO: 6 to 10 or SEQ ID NO: 16 to 20.

39. The polynucleotide according to claim 37, comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 6 to 10 or SEQ ID NO: 16 to 20.

40. A polynucleotide according to any one of claims 37 to 39, which is RNA, mRNA, or DNA.

41. A composition comprising a polynucleotide according to any one of claims 37 to 40 and a pharmaceutically acceptable carrier, diluent, or excipient.

42. An isolated, non-natural hemagglutinin (HA) polypeptide antigen of an H3 influenza virus (H3 virus) according to any one of claims 1 to 3, which is a recombinant protein and / or produced by recombinant protein.

43. An immunogenic composition or vaccine comprising a polynucleotide encoding an H3 virus polypeptide antigen according to any one of claims 1 to 3.

44. An immunogenic composition or vaccine comprising a polynucleotide encoding the H3 virus HA polypeptide antigen according to claim 42.

45. The immunogenic composition or vaccine according to claim 43 or 44, wherein the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 6 to 10 or SEQ ID NO: 16 to 20.

46. The immunogenic composition or vaccine according to any one of claims 43 to 45, wherein the polynucleotide is RNA, mRNA, or DNA.

47. A pharmaceutical composition comprising an immunogenic composition or vaccine according to any one of claims 43 to 46, and a pharmaceutically or physiologically acceptable carrier, diluent, or excipient.

48. A method for inducing an immune response to the hemagglutinin (HA) protein of the H3N2 influenza virus in a subject, comprising the step of administering an effective amount of the pharmaceutical composition according to claim 47 to the subject.

49. The method according to claim 48, wherein the immune response in the subject is in response to a disease or condition and / or symptoms thereof resulting from infection by the H3 influenza virus or its subtype.

50. The method according to claim 48 or 49, wherein the immune response comprises the production of antibodies having activity against current and future H3 influenza (H3) virus lineages and / or their HA polypeptides.

51. The method according to any one of claims 48 to 50, wherein the immune response comprises the production of an antibody having hemagglutinin (HA) inhibitory activity.

52. The method according to any one of claims 48 to 51, wherein the adjuvant is administered simultaneously to the subject.

53. The method according to claim 52, wherein the adjuvant is formulated together with the pharmaceutical composition.

54. The method according to claim 52 or 53, wherein the adjuvant comprises a squalene oil-in-water emulsion adjuvant or a cationic lipid nanoparticle adjuvant.

55. A composition comprising two combinations of isolated non-natural hemagglutinin (HA) polypeptide antigens of H3 influenza virus (H3 virus) as described in any one of claims 1 to 3.

56. The composition according to claim 55, wherein the isolated non-natural H3 influenza virus HA polypeptide antigen comprises NG-7 with SEQ ID NO:4 or SEQ ID NO:14 and NG-8 with SEQ ID NO:5 or SEQ ID NO:

15.

57. The composition according to claim 55 or 56, wherein the isolated non-natural H3 influenza virus HA polypeptide antigen is recombinant and / or produced by recombinant.

58. The composition according to any one of claims 55 to 57, further comprising a pharmaceutically or physiologically acceptable carrier, diluent, or excipient.

59. The composition according to claim 58, further comprising an adjuvant.

60. The composition according to claim 59, wherein the adjuvant comprises a squalene oil-in-water emulsion adjuvant or a cationic lipid nanoparticle adjuvant.

61. A method for inducing an immune response to the hemagglutinin (HA) protein of the H3N2 influenza virus in a subject, comprising the step of administering an effective amount of the composition according to any one of claims 58 to 60 to the subject.

62. The aforementioned immune response, (i) Production of antibodies that are active against current and future H3 influenza (H3) virus strains; (ii) Production of antibodies having hemagglutinin inhibitory activity; (iii) Production of either or both viral neutralizing antibodies and T lymphocytes; and / or (iv) Production of antibodies active against hemagglutinin (HA) protein of historical influenza vaccines in hemagglutinin inhibition assays. The method according to claim 61, including the method described in claim 61.

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