Broadly reactive viral antigens as immunogens, compositions thereof and methods of use

Abstract

Provided herein are non-natural broadly reactive antigens derived from influenza viruses that are immunogenic and capable of eliciting a broadly reactive immune response, e.g., a broadly reactive neutralizing antibody response, directed against influenza virus antigens after introduction into a subject. Also provided are non-natural broadly reactive immunogens, vaccines, virus particles, virus-like particles (VLPs), and compositions comprising the immunogens and vaccines. Methods of generating an immune response in a human or non-human subject by administering the immunogens, vaccines, VLPs, or compositions thereof are provided. In particular, the immunogen comprises a broadly reactive hemagglutinin (HA) protein antigen or a soluble HA protein antigen of an influenza virus strain, such as H1 or H3.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 209,209, filed June 10, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] background Influenza virus infection is a significant cause of acute respiratory illness seen in medical clinics each year, imposing a significant amount of morbidity, mortality, and economic burden both in the United States and worldwide. Just prior to the 2009 influenza pandemic, influenza-related mortality was responsible for over 611,000 years of life lost annually, with an estimated cost to society of $87 billion annually. The US Centers for Disease Control and Prevention estimated that the effectiveness of seasonal influenza (flu) vaccines was only 42% in 2017. This limited effectiveness was attributed to mutations that occurred in the influenza A H3N2 vaccine strain that causes influenza in infected individuals. In addition, there has been an increase in cases of influenza caused by influenza type B viruses from 2017 to 2018. Because a bad flu season can kill as many as 50,000 people in the United States alone, there is an urgent need for new and improved immunogens and vaccines that provide broad protection from viruses, especially influenza virus strains, in current and future epidemics.

[0003] Infections from other virus types, such as coronaviruses, also cause severe disease and lesions in humans and other species, such as avian species, such as poultry. Such infections and diseases adversely affect the health and performance of meat-producing and egg-producing avian animals, such as chickens, resulting in significant economic losses in the industry. Thus, new and improved immunogens and vaccines that provide broad protection from other virus strains, such as avian viruses and their strains, in current and future epidemics are also needed. Summary of the Invention

[0004] overview As described herein, provided are non-naturally occurring broadly reactive antigens and antigenic sequences derived from influenza viruses, e.g., influenza H1 or H3 viruses (also referred to herein as "H1 or H3 influenza," "H1 or H3 influenza viruses," or simply "H1 or H3"). In embodiments, the non-naturally occurring broadly reactive antigens and antigenic sequences comprise the amino acid sequences of HA from H1 and H3 influenza viruses as provided herein in SEQ ID NOs: 1-17. In some embodiments, the HA antigen comprises a full-length HA polypeptide. In some embodiments, the HA antigen comprises a soluble HA (sHA) that lacks the transmembrane and tail domains of the HA polypeptide.

[0005] Influenza virus antigens described herein may be structural proteins (polypeptides) or peptides, including, for example, the hemagglutinin (HA) protein, and / or the HA1 (head) or HA2 (tail or stalk) portions (domains) of the HA protein, which are potent immunogens that induce broadly reactive immune responses in subjects against the HA protein and ultimately against current and future virus strains in the subject. In one embodiment, the HA antigen comprises a full-length HA protein or a soluble HA (sHA) that lacks the transmembrane (TM) and tail domains of the HA protein. As referred to herein, a viral antigen or antigen sequence (e.g., an influenza virus antigen) that induces an immune response in a subject is an immunogenic antigen (i.e., an immunogen). These influenza virus immunogens are referred to as broadly reactive because they can induce the production of broadly reactive antibodies directed against different subtypes or strains of influenza virus that have both sequence similarity and variability, and epitope (antigenic determinant) diversity in their protein antigens and their sequences, particularly the HA antigens of influenza viruses.

[0006] There are four different types of influenza viruses, three of which (influenza A, B, and C) infect people. Of these three infectious viruses, influenza A and B subsets are the most common types, and each of these subsets produces different strains or subtypes. Influenza A and B viruses routinely transmit to humans and cause seasonal influenza epidemics. As a non-limiting example, H1N1, H2N2, H3N2, and H5N1 strains are subtypes of influenza A that typically cause severe influenza disease and adapt to avoid eradication by constantly changing their surface proteins, e.g., the hemagglutinin (HA) protein. Influenza virus strains have been particularly difficult to treat due to their unusually high mutation rates and the inability to generate effective vaccines against the relatively rapid changes that have occurred in the HA surface protein.

[0007] In some embodiments, the immunogenic antigen (antigen sequence) is derived from an influenza A virus. In particular embodiments, the immunogen is derived from an H1 or H3 influenza virus strain or type. In some embodiments, the immunogen is derived from an influenza B virus. In some embodiments, the antigen is a viral structural protein. In particular embodiments, the influenza antigen is hemagglutinin (HA). In some embodiments, the HA antigen is full length HA. In some embodiments, the HA antigen is soluble HA (sHA). In other embodiments, the influenza antigen is neuraminidase (NA).

[0008] In certain aspects, non-natural influenza virus amino acid sequences and antigens (e.g., structural antigens) comprising the sequences described herein contain broadly reactive epitopes that reflect the sequence similarity and variability of past, present, and future influenza virus antigens. Thus, such antigen sequences and antigens comprising the sequences are "non-natural broadly reactive" antigens. The antigens are immunogenic and, when introduced or administered to a subject, elicit broadly reactive antibodies, e.g., neutralizing antibodies, in the subject that are directed against influenza virus, particularly H1 or H3 influenza virus protein antigens, e.g., HA, or antibody-binding portions thereof. In certain embodiments, the elicited antibodies are also reactive against related but non-identical H1 or H3 influenza virus types. In certain embodiments, such influenza virus sequences are amino acid sequences. In certain embodiments, the influenza virus sequences are polynucleotide sequences, e.g., polynucleotide sequences that encode the amino acid sequences of the antigens described herein. For ease of reference, the "non-natural broadly reactive" antigens of influenza viruses described herein are referred to as "broadly reactive antigens."

[0009] Thus, the broadly reactive influenza virus 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, e.g., neutralizing antibodies that are specifically directed against the HA antigen of the influenza virus and neutralize the activity of the HA protein. Thus, also provided are immunogens and immunogenic compositions containing the broadly reactive influenza virus antigens described herein, including immunogenic compositions, e.g., vaccines (e.g., polypeptide or polynucleotide products), that induce an immune response in a subject against influenza, e.g., against the HA protein of the influenza virus. For ease of reference, the "non-natural broadly reactive influenza virus immunogens" described herein are referred to as "broadly reactive immunogens."

[0010] Also provided are methods of inducing an immune response against influenza infection, disease, and / or symptoms thereof in a subject using the immunogens described herein. In particular embodiments, the influenza virus antigen is the HA, HA1, or HA2 protein of an influenza virus type or subtype, e.g., H1 or H3 influenza virus type, or a related virus type, or an antibody-binding portion thereof. In other embodiments, the HA protein is full-length or soluble HA (sHA). Also provided are methods of inducing an immune response in a subject using the immunogens.

[0011] In one embodiment, the HA immunogenic antigen has an amino acid sequence that is at least 85% or equal, at least 90% or equal, at least 91% or equal, at least 92% or equal, at least 93% or equal, at least 94% or equal, at least 95% or equal, at least 96% or equal, at least 97% or equal, at least 98% or equal, or at least 99% or equal identical to the HA amino acid sequence of one or more of the HA proteins of SEQ ID NOs: 1-17 set forth in Example 1, below.

[0012] In one aspect, a non-naturally occurring and immunogenic influenza virus antigen, or an immunogenic portion thereof, is provided, the influenza virus antigen comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of a hemagglutinin (HA) antigen of any one of SEQ ID NOs: 1-17 as set forth in Example 1. In one embodiment, the influenza virus antigen comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of a HA antigen of any one of SEQ ID NOs: 1-17. In one embodiment, the influenza virus antigen comprises the amino acid sequence of a HA antigen of any one of SEQ ID NOs: 1-17. In one embodiment, the influenza virus antigen consists of the amino acid sequence of a HA antigen of any one of SEQ ID NOs: 1-17. In one embodiment, the HA antigen is a full-length HA protein. In one embodiment, the HA antigen is a soluble HA protein, e.g., lacking the TM and tail portions of the HA protein. In one embodiment, the influenza virus antigen comprises the amino acid sequence of a full-length HA (sHA) protein antigen of any one of SEQ ID NOs: 1-8. In one embodiment, the influenza virus antigen comprises the amino acid sequence of a soluble HA (sHA) protein antigen of any one of SEQ ID NOs: 9-17. In certain embodiments, the influenza virus is an H1 or H3 influenza virus.

[0013] In another aspect, there is provided a virus-like particle (VLP) comprising an influenza virus immunogenic antigen according to any one of the above aspects. In an embodiment, the VLP comprises a polynucleotide encoding an influenza virus antigen. In an embodiment, the VLP comprises a polynucleotide encoding an influenza virus HA or sHA antigen. In an embodiment, the VLP comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 1-17 as provided in Example 1 below. In an embodiment, the influenza virus is an H1 or H3 influenza virus. In an embodiment, the polynucleotide is RNA or DNA. In an embodiment, the RNA is mRNA.

[0014] In another aspect, a non-naturally occurring immunogen capable of generating an immune response against current and future influenza virus strains is provided, the immunogen comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of a hemagglutinin (HA) antigen or sHA antigen set forth in SEQ ID NOs: 1-17 as provided in Example 1. In one embodiment, the immunogen comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of a hemagglutinin (HA) antigen or sHA antigen set forth in SEQ ID NOs: 1-17 as provided in Example 1.

[0015] In certain embodiments of any of the aspects delineated herein above, the viral antigen, VLP, or immunogen elicits an immune response that includes the production of neutralizing antibodies. In certain embodiments, the immune response includes the production of antibodies that have hemagglutinin-inhibiting activity and / or neuraminidase-inhibiting activity. In certain embodiments, the immune response further includes a cellular immune response, e.g., the production of antigen-responsive T lymphocytes.

[0016] In another aspect, there is provided an immunogenic composition or vaccine comprising an influenza virus immunogen or VLP of any of the above-depicted aspects and / or embodiments. In some embodiments, the immunogenic composition or vaccine comprises a pharma- ceutically acceptable carrier, diluent, or excipient. In some embodiments, the immunogenic composition or vaccine further comprises an adjuvant.

[0017] In another aspect, a pharma- ceutically acceptable composition is provided comprising an influenza virus antigen, immunogen, or VLP of any of the aspects and / or embodiments delineated above and a pharma- ceutically acceptable carrier, diluent, or excipient. In some embodiments, the composition further comprises an adjuvant. In some embodiments, the virus or viral antigen is derived from an H1 or H3 influenza virus. In some embodiments, the HA antigen or immunogen is a full-length HA or a soluble influenza HA antigen or immunogen. In some embodiments, the full-length HA or a soluble HA influenza antigen or immunogen comprises an amino acid sequence set forth in SEQ ID NOs: 1-17, as provided in Example 1.

[0018] In another aspect, there is provided a pharma- ceutically acceptable composition comprising the immunogenic composition or vaccine of the above depicted aspect and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0019] In another aspect, there is provided a method of generating an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a viral antigen, VLP, immunogen, immunogenic composition, vaccine, or pharmaceutical composition of any of the aspects and / or embodiments delineated above.

[0020] In another aspect, there is provided a method of treating or protecting a subject from disease caused by influenza virus infection and / or symptoms thereof, the method comprising administering to the subject an effective amount of a viral antigen, VLP, immunogen, immunogenic composition, vaccine, or pharmaceutical composition of any of the aspects and / or embodiments depicted above. In some embodiments of the method, the subject is infected with influenza virus, or at risk of infection by influenza virus, or susceptible to influenza virus infection. In some embodiments of the method, the immune response elicited comprises production of neutralizing antibodies and / or cellular immune responses, e.g., production of T lymphocytes. In some embodiments of the method, an adjuvant is administered to the subject simultaneously. In some embodiments of the method, the immune response is prophylactic or therapeutic. In some embodiments of the method, an adjuvant or one or more antiviral agents are administered to the subject. In some embodiments of the method, the subject is a human subject. In some embodiments of the method, the subject is a non-human subject or a veterinary subject.

[0021] In another aspect, a polynucleotide is provided that encodes a viral antigen, in particular an influenza HA antigen (either full length or soluble HA) of any of the aspects and embodiments described above. In certain embodiments, the polynucleotide is DNA or RNA. In certain embodiments, the polynucleotide is mRNA. In certain embodiments, the viral antigen is an HA protein antigen. In particular embodiments, the HA protein antigen comprises the amino acid sequence of any one of SEQ ID NOs: 1-17 provided in Example 1. In some embodiments, the HA protein antigen is full length HA or soluble HA. In certain embodiments, the polynucleotide encodes an influenza virus antigen comprising the amino acid sequence of a full length HA (sHA) protein antigen as set forth in SEQ ID NOs: 1-8. In certain embodiments, the polynucleotide encodes an influenza virus antigen comprising the amino acid sequence of a soluble HA (sHA) protein antigen as set forth in SEQ ID NOs: 9-17. In certain embodiments, the virus is an influenza virus. In certain embodiments, the influenza virus is an H1 or H3 influenza virus. In certain embodiments, the polynucleotide depicted above is contained in a composition comprising a pharma- ceutically acceptable carrier, diluent, or excipient. In certain embodiments, the above depicted polynucleotides are contained in a virus-like particle (VLP).

[0022] In another aspect, there is provided a monovalent immunogen comprising a non-naturally occurring and immunogenic influenza virus antigen of the aspect and / or embodiment thereof delineated above. In an embodiment, the immunogenic influenza virus antigen comprises the following non-naturally occurring broadly reactive influenza polypeptide immunogens described herein: Y2 comprising the sequence set forth in SEQ ID NO: 15, J1 comprising the sequence set forth in SEQ ID NO: 3, 7, or 9, J2 comprising the sequence set forth in SEQ ID NO: 4, J3 comprising the sequence set forth in SEQ ID NO: 5, J4 comprising the sequence set forth in SEQ ID NO: 6 or 8, NG1 comprising the sequence set forth in SEQ ID NO: 11, NG2 comprising the sequence set forth in SEQ ID NO: 2 or 12, or NG3 comprising the sequence set forth in SEQ ID NO: 13.

[0023] In another aspect, a multivalent immunogen is provided that comprises at least two of the non-natural and immunogenic influenza virus antigens of the aspects and / or embodiments thereof depicted above. In an embodiment, the immunogen comprises two of the non-natural and immunogenic influenza virus antigens. In embodiments, the immunogen is bivalent and comprises a combination of Y2 comprising the sequence set forth in SEQ ID NO: 15 and J4 comprising the sequence set forth in SEQ ID NO: 6 or 8, or a combination of Y2 comprising the sequence set forth in SEQ ID NO: 15 and NG2 comprising the sequence set forth in SEQ ID NO: 2 or 12. In an embodiment, the multivalent immunogen comprises eight of the non-natural and immunogenic influenza virus antigens described herein. In an embodiment of the aforementioned monovalent or multivalent immunogen, the immunogen comprises a recombinant influenza hemagglutinin (rHA) polypeptide. In an embodiment of the aforementioned monovalent or multivalent immunogen, the immunogen comprises a recombinant influenza neuraminidase (rNA) polypeptide.

[0024] In another aspect, there is provided a virus or virus-like particle (VLP) comprising one or more polynucleotides encoding an immunogenic influenza virus antigen of any of the aspects and / or embodiments thereof delineated above.

[0025] In another aspect, there is provided a composition comprising the monovalent or multivalent immunogen of any one of its aspects and / or embodiments depicted above, hi certain embodiments, the composition further comprises a pharma- ceutically acceptable carrier, excipient, or vehicle, i.e., a pharmaceutical composition.

[0026] In one aspect, there is provided a composition comprising a viral particle or VLP of the aspects and / or embodiments thereof depicted above, hi one embodiment, the composition further comprises a pharma- ceutically acceptable carrier, excipient, or vehicle, i.e., a pharmaceutical composition.

[0027] In one aspect, there is provided a method of treating or protecting a subject against disease caused by influenza virus infection and / or symptoms thereof, the method comprising administering to the subject an effective amount of a pharma- ceutically acceptable composition as depicted above comprising a monovalent or polyvalent immunogen of any of the aspects and / or embodiments thereof as depicted above.

[0028] In one aspect, there is provided a method of treating or protecting a subject against disease caused by influenza virus infection and / or symptoms thereof, the method comprising administering to the subject an effective amount of a pharma- ceutically acceptable composition as depicted above comprising a virus particle or virus-like particle (VLP) of the aspect and / or embodiment as depicted above.

[0029] In one aspect, a method of generating an immune response in a subject is provided, the method comprising administering to the subject an effective amount of a pharma- ceutically acceptable composition comprising a monovalent or polyvalent immunogen, virus particle or virus-like particle of any of the aspects and / or embodiments thereof delineated above.

[0030] definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the aspects and embodiments described herein pertain. The following references provide those of ordinary skill in the art with general definitions of many of the terms used in the aspects and embodiments described 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 ascribed to them below, unless otherwise specified.

[0031] "Adjuvant" refers to a substance or vehicle that nonspecifically enhances the immune response to an antigen. Adjuvants may include suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphates) to which antigens are adsorbed, or water-in-oil emulsions in which antigen solutions are emulsified in mineral oil (e.g., Freund's incomplete adjuvant), and in some cases, killed mycobacteria to further enhance antigenicity (Freund's complete adjuvant). Immunostimulatory oligonucleotides (such as those containing CpG motifs) can also be used as adjuvants (see, e.g., U.S. Patent Nos. 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). Adjuvants also include biological molecules, such as costimulatory molecules. Exemplary biological adjuvants include, but are not limited to, interleukin-1 (IL-2), 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), cluster of differentiation antigen 72 (CD72), (negative regulator of B cell responsiveness), peripheral membrane protein B7-1 (B7-1, also known as CD80), peripheral membrane protein B7-2 (B7-2, also known as CD86), TNF ligand superfamily member 4 ligand (OX40L), or type 2 membrane glycoprotein receptor belonging to the TNF superfamily (4-1BBL).

[0032] "Administering" means giving, providing, distributing, delivering, or applying a composition, drug, therapeutic agent, etc. to a subject, or applying or contacting a subject with a composition, etc. Administering, or administration, can be accomplished by any of a number of routes, including, for example, but not limited to, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous (IV), (injection), intrathecal, intramuscular, dermal, intradermal, intracranial, inhalation, rectal, intravaginal, or intraocular.

[0033] By "agent" is meant any small molecule, small molecule chemical compound, antibody, nucleic acid molecule, peptide, polypeptide, or fragment thereof.

[0034] By "alteration" is meant a change (increase or decrease) in expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, alteration includes a 5% change in expression levels, a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.

[0035] By "ameliorate" is meant to decrease, reduce, decrease, inhibit, attenuate, arrest, or stabilize the onset or progression of a disease or pathological condition.

[0036] "Analog" refers to a molecule that is not identical but has similar functional or structural characteristics. For example, a polypeptide analog retains the biological activity of the corresponding naturally occurring polypeptide while possessing certain biochemical modifications that enhance the function of the analog compared to the naturally occurring polypeptide. Such biochemical modifications can, for example, increase the analog's protease resistance, membrane permeability, or half-life without altering ligand binding. Analogs can include unnatural amino acids.

[0037] "Antibody" refers to an immunoglobulin (Ig) molecule produced by B lymphocyte cells and having a specific amino acid sequence. Antibodies are raised or induced in a subject (human or other animal or mammal) after exposure to a specific antigen (immunogen). A subject capable of generating antibodies / immunoglobulins (i.e., an immune response) directed against a particular antigen / immunogen is said to be immunocompetent. Antibodies are characterized by specifically reacting with (e.g., binding to) an antigen or immunogen in some demonstrable way, and the antibody and antigen / immunogen are each defined in terms of the other.

[0038] "Eliciting 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. The antibody / immunoglobulin response elicited in a subject can neutralize a pathogenic (e.g., infectious or disease-causing) agent by binding to an epitope (antigenic determinant) on the agent, blocking or inhibiting the activity of the agent, and / or by forming a binding complex with the agent that is cleared from the subject's system (or body), e.g., via the liver.

[0039] As used herein, "broadly reactive" means that an immune response is elicited in a subject against antigenic proteins (e.g., viral protein sequences such as HA or NA) from a pathogen that is sufficient to block, inhibit, impede, neutralize, or prevent infection with a broad range of relevant pathogens (such as most or all influenza viruses within a particular subtype). In some embodiments, the subject is a mammalian subject. In some embodiments, the subject is an avian subject.

[0040] "Antigen" refers to a compound, composition, or substance capable of stimulating the production of an antibody or T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. In some embodiments of the disclosed compositions and methods, the antigen is influenza hemagglutinin (HA) protein. In certain embodiments, the HA protein is full-length HA. In certain embodiments, the HA protein is sHA that is truncated and does not include the transmembrane and tail domains. An antigen that induces or stimulates an immune response in a subject is often referred to as an "immunogen." In specific embodiments, the HA protein comprises the amino acid sequence provided in SEQ ID NOs: 1-17 (Example 1).

[0041] The term "antigenic drift" refers to a mechanism of mutation in organisms or microorganisms such as viruses that involves the accumulation of mutations in genes that code for antibody binding sites (also called antigenic determinants or epitopes). This process results in new strains of virus / virus particles that are not effectively inhibited or blocked by antibodies originally generated against the antigens of the viral strain before the mutation, thus allowing the virus to spread more easily throughout sub-immune populations. By way of example, antigenic drift occurs in both influenza A and influenza B viruses.

[0042] In the context of a live virus, the term "attenuated" refers to a virus that is attenuated when its ability to infect a cell or subject and / or its ability to cause disease is reduced (e.g., decreased, abolished, or eliminated) compared to the ability of the wild-type virus to cause disease in a subject. Typically, an attenuated virus retains at least some ability to induce an immune response after administration to an immunocompetent subject. In some cases, the attenuated virus can induce a protective immune response without causing signs or symptoms of infection. In some embodiments, the ability of the attenuated virus to cause disease or pathology in a subject is reduced by at least about 5% or more, or at least about 10% or more, or at least about 25% or more, at least about 50% or more, at least about 75% or more, or at least about 80% or more, or at least about 85% or more, or at least about 90% or more, or at least about 95% or more, or more, compared to the ability of the wild-type virus to cause disease or pathology in a subject.

[0043] The term "clade" refers to different classifications (often called subtypes) of known influenza viruses, such as, for example, influenza A H3N2 viruses. By way of example, viruses within the H3N2 clade are genetically related but do not share the exact viral genome. As will be appreciated by those skilled in the art, there are many clades and subclades of H3N2 virus subtypes designated in the art. By way of example, one clade is 3C.2a, and subclades of this clade include 3C.2a.1, 3C.2a.2, 3C.2a.3, and 3C.2a.4. In addition, 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 clade 2.1, clade 2.2, clade 2.3, clade 2.4 and clade 2.5.

[0044] "Codon-optimized" nucleic acid refers to a nucleic acid sequence in which the codons have been altered to be optimal for expression in a particular system, such as a particular species or group of species. For example, a nucleic acid sequence can be optimized for expression in mammalian cells. Codon optimization does not change the amino acid sequence of the encoded protein.

[0045] In this disclosure, "comprises," "comprising," "containing," "having," and the like can have the meaning ascribed to them in U.S. patent law, and may mean "includes," "including," "consisting essentially of" or "consists essentially," and the like can have the meaning ascribed to them in U.S. patent law, and the terms are open-ended, permitting the presence of more than what is recited so long as the basic or novel characteristics of the recited items are not altered by the presence of more than what is recited, but prior art embodiments are excluded.

[0046] "Detecting" refers to identifying the presence, absence, or amount of an analyte, compound, agent, or substance being detected. "Detectable label" refers to a composition that, when attached to a molecule of interest, renders the latter 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, electron-dense reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, or haptens.

[0047] "Disease" refers to any condition, disorder, or lesion that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include diseases caused by influenza virus infection and symptoms and adverse effects caused by infection of the body with H1 or H3 influenza viruses. Influenza viruses cause the flu and its symptoms in infected individuals.

[0048] By "effective amount" is meant the amount of active therapeutic agent, composition, compound, biological (e.g., vaccine or therapeutic peptide, polypeptide, or polynucleotide) required to ameliorate, alleviate, ameliorate, neutralize, reduce, or eliminate the symptoms and / or effects of a disease, condition, or lesion, as compared to an untreated patient. In one embodiment, the effective amount is the amount of antigen required to elicit an immune response. The effective amount of an immunogen or composition comprising an immunogen as used to practice the method of therapeutic treatment of a disease, condition, or lesion varies depending on the method of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosing regimen. Such an amount is referred to as an "effective" amount.

[0049] A "therapeutically effective amount" refers to an amount of a particular agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, a therapeutically effective amount can be an amount of an influenza virus immunogen or vaccine useful for inducing an immune response in a subject and / or preventing infection by influenza virus. Ideally, in the context of the present disclosure, a therapeutically effective amount of an influenza vaccine or immunogenic composition is an amount sufficient to increase resistance to, prevent, ameliorate, reduce, and / or treat infection caused by influenza virus in a subject without causing substantial cytotoxic effects in the subject. As described above, an effective amount of an immunogenic composition (or vaccine) useful for increasing resistance to, preventing, ameliorating, reducing, and / or treating infection in a subject depends, for example, on the subject being treated, the method of administration of the therapeutic composition, and other factors.

[0050] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. The portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full 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 portion or fragment of a polypeptide may be a peptide. In the case of an antibody or immunoglobulin fragment, the fragment typically binds to a target antigen.

[0051] "Fusion protein" refers to a protein produced by expression of a nucleic acid (polynucleotide) sequence engineered from nucleic acid sequences encoding at least a portion of two different (heterologous) proteins or peptides. To create a fusion protein, the nucleic acid sequences must be in the same reading frame and contain no internal stop codons. For example, a fusion protein includes an influenza HA protein or NA protein fused to a heterologous protein.

[0052] By "genetic vaccine" is meant an immunogenic composition that includes a polynucleotide encoding an antigen.

[0053] "Viral polypeptide", e.g., an H1 or H3 influenza virus, means an amino acid sequence that is at least 85% identical, or at least 95% or more identical, to an antigenic amino acid sequence, e.g., an HA protein set forth in SEQ ID NOs: 1-17 below, or a fragment thereof, that is capable of inducing an immune response against the virus, viral infection, and / or symptoms thereof in an immunized subject. In embodiments, the influenza virus polypeptide comprises or consists of the amino acid sequence described herein and provided in SEQ ID NOs: 1-17 below, or a fragment thereof.

[0054] "Viral polynucleotide" refers to a nucleic acid molecule that encodes an influenza virus polypeptide (antigen or antigenic protein), such as an H1 or H3 influenza virus, as described herein. In certain embodiments, the polynucleotide is a DNA or RNA polynucleotide. In certain embodiments, the polynucleotide is an mRNA.

[0055] The term "hemagglutinin (HA)" refers to a surface glycoprotein expressed by influenza viruses. HA mediates the binding of viral particles to and subsequent entry of the virus into the host cell. Numerous influenza HA protein nucleotide and amino acid sequences are known in the art and publicly available, such as those deposited in the publicly accessible GenBank (NCBI) and UniProtKB databases. As a non-limiting example, a list of GenBank accession numbers for H5N1 HA sequences can be found in U.S. Patent Application Publication No. US2015 / 0030628. A non-limiting example of the amino acid sequence of the HA protein of influenza A virus (strain A / Puerto Rico / 8 / 1934H1N1) is provided in UniProtKB accession number P03452 (HEMA_134A1). A non-limiting example of the amino acid sequence of the HA protein of influenza A H3N2 virus (A / Hong Kong / 1-4 / 1968 (H3N2)) is provided in Accession No. CY033017. HA (along with neuraminidase (NA)) is one of the two major influenza virus antigenic proteins that carry the antigenic determinants (epitopes) recognized and bound by antibodies / immunoglobulins. In embodiments, HA is HA1 (H1) or HA2 (H2).

[0056] In embodiments, the HA protein or fragment thereof may have at least 85% or equal thereto, or at least 90%, 95%, 98%, 99% or equal thereto, or greater, amino acid sequence identity to the amino acid sequence of a representative influenza A virus HA protein or fragment thereof.

[0057] In one embodiment, the HA immunogenic antigen is an H1 or H3 HA protein, which may be full-length or a soluble form thereof, and which comprises or consists of the amino acid sequence set forth in Example 1 below.

[0058] "Hybridization" refers to hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, in DNA, adenine and thymine, and cytosine and guanine, respectively, are complementary nucleobases that pair through the formation of hydrogen bonds.

[0059] The term "immune response" refers to any response mediated by immunocompetent cells. One example of an immune response recruits white blood cells to perform a variety of different specific functions in response to exposure to an antigen (e.g., a foreign substance). The immune response is a multifactorial process that differs depending on the type of cells involved. Immune responses include cell-mediated responses (e.g., T cell responses), humoral responses (B cell / antibody responses), innate responses, and combinations thereof.

[0060] "Immunogen" means a compound, composition, or substance that, under appropriate conditions, can induce or stimulate an immune response in an animal, e.g., the production of antibodies and / or a T cell response, including compositions that are injected or otherwise delivered to an animal. As used herein, an "immunogenic composition" is a composition that includes an immunogen (an HA polypeptide or peptide (e.g., a full-length or soluble form of an HA polypeptide) or a polynucleotide encoding such an immunogen) or a vaccine that includes an HA polypeptide or peptide (e.g., a full-length or soluble form of an HA polypeptide) or a polynucleotide encoding such an immunogen. As can be appreciated by one of skill in the art, an immunogenic composition can be prophylactic and induce an immune response, e.g., a neutralizing antibody and / or cellular immune response, in the subject when administered to a subject in need thereof before the subject contracts or experiences full-blown disease, and can protect against disease or prevent a more severe disease or condition, and / or symptoms thereof. When administered to a subject in need thereof after the subject has contracted a disease, the immunogenic composition may be therapeutic, e.g., capable of eliciting an immune response, e.g., a neutralizing antibody and / or cellular immune response, in the subject to treat the disease by, e.g., alleviating, reducing, neutralizing, ameliorating, mitigating, alleviating, or eliminating the disease and / or its symptoms. In some embodiments, the immune response is a B cell response, which results in the production of antibodies, e.g., neutralizing antibodies, directed against the immunogen or immunogenic composition comprising the antigen or antigen sequence. As above, in some embodiments, the immunogen, immunogenic composition, or vaccine may be prophylactic. In some embodiments, the immunogen, immunogenic composition, or vaccine may be therapeutic. In some embodiments, the disease is influenza. In some embodiments, the disease is infectious bronchitis. In some instances herein, the terms immunogen and vaccine are used interchangeably.

[0061] By "immunogenic composition" is meant a composition that includes an antigen, antigenic sequence, or immunogen, which composition elicits an immune response in an immunized subject.

[0062] The term "immunize" (or immunization) refers to protecting a subject against or rendering them immunologically responsive to a disease or lesion caused by a pathogen, e.g., a pathogenic agent, e.g., an infectious disease caused by a virus (e.g., influenza virus H1 or H3), e.g., by vaccination. In some cases herein, the terms "immunization" and "vaccination" may be used interchangeably (e.g., immunization / vaccination).

[0063] The term "influenza virus" refers to a segmented negative-stranded RNA virus that belongs to the Orthomyxoviridae family of viruses. There are three types of influenza viruses: A, B, and C. Influenza A viruses infect a wide variety of birds and mammals, including 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, but not limited to, H5N1, H5N2, H5N6, H5N8, H7N9, H9N2, H1N1, H1N2, H2N1, H2N2, H2N3, H7N3, H7N7, H3N2, H3N1, and related viruses, can cause systemic infections in poultry with mortality rates approaching 100%. H5N1 is also referred to as "avian influenza."

[0064] "Inhibitory nucleic acid" refers to a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, which, when administered to a mammalian cell, results in a reduction (e.g., 5%, 10%, 25%, 50%, 75%, or 90-100%) in expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule or its ortholog, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids depicted herein.

[0065] The terms "isolated," "purified," or "biologically pure" refer to material that is free or to varying degrees free of components that normally accompany it as found in its native state. "Isolate" refers to a degree of separation from the original source or surroundings. "Purify" refers to a degree of separation that is greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid, protein, or peptide is purified when it is substantially free of cellular material, debris, unrelated viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. 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 the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For proteins that may be subject to modifications, such as phosphorylation or glycosylation, different modifications may result in different isolated proteins that may be purified separately. The term "isolated" also encompasses recombinant nucleic acids, proteins or viruses, as well as chemically synthesized nucleic acids or peptides.

[0066] "Isolated polynucleotide" refers to a nucleic acid (e.g., a DNA molecule) that is free of genes that flank the genes in the native genome of the organism from which the nucleic acid molecule of the described aspects and embodiments is derived. Thus, the term includes recombinant DNA that is, for example, incorporated into a vector, incorporated into an autonomously replicating plasmid or virus, or incorporated into the genomic DNA of a prokaryotic or eukaryotic organism, or exists as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion). In addition, the term includes RNA molecules (e.g., mRNA) that are transcribed from a DNA molecule, as well as recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

[0067] "Isolated polypeptide" refers to a polypeptide as described herein that has been separated from components that naturally accompany it. Typically, a polypeptide is isolated when it is at least 30%, at least 40%, at least 50%, or at least 60%, by weight, free of proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, an isolated polypeptide preparation is at least 75%, at least 90%, or at least 99%, by weight, free of proteins and naturally occurring organic molecules with which it is naturally associated. An isolated polypeptide can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide, or by chemically synthesizing the protein. Purity can be measured by any standard and appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. An isolated polypeptide can refer to a broadly active viral immunogen polypeptide produced by the methods described herein.

[0068] By "linker" is meant one or more amino acids that function as a spacer between two polypeptides or peptides of a fusion protein.

[0069] By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity (eg, an increase or decrease) that is associated with a disease, condition, pathology, or disorder.

[0070] "Matrix (M1) protein" refers to an influenza virus structural protein found within the viral envelope. M1 is thought to function in virus assembly and budding after infection of a cell.

[0071] As used herein, "obtaining," as in "obtaining an agent," includes synthesizing, isolating, purifying, purchasing, or otherwise acquiring the agent.

[0072] The term "operably linked" refers to a nucleic acid sequence as used herein. By way of example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects (enables) the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to connect two protein coding regions, in the same reading frame.

[0073] Nucleotide sequences encoding broadly reactive HA proteins (so that an immune response (e.g., an antibody response) can be generated against them as well as against other, possibly antigenically shifting, yet related viruses) can be optimized for expression in mammalian cells through codon optimization and RNA optimization (e.g., to increase RNA stability) using procedures and techniques practiced in the art.

[0074] A (non-natural) broadly reactive immunogenic antigen, such as influenza (e.g., H1 or H3 influenza virus) hemagglutinin (HA) protein, for eliciting an immune response in a subject, has a collective set of strongly immunogenic epitopes (also called antigenic determinants). The influenza virus HA protein described herein is suitable for use as an immune response-eliciting immunogen or vaccine that, when introduced into a host subject, particularly a human subject infected with influenza H1 or H3 virus, elicits a broadly reactive immune response, e.g., a neutralizing antibody response, against other related but non-identical virus types that express the HA protein on the viral surface. The immunogenic antigen (or vaccine) provides an anti-viral immunogen (or vaccine) that elicits a broadly active immune response against other influenza virus HA antigens, such as H1 or H3, with antigenic variability and similarity, and is advantageous for treating or preventing infection and disease caused by more than one H1 or H3 influenza virus subtype.

[0075] "Open reading frame (ORF)" means a series of nucleotide triplets (codons) coding for amino acids without a termination codon. These sequences are usually translatable into a peptide or polypeptide.

[0076] The term "pharmaceutical acceptable vehicle" refers to conventional carriers (vehicles) and excipients that are physiologically and pharmaceutical acceptable, especially for use in mammals, e.g., human subjects, as well as other animal or avian subjects. Such pharmaceutical acceptable vehicles are known to those skilled in the art and can be readily found in Remington's Pharmaceutical Sciences by E.W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975) and updates thereto, which describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions, such as one or more HA-type influenza immunogens (vaccines), and additional pharmaceutical agents. In general, the nature of the pharmaceutical acceptable carrier depends on the particular mode of administration used. For example, parenteral formulations usually contain an injectable fluid / liquid, including pharmaceutical and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc., as a vehicle or diluent. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate, which typically stabilize the composition or drug and / or increase its half-life. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0077] By "plasmid" (or "vector") is meant a circular nucleic acid molecule capable of autonomous replication in a host cell.

[0078] "Polypeptide" (or protein) refers to a polymer comprising amino acid residues in which the monomers are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used. As used herein, the term "polypeptide" or "protein" is intended to encompass any amino acid sequence, including modified sequences such as glycoproteins. The term "polypeptide" is specifically intended to encompass naturally occurring proteins, and proteins that are recombinantly or synthetically produced. The term "residue" or "amino acid residue" also refers to an amino acid that is incorporated into a protein, polypeptide, or peptide.

[0079] Conservative amino acid substitution is a substitution that, when made, causes minimal interference with the properties of the original protein, i.e., the structure, particularly the function, of the protein is preserved and does not change significantly due to such substitution.Examples of conservative amino acid substitution are known in the art, for example, as described in US Publication No. 2015 / 0030628.Conservative substitution generally maintains (a) the structure of the polypeptide backbone in the substituted region, for example, sheet or helix structure, (b) the charge or hydrophobicity of the molecule at the target site, and / or (c) the bulkiness of the side chain.

[0080] Substitutions that are generally expected to result in the most significant changes to protein properties are non-conservative changes, for example, (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine ​​or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylanine, is substituted for (or by) a residue having no side chain, e.g., in this case, glycine.

[0081] "Primer set" refers to a set of oligonucleotides that can be used, for example, in PCR. A primer set consists 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.

[0082] "Promoter" refers to an array of nucleic acid control sequences that directs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor sequence elements. A "constitutive promoter" is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In contrast, the activity of an "inducible promoter" is regulated by external signals or molecules (e.g., transcription factors). By way of example, the promoter can be a CMV promoter.

[0083] As will be understood by those skilled in the art, the term "purified" does not require absolute purity, but rather is intended as a relative term. Thus, for example, a purified peptide, protein, virus, polynucleotide, or other active compound is one that is wholly or partially isolated from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to a peptide, protein, virus, polynucleotide, or other active compound that has been isolated from cells, cell culture medium, or other crude preparations and subjected to routine methods, such as, but not limited to, fractionation, chromatography, or electrophoresis, to remove various components of the initial preparation, such as proteins, cell debris, and other components.

[0084] A "recombinant" nucleic acid, protein, or virus is one that has a non-naturally occurring sequence or that has a sequence that is otherwise created by the artificial combination of two isolated segments of sequence. This artificial combination is often accomplished by either chemical synthesis or the artificial manipulation of isolated nucleic acid segments (e.g., genetic engineering techniques). A "non-naturally occurring" nucleic acid, protein, or virus is one that can be created through recombinant techniques, artificial manipulation, genetic or molecular biological manipulation, or molecular synthesis procedures and techniques such as those commonly practiced in the art.

[0085] By "reduce" it is meant a negative change (e.g., a decrease or reduction) of at least 5%, 10%, 25%, 30%, 40%, 50%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%.

[0086] "Reference" means a standard or control condition, e.g., a wild-type or non-mutated protein or polynucleotide. In some cases, the reference can be a healthy, uninfected subject or cell, e.g., a subject or cell that is not infected with influenza virus.

[0087] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference genome may be a subset or the entirety of a particular sequence, for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is generally at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably 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 at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides, or about 300 nucleotides, or any integer number about or between.

[0088] A "specifically binding" compound or antibody refers to a compound or antibody that recognizes and binds to a polypeptide, such as a viral polypeptide, a peptide, or a vaccine product, but does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample that naturally contains the polypeptide or peptide, such as a viral polypeptide.

[0089] Nucleic acid molecules useful in the methods described herein include any nucleic acid molecule that encodes a polypeptide or a fragment thereof as described. Such nucleic acid molecules do not need to be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" refers to pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes), or portions thereof, under various stringency conditions. (See, e.g., Wahl, GM and SL Berger, (1987), Methods Enzymol., 152:399; Kimmel, AR, (1987), Methods Enzymol. 152:507.)

[0090] As an example, stringent salt concentrations are usually less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, 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 usually include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, concentration of detergent (e.g., sodium dodecyl sulfate (SDS)), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are achieved by combining these various conditions as needed. In a preferred embodiment, hybridization is performed at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization is performed 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 a most preferred embodiment, hybridization is performed at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to one of skill in the art.

[0091] In most applications, the washing steps following hybridization are also of various stringencies. Wash stringency conditions can be defined by salt concentration and temperature. As mentioned above, washing stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentrations of the washing steps are preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions of the washing steps usually include a temperature 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 steps are performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing steps are performed 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 carried out at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations of these conditions will 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.

[0092] The term "substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% or greater identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, or at least 80%, or 85%, or at least 90%, 93%, 95%, or equal thereto, or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0093] "Sequence identity" refers to the similarity between amino acid or nucleic acid sequences expressed in terms of the similarity between the sequences. Sequence identity may often be measured in terms of percentage of identity (similarity or homology), the higher the percentage, the more identical the sequences are. Homologs or variants of a given gene or protein will have a relatively high degree of sequence identity when aligned using standard methods. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, 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. In an exemplary approach to determine the degree of identity, the BLAST program may be used, e.g. -3 ~e -100A probability score of 0.05 indicates closely related sequences. In addition, other programs and alignment algorithms are described, 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 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 on the Internet from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.), and for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0094] "Subject" refers to a vertebrate, e.g., a mammal, including but not limited to a human, a non-human primate, or a non-human animal or mammal, such as a cow, horse, dog, sheep, or feline mammal, or a sheep, goat, llama, camel, ferret, or rodent (rat, mouse), gerbil, or hamster, etc. "Subject" may also refer to a non-human animal, or an avian vertebrate. A non-human subject or a non-human animal subject may also be referred to as a "veterinary subject." In a non-limiting example, the subject is a subject infected with a pathogen, such as an influenza virus, e.g., an H1 or H3 virus, or at risk of infection by such a virus, or susceptible to such infection. In some embodiments described herein, the subject is a human subject, such as a patient. In some embodiments described herein, the subject is a non-human subject, e.g., a non-human animal subject or a veterinary subject.

[0095] Ranges provided herein are understood to be shorthand for all values ​​within that range, for example, a range of 1 to 50 is understood to include 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, e.g., any number, combination of numbers, or subranges (including the first and last values, and any values ​​therebetween) from 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, e.g., consecutively, e.g., 100 or more.

[0096] As used herein, the terms "treat", "treating", "treatment" and the like refer to reducing, lowering, reducing, neutralizing, alleviating, ameliorating, or eliminating a disease, condition, disorder, or pathology, and / or symptoms associated therewith. Without intending to be limiting, "treating" typically relates to a therapeutic intervention that may be performed after a disease, condition, disorder, or pathology, and / or symptoms associated therewith, has begun to develop, such as reducing the severity of the disease, and the like, and associated signs and symptoms. It is understood that, without precluding, treating a disorder or condition does not require the complete elimination of the disease, condition, disorder, pathology, or symptoms associated therewith.

[0097] As used herein, the terms "prevent", "preventing", "prevention", "prophylactic treatment" and the like refer to inhibiting or blocking the full or full-blown development of a disease state or disease in a subject, or reducing the likelihood of developing a disease, disorder, or condition, in a subject who does not have the disease, disorder, or condition, but is at risk of or susceptible to developing it.

[0098] As referred to herein, a "transformed" cell is a cell into which a nucleic acid molecule or polynucleotide sequence has been introduced by molecular biology techniques. As used herein, the term "transformation" encompasses all techniques by which a nucleic acid molecule or polynucleotide can be introduced into such a cell, including transfection with a viral vector, transformation with a plasmid vector, and introduction of naked nucleic acid (DNA or RNA, e.g., mRNA) by electroporation, lipofection, particle gun acceleration, or other methods known and practiced in the art.

[0099] "Vaccine" refers to a preparation of immunogenic material (e.g., protein or nucleic acid), such as a protein or peptide antigen, capable of stimulating (eliciting) an immune response, administered to a subject to treat a disease, condition, or pathology, or to prevent or protect against a disease, condition, or pathology, such as an infectious disease, e.g., viral infection. The immunogenic material may include, for example, attenuated or killed microorganisms (such as attenuated viruses), or antigenic proteins, peptides, DNA, or RNA derived from such microorganisms. Vaccines may induce a prophylactic (preventative) immune response in a subject. They may also induce a therapeutic immune response in a subject. As discussed above, vaccine administration methods vary depending on the vaccine and may include routes or means such as inoculation (intravenous or subcutaneous injection), ingestion, inhalation, or other forms of administration known and practiced in the medical arts. Inoculation may be delivered by any of several routes, including parenterally, such as intravenously, subcutaneously, or intramuscularly. The vaccine may also be administered with an adjuvant to boost the immune response. The vaccine may be administered to a human subject, a non-human subject, or a veterinary subject. The terms vaccine and immunogen are used interchangeably herein. In an embodiment, a monovalent immunogen / vaccine contains one non-natural broadly reactive influenza HA or NA immunogenic polypeptide antigen as described herein, or one HA or NA influenza virus antigen of a particular type or subtype. In an embodiment, a bivalent immunogen / vaccine contains two of the same or different non-natural broadly reactive influenza HA or NA immunogenic polypeptide antigens as described herein, and / or two of the same or different HA or NA influenza virus antigens of a particular type or subtype. In an embodiment, a multivalent immunogen / vaccine contains at least two (or more than two) non-natural broadly reactive influenza HA or NA immunogenic polypeptide antigens as described herein, and / or at least two HA or NA influenza virus antigens of a particular type or subtype.In some embodiments, the multivalent immunogen / vaccine contains a mixture of different influenza immunogenic polypeptide antigens as described and exemplified herein, and / or 2, 3, 4, 5, 6, 7, 8, 9, or 10 influenza polypeptide antigens as described and exemplified herein, including HA or NA influenza virus antigens of a particular type or subtype. In some embodiments, the influenza immunogen / vaccine preparation is bivalent. In some embodiments, the influenza immunogen / vaccine preparation is multivalent, e.g., octavalent.

[0100] As used herein, a "vector" refers to a nucleic acid (polynucleotide) molecule into which a foreign nucleic acid can be inserted without destroying the vector's ability to replicate and / or integrate in a host cell. A vector may contain a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. An insertion vector is capable of inserting itself into a host nucleic acid. A vector may also contain one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of the inserted gene or genes in a host cell. In some embodiments of the present disclosure, the vector encodes an influenza HA, NA, or M1 protein. 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).

[0101] "Virus-like particle (VLP)" refers to a viral particle that is composed of one of many viral structural proteins, but lacks the viral genome. Because VLPs lack the viral genome, they are non-infectious, resulting in safer and potentially more economical vaccines and vaccine products. In addition, VLPs can often be produced by heterologous expression and can be easily purified. Most VLPs contain at least a viral core protein that facilitates budding and release of the particle from the host cell. One example of such a core protein is influenza M1. In some embodiments herein, influenza VLPs contain HA, NA and M1 proteins. In some cases, influenza VLPs can be produced by transfection of host cells with plasmids encoding HA, NA and M1 proteins. After incubating the transfected cells for an appropriate time to allow protein expression (e.g., approximately 72 hours), the VLPs can be isolated from the cell culture supernatant. By way of example, a protocol for purifying or isolating influenza VLPs from cell supernatant involves low speed centrifugation (to remove cell debris), vacuum filtration, and ultracentrifugation of the VLPs through 20% glycerol. Virus-like particles may also include subviral particles (SVPs), which constitute particles that are typically smaller in size than viruses and do not contain the viral capsid or genome.

[0102] As used herein, the term "or" is understood to be inclusive unless specifically stated or clear from the context. As used herein, the terms "a," "an," and "the" are understood to be singular or plural, unless specifically stated or clear from the context. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Thus, "comprising A or B" means including A, or B, or A and B. It should be further understood that all base sizes or amino acid sizes and all molecular weights or molecular weight values ​​given for nucleic acids or polypeptides are approximate and are provided for illustration purposes.

[0103] As used herein, unless specifically stated or clear from the context, the term "about" is understood to be within normal tolerances in the art, e.g., within 2 standard deviations of the mean. "About" may be understood to be 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 clear from the context, all numerical values ​​provided herein are modified by the term about.

[0104] The recitation of a list of chemical groups in any definition of a variable herein includes a definition of that variable as any single group or combination of the listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0105] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein. [Brief description of the drawings]

[0106] [Figure 1]1 shows an illustrative timeline of a study conducted to evaluate the efficacy of the polypeptides as immunogens (e.g., vaccines) to protect against and / or reduce the effects of post-immunization viral challenge and severe disease using pre-immunized naive ferrets immunized (vaccinated) with a combination of broadly reactive influenza HA polypeptide immunogens described herein (Example 2). As shown in the timeline, groups of animals were pre-immunized by infecting them with influenza virus strains A / California / 2009 (H1N1), A / Panama / 1999 (H3N2), and B / Hong Kong / 2001 (IBV, influenza B virus) 60 days (day -60) prior to immunization / vaccination of the animals with the broadly reactive influenza HA polypeptide immunogens. These strains have been found historically in normal human populations and mimic in the animals the typical viral load or viral components that would be found in a normal pre-immunized human population prior to immunizing / vaccinating the animals with the broadly reactive HA polypeptide immunogens described herein. On day -30, animals were bled to confirm seroconversion. On days 0 and 28 of the study, ferrets were bled and immunized / vaccinated with broadly reactive HA polypeptide immunogen and c-di-AMP adjuvant (15 μg + 50 μg c-di-AMP per antigen) as described herein (Example 2). Four weeks after the second vaccination, animals were challenged with influenza viruses A / Brisbane / 02 / 2018 (H1N1), B / Washington / 02 / 2019 (IBV), or A / Vietnam / 1203 / 2004 (H5N1) (study day 56). Nasal washes were performed on days 1, 3, 5, and 7 post-infection between challenge (day 56) and the end of the study (day 60). Animals were monitored daily for clinical signs and body weights during this period. [Figure 2A]A graph is presented showing the results of an ELISA analysis performed on day 56 of the study described in Example 2 and illustrated in FIG. 1 using serum obtained after obtaining blood from immunized ferrets. Sera were tested for the presence of a total IgG antibody response in ferrets after the second vaccination. The amount of antibody binding to different immunogenic HA polypeptide antigens coated on a microtiter plate was evaluated. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as the adjuvant. The immunized / vaccinated animal group shown is a pre-immunized ferret that received an octavalent Cobra vaccine. Total IgG antibody titers were determined against each of the eight HA immunogenic polypeptide antigens (Cobra antigens) as shown on the x-axis. The y-axis shows OD414 nm values. Each point represents an individual ferret. For each independent experiment, serum was assayed in duplicate. The results demonstrated that immunization with the octavalent HA immunogenic polypeptide antigen (Cobra antigen) described herein induced an immune response and the production of antibodies that bound to all components of the HA immunogenic polypeptide / vaccine (A and C). An OD value of 1 was chosen as the minimum value due to non-specific binding to the his-tag present on the polypeptide. [Figure 2B]A graph is presented showing the results of an ELISA analysis performed on day 56 of the study described in Example 2 and illustrated in Figure 1, using serum obtained after obtaining blood from immunized ferrets. Sera were tested for the presence of a total IgG antibody response in ferrets after the second vaccination. The amount of antibodies binding to the different immunogenic HA polypeptide antigens coated on a microtiter plate was evaluated. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as adjuvant. The immunized / vaccinated animal group shown is a pre-immunized ferret that received a mock vaccine. Total IgG antibody titers were determined against each of the eight HA immunogenic polypeptide antigens (Cobra antigens) as shown on the x-axis. The y-axis shows the OD414 nm value. Each point represents an individual ferret. For each independent experiment, serum was assayed in duplicate. Antibodies from pre-immunized mock animals bound to H1, N1 and N2 HA antigens. An OD value of 1 was chosen as the minimum value due to non-specific binding to the his-tag present on the polypeptide. [Figure 2C]A graph is presented showing the results of an ELISA analysis performed on day 56 of the study described in Example 2 and illustrated in FIG. 1 using serum obtained after obtaining blood from immunized ferrets. Sera were tested for the presence of a total IgG antibody response in ferrets after the second vaccination. The amount of antibody binding to different immunogenic HA polypeptide antigens coated on a microtiter plate was evaluated. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as the adjuvant. The immunized / vaccinated animal group shown is naive ferrets administered the octavalent Cobra vaccine. Total IgG antibody titers were determined against each of the eight HA immunogenic polypeptide antigens (Cobra antigens) as shown on the x-axis. The y-axis shows OD414 nm values. Each point represents an individual ferret. For each independent experiment, serum was assayed in duplicate. The results demonstrated that immunization with the octavalent HA immunogenic polypeptide antigen (Cobra antigen) described herein induced an immune response and the production of antibodies that bound to all components of the HA immunogenic polypeptide / vaccine (A and C). An OD value of 1 was chosen as the minimum value due to non-specific binding to the his-tag present on the polypeptide. [Figure 2D]A graph is presented showing the results of an ELISA analysis performed on day 56 of the study described in Example 2 and illustrated in Figure 1, using serum obtained after obtaining blood from immunized ferrets. Sera were tested for the presence of a total IgG antibody response in ferrets after the second vaccination. The amount of antibodies binding to the different immunogenic HA polypeptide antigens coated on a microtiter plate was evaluated. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as adjuvant. The immunized / vaccinated animal group shown is naive ferrets that received a mock vaccine. Total IgG antibody titers were determined against each of the eight HA immunogenic polypeptide antigens (Cobra antigens) as shown on the x-axis. The y-axis shows OD414 nm values. Each point represents an individual ferret. For each independent experiment, serum was assayed in duplicate. Antibodies from naive mock animals showed no binding to the HA antigen. An OD value of 1 was chosen as the minimum value due to non-specific binding to the his-tag present on the polypeptide. [Figure 3A]Graphs are presented of the results of ELISA analyses performed to compare pre- and post-vaccination total IgG antibody responses in pre-immunized groups of ferrets on days 0 and 56 of the study described in FIG. 1 using sera obtained from pre-immunized ferrets immunized with HA immunogenic polypeptide antigen or from pre-immunized mock immunized ferrets. Sera were collected for each pre-immunized ferret immunized with HA polypeptide immunogen (Cobra) prior to immunization / vaccination (day 0 (d0)) and after the final immunization / vaccination (day 56 (d56)). The results demonstrated that pre-immunized ferrets immunized with the HA immunogenic polypeptide antigen described herein had an increase in antibodies against all HA components of the immunogenic polypeptide antigen / vaccine and had a statistically significant increase in antibodies generated against the Z1, NG3, IAN8, Q6, and BC2 immunogens. One-way ANOVA was used to analyze statistical differences between ELISA results on d0 and d56 for each group (Figures 2A-D) by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 3B]Graphs are presented showing the results of ELISA analyses performed to compare total IgG antibody responses before and after vaccination in pre-immunized groups of ferrets on days 0 and 56 of the study described in FIG. 1 using serum obtained from pre-immunized ferrets immunized with HA immunogenic polypeptide antigens or from pre-immunized mock immunized ferrets. Serum was collected for mock vaccinated pre-immunized ferrets before immunization / vaccination (day 0 (d0)) and after the final immunization / vaccination (day 56 (d56)). Results demonstrated that pre-immunized ferrets immunized with HA immunogenic polypeptide antigens described herein had an increase in antibodies against all HA components of the immunogenic polypeptide antigen / vaccine, and a statistically significant increase in antibodies generated against the Z1, NG3, IAN8, Q6, and BC2 immunogens. One-way ANOVA was used to analyze statistical differences between d0 and d56 ELISA results (FIGS. 2A-D) for each group by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p-value less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 4A]A graph showing the results of ferret serum HAI antibody titers against influenza H1N1 viruses before and after vaccination is presented. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as the adjuvant. Serum was collected before vaccination to perform HAI assays against a panel of six H1N1 influenza viruses. Viruses listed on the x-axis are: A / Solomon Island / 03 / 2006 (SI / 06), A / Brisbane / 59 / 2007 (Bris / 07), A / California / 07 / 2009 (CA / 09), A / Michigan / 45 / 2015 (Mich / 15), A / Brisbane / 02 / 2018 (Bris / 18), and A / Guangdong-Monan / SWL1536 / 2019 (GD / 19). The y-axis shows the log2 HAI titers for each vaccination group, which are presented as absolute mean ± SEM. Dotted lines indicate HAI titers ranging from 1:40 (lower line) and 1:80 (upper line). Non-parametric one-way ANOVA was used to analyze statistical differences between HAI titers on days 0 and 56 for each vaccine group by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p-value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). The results showed that the pre-immunized mock animal group had statistically significantly lower HAI titers for Mich / 15 and Bris / 18. Pre-immunized animals immunized with HA immunogenic polypeptides (COBRA antigens) maintained high HAI titers, and naive animals immunized with HA immunogenic polypeptides (COBRA antigens) reached HAI titers of 1:40 against pandemic-like virus strains, except for Guangdong / 19. [Figure 4B]A graph showing the results of ferret serum HAI antibody titers against influenza H1N1 viruses before and after vaccination is presented. Ferrets were vaccinated intranasally twice, 4 weeks apart, with c-di-AMP as the adjuvant. Serum was collected 4 weeks after the second vaccination to perform HAI assays against a panel of six H1N1 influenza viruses. Viruses listed on the x-axis are: A / Solomon Island / 03 / 2006 (SI / 06), A / Brisbane / 59 / 2007 (Bris / 07), A / California / 07 / 2009 (CA / 09), A / Michigan / 45 / 2015 (Mich / 15), A / Brisbane / 02 / 2018 (Bris / 18), and A / Guangdong-Monan / SWL1536 / 2019 (GD / 19). The y-axis shows the log2 HAI titers for each vaccination group, which are presented as absolute mean ± SEM. Dotted lines indicate HAI titers ranging from 1:40 (lower line) and 1:80 (upper line). Non-parametric one-way ANOVA was used to analyze statistical differences between HAI titers on days 0 and 56 for each vaccine group by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p-value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). The results showed that the pre-immunized mock animal group had statistically significantly lower HAI titers for Mich / 15 and Bris / 18. Pre-immunized animals immunized with HA immunogenic polypeptides (COBRA antigens) maintained high HAI titers, and naive animals immunized with HA immunogenic polypeptides (COBRA antigens) reached HAI titers of 1:40 against pandemic-like virus strains, except for Guangdong / 19. [Figure 5A]Graphs are presented showing the results of ferret serum HAI antibody titers against influenza H3N2 viruses before and after vaccination. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as the adjuvant. Vaccine groups were pre-immunized ferrets receiving octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black bars), naive ferrets receiving octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (white bars), or pre-immunized ferrets receiving mock vaccination (gray bars). Serum was collected prior to vaccination for HAI assay against a panel of six H3N2 influenza viruses. Viruses listed on the x-axis are: A / Switzerland / 9715293 / 2013 (Switzerland / 13), A / Hong Kong / 4801 / 2014 (HK / 14), A / Singapore / IFNIMH-16-0019 / 2016 (Sing / 16), A / Kansas / 14 / 2017 (KS / 17), A / South Australia / 34 / 2019 (SA / 19), and A / Hong Kong / 2671 / 2019 (HK / 19). The y-axis shows the log2 HAI titers for each vaccination group, which are presented as absolute mean ± SEM. The dotted lines indicate HAI titers in the range of 1:40 (lower line) and 1:80 (upper line). Non-parametric one-way ANOVA was used to analyze statistical differences between HAI titers on days 0 and 56 for each vaccinated animal group by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p-value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 5B]Graphs are presented showing the results of ferret serum HAI antibody titers against influenza H3N2 viruses before and after vaccination. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as the adjuvant. Vaccine groups were pre-immunized ferrets receiving octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black bars), naive ferrets receiving octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (white bars), or pre-immunized ferrets receiving mock vaccination (gray bars). Serum was collected 4 weeks after the second vaccination for HAI assay against a panel of six H3N2 influenza viruses. Viruses listed on the x-axis are: A / Switzerland / 9715293 / 2013 (Switzerland / 13), A / Hong Kong / 4801 / 2014 (HK / 14), A / Singapore / IFNIMH-16-0019 / 2016 (Sing / 16), A / Kansas / 14 / 2017 (KS / 17), A / South Australia / 34 / 2019 (SA / 19), and A / Hong Kong / 2671 / 2019 (HK / 19). The y-axis shows the log2 HAI titers for each vaccination group, which are presented as absolute mean ± SEM. The dotted lines indicate HAI titers in the range of 1:40 (lower line) and 1:80 (upper line). Non-parametric one-way ANOVA was used to analyze statistical differences between HAI titers on days 0 and 56 for each vaccinated animal group by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p-value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 6A]Graphs are presented showing ferret serum HAI antibody titer results for influenza B virus (IBV) before and after vaccination. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as the adjuvant. Vaccine groups were pre-immunized ferrets receiving octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black bars), naive ferrets receiving octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (white bars), or pre-immunized ferrets receiving mock vaccination (gray bars). Serum was collected prior to vaccination for HAI assay against a panel of six IBV influenza viruses. Viruses listed on the x-axis were B / Florida / 04 / 2006 (B / FL / 06), B / Massachusetts / 02 / 2012 (B / Mass / 12), B / Phuket / 3073 / 2013 (B / Phuk / 13) for the Yamagata-like lineage, and B / Brisbane / 60 / 2008 (B / Bris / 08), B / Colordado / 06 / 2017 (B / CO / 17), and B / Washington / 02 / 2019 (B / WA / 19) for the Victoria-like lineage. The y-axis shows the log2 HAI titers for each vaccination group, which are presented as absolute mean ± SEM. Dotted lines indicate HAI titers ranging from 1:40 (lower line) to 1:80 (upper line). Non-parametric one-way ANOVA was used to analyze statistical differences between HAI titers on days 0 and 56 for each vaccine group by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p-value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 6B]Graphs are presented showing ferret serum HAI antibody titer results for influenza B virus (IBV) before and after vaccination. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as adjuvant. Vaccine groups were pre-immunized ferrets receiving octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black bars), naive ferrets receiving octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (white bars), or pre-immunized ferrets receiving mock vaccination (gray bars). Serum was collected 4 weeks after the second vaccination for HAI assay against a panel of six IBV influenza viruses. Viruses listed on the x-axis were B / Florida / 04 / 2006 (B / FL / 06), B / Massachusetts / 02 / 2012 (B / Mass / 12), B / Phuket / 3073 / 2013 (B / Phuk / 13) for the Yamagata-like lineage, and B / Brisbane / 60 / 2008 (B / Bris / 08), B / Colordado / 06 / 2017 (B / CO / 17), and B / Washington / 02 / 2019 (B / WA / 19) for the Victoria-like lineage. The y-axis shows the log2 HAI titers for each vaccination group, which are presented as absolute mean ± SEM. Dotted lines indicate HAI titers ranging from 1:40 (lower line) to 1:80 (upper line). Non-parametric one-way ANOVA was used to analyze statistical differences between HAI titers on days 0 and 56 for each vaccine group by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p-value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 7A]Serum HAI antibody titers in test ferrets against influenza H5 viruses before and after vaccination are shown. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as the adjuvant. Vaccine groups were pre-immunized ferrets that received octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black bars), naive ferrets that received octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (white bars), or pre-immunized ferrets that received a mock vaccination (gray bars). Serum was collected prior to vaccination for HAI assay against a panel of six H5 influenza viruses: A / Vietnam / 1203 / 2004 (H5N1, Vn / 04), A / Wu / Mongolia / 244 / 2005 (H5N1, ws / Mo / 05), A / Egypt / 321 / 2007 (H5N1, Eg / 07), A / Hupei / 01 / 2010 (H5N1, Hu / 10), A / Guizhou / 01 / 2013 (H5N1, Gu / 13). A / Sichuan / 26221 / 2014 (H5N6, Si / 14). The y-axis shows the log2 HAI titers for each vaccination group, which are presented as absolute mean ± SEM. The dotted lines indicate HAI titers in the range of 1:40 (lower line) and 1:80 (upper line). Non-parametric one-way ANOVA was used to analyze statistical differences between HAI titers on days 0 and 56 for each vaccine group by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p-value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 7B]Serum HAI antibody titers in test ferrets against influenza H5 viruses before and after vaccination are shown. Ferrets were vaccinated intranasally twice, 4 weeks apart, with c-di-AMP as the adjuvant. Vaccine groups were pre-immunized ferrets that received octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black bars), naive ferrets that received octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (white bars), or pre-immunized ferrets that received a mock vaccination (gray bars). Serum was collected 4 weeks after the second vaccination for HAI assays against a panel of six H5 influenza viruses: A / Vietnam / 1203 / 2004 (H5N1, Vn / 04), A / Wu / Mongolia / 244 / 2005 (H5N1, ws / Mo / 05), A / Egypt / 321 / 2007 (H5N1, Eg / 07), A / Hupei / 01 / 2010 (H5N1, Hu / 10), A / Guizhou / 01 / 2013 (H5N1, Gu / 13). A / Sichuan / 26221 / 2014 (H5N6, Si / 14). The y-axis shows the log2 HAI titers for each vaccination group, which are presented as absolute mean ± SEM. The dotted lines indicate HAI titers in the range of 1:40 (lower line) and 1:80 (upper line). Non-parametric one-way ANOVA was used to analyze statistical differences between HAI titers on days 0 and 56 for each vaccine group by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p-value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 8A]Figure 8 shows the weight and survival curves of ferrets after challenge with influenza H5N1 virus. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as adjuvant. Vaccine groups were pre-immunized ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black line), naive ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black line with circles), or pre-immunized mock-immunized ferrets (gray line). Four weeks after the final immunization / vaccination, ferrets were infected intranasally with a lethal dose of virus A / Vietnam / 1203 / 2004 (105 PFU) in a volume of 1 mL. Animals were observed for clinical signs and their weights were recorded daily after infection (Figure 8A). [Figure 8B] Figure 1 shows the weight and survival curves of ferrets after challenge with influenza H5N1 virus. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as adjuvant. Vaccine groups were pre-immunized ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black line), naive ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black line with circles), or pre-immunized mock-immunized ferrets (gray line). Four weeks after the final immunization / vaccination, ferrets were infected intranasally with a lethal dose of virus A / Vietnam / 1203 / 2004 (105 PFU) in a volume of 1 mL. Post-infection survival curve data show that all animals survived the lethal virus challenge. [Figure 9A]Figure 1 shows the weight curves of ferrets after challenge with influenza virus. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as adjuvant. Vaccine groups were pre-immunized ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black line), naive ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black line with circles), pre-immunized ferrets administered a mock vaccination (gray line), or naive ferrets administered a mock vaccination (dashed black line). Four weeks after the final vaccination, ferrets were infected intranasally with influenza virus A / Brisbane / 02 / 2018 (108 PFU) in a volume of 1 mL. Animals were observed for clinical signs and their weights were recorded daily after infection. The statistical differences in weight loss are shown in the tables of Figure 10A and Figure 10B. [Figure 9B] Figure 1 shows the weight curves of ferrets after challenge with influenza virus. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as adjuvant. Vaccine groups were pre-immunized ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black line), naive ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black line with circles), pre-immunized ferrets administered a mock vaccination (gray line), or naive ferrets administered a mock vaccination (dashed black line). Four weeks after the final vaccination, ferrets were infected intranasally with influenza virus (b)B / Washington / 02 / 2019 (107 PFU) in a volume of 1 mL. Animals were observed for clinical signs and their weights were recorded daily after infection. The statistical differences in weight loss are shown in the tables of Figure 10A and Figure 10B. [Figure 10A]A table showing the statistical differences in weight loss between groups of immunized ferrets challenged with different virus strains (H1N1 vs. IBV) is presented. Weight loss values ​​from ferrets challenged with H1N1 A / Brisbane / 02 / 2018 (FIG. 10A), as described above in FIG. 9A and FIG. 9B, were analyzed for statistical differences on each infection day by two-way ANOVA with multiple comparisons by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p-value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant). [Figure 10B] A table showing the statistical difference in weight loss between groups of immunized ferrets challenged with different virus strains (H1N1 vs. IBV) is presented. Weight loss values ​​from ferrets challenged with IBV B / Washington / 02 / 2019, as described above in Figures 9A and 9B, were analyzed for statistical differences on each infection day by two-way ANOVA with multiple comparisons by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p-value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant). [Figure 11A] Figure 1 shows a graph of viral titers in the upper respiratory tract of ferrets following infection with influenza virus A / Brisbane / 02 / 2018 (H1N1). Ferrets were vaccinated intranasally twice, 4 weeks apart, with c-di-AMP as the adjuvant. Vaccine groups are indicated on the x-axis. Four weeks after the second vaccination, animal groups were challenged with H1N1 A / Brisbane / 02 / 2018 virus. Nasal washes were taken from animals on day 1 post-infection and viral titers were determined. Viral titers in the nasal washes are presented as PFU / mL, shown on the y-axis. Each point represents an individual ferret. [Figure 11B]Figure 1 shows a graph of viral titers in the upper respiratory tract of ferrets following infection with influenza virus A / Brisbane / 02 / 2018 (H1N1). Ferrets were vaccinated intranasally twice, 4 weeks apart, with c-di-AMP as the adjuvant. Vaccine groups are indicated on the x-axis. Four weeks after the second vaccination, animal groups were challenged with H1N1 A / Brisbane / 02 / 2018 virus. Nasal washes were taken from animals 3 days post-infection and viral titers were determined. Viral titers in the nasal washes are presented as PFU / mL, shown on the y-axis. Each point represents an individual ferret. [Figure 11C] Figure 1 shows a graph of viral titers in the upper respiratory tract of ferrets following infection with influenza virus A / Brisbane / 02 / 2018 (H1N1). Ferrets were vaccinated intranasally twice, 4 weeks apart, with c-di-AMP as the adjuvant. Vaccine groups are indicated on the x-axis. Four weeks after the second vaccination, animal groups were challenged with H1N1 A / Brisbane / 02 / 2018 virus. Nasal washes were taken from animals 5 days post-infection and viral titers were determined. Viral titers in the nasal washes are presented as PFU / mL, shown on the y-axis. Each point represents an individual ferret. [Figure 12A] Figure 1 shows a graph of titers in nasal washes of ferrets following infection with influenza virus B / Washington / 02 / 2019 (IBV). Ferrets were vaccinated intranasally twice, 4 weeks apart, with c-di-AMP as the adjuvant. Vaccine groups are indicated on the x-axis. Four weeks after the second vaccination, animal groups were challenged with IBV B / Washington / 02 / 2019 virus. Nasal washes were performed 1 day post-infection and viral titers were determined. Viral titers in nasal washes are presented as PFU / mL, shown on the y-axis. Each point represents an individual ferret. [Figure 12B]Figure 1 shows a graph of titers in nasal washes of ferrets following infection with influenza virus B / Washington / 02 / 2019 (IBV). Ferrets were vaccinated intranasally twice, 4 weeks apart, with c-di-AMP as the adjuvant. Vaccine groups are indicated on the x-axis. Four weeks after the second vaccination, animal groups were challenged with IBV B / Washington / 02 / 2019 virus. Nasal washes were performed 3 days post-infection and viral titers were determined. Viral titers in nasal washes are presented as PFU / mL, shown on the y-axis. Each point represents an individual ferret. [Figure 12C] Figure 1 shows a graph of titers in nasal washes of ferrets following infection with influenza virus B / Washington / 02 / 2019 (IBV). Ferrets were vaccinated intranasally twice, 4 weeks apart, with c-di-AMP as the adjuvant. Vaccine groups are indicated on the x-axis. Four weeks after the second vaccination, animal groups were challenged with IBV B / Washington / 02 / 2019 virus. Nasal washes were performed 5 days post-infection and viral titers were determined. Viral titers in nasal washes are presented as PFU / mL, shown on the y-axis. Each point represents an individual ferret. [Figure 13A]Figure 1 shows a graph of the results of a hemagglutinin inhibition (HAI) assay performed on serum from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination. Serum collected from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination was assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated between 2012-2019 (x-axis). Log2 HAI titers are reported as absolute mean 6 standard error of the mean (SEM) (y-axis). The lower dotted line represents an HAI titer of 1:40 and the upper dotted line represents an HAI titer of 1:80. Mice were vaccinated with 3 mg of recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen as follows: mock vaccinated; HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) with Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). The H3N2 viruses belong to the following clades: Tx / 12 (3c2), Switz / 13 (3c3.a), HK / 14 (3c.2a), Sing / 16 (3c2.a1), Kan / 17 (3c3.a), Tx17 (3c3.a), Switz / 17 (3c3.a2), SA / 19 (3c2.ab / 131K), and HK / 19 (3c2.a1b / 137F). [Figure 13B]Figure 1 shows a graph of the results of a hemagglutinin inhibition (HAI) assay performed on serum from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination. Serum collected from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination was assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated between 2012-2019 (x-axis). Log2 HAI titers are reported as absolute mean 6 standard error of the mean (SEM) (y-axis). The lower dotted line represents an HAI titer of 1:40 and the upper dotted line represents an HAI titer of 1:80. Mice were vaccinated with 3 mg of recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen as follows: J1. HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) with Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). The H3N2 viruses belong to the following clades: Tx / 12 (3c2), Switz / 13 (3c3.a), HK / 14 (3c.2a), Sing / 16 (3c2.a1), Kan / 17 (3c3.a), Tx17 (3c3.a), Switz / 17 (3c3.a2), SA / 19 (3c2.ab / 131K), and HK / 19 (3c2.a1b / 137F). [Figure 13C]Graph showing results of hemagglutinin inhibition (HAI) assays performed on serum from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination. Serum collected from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination was assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated between 2012-2019 (x-axis). Log2 HAI titers are reported as absolute mean 6 standard error of the mean (SEM) (y-axis). The lower dotted line represents an HAI titer of 1:40 and the upper dotted line represents an HAI titer of 1:80. Mice were vaccinated with 3 mg of recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen as follows: J2. HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) with Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). The H3N2 viruses belong to the following clades: Tx / 12 (3c2), Switz / 13 (3c3.a), HK / 14 (3c.2a), Sing / 16 (3c2.a1), Kan / 17 (3c3.a), Tx17 (3c3.a), Switz / 17 (3c3.a2), SA / 19 (3c2.ab / 131K), and HK / 19 (3c2.a1b / 137F). [Figure 13D]Graph showing results of hemagglutinin inhibition (HAI) assays performed on serum from 75 influenza naive BALB / c mice (n=5 / group) 70 days after first vaccination. Serum collected from 75 influenza naive BALB / c mice (n=5 / group) 70 days after first vaccination was assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated between 2012-2019 (x-axis). Log2 HAI titers are reported as absolute mean 6 standard error of the mean (SEM) (y-axis). The lower dotted line represents an HAI titer of 1:40 and the upper dotted line represents an HAI titer of 1:80. Mice were vaccinated with 3 mg of recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen as follows: J3. HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) with Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). The H3N2 viruses belong to the following clades: Tx / 12 (3c2), Switz / 13 (3c3.a), HK / 14 (3c.2a), Sing / 16 (3c2.a1), Kan / 17 (3c3.a), Tx17 (3c3.a), Switz / 17 (3c3.a2), SA / 19 (3c2.ab / 131K), and HK / 19 (3c2.a1b / 137F). [Figure 13E]Figure 1 shows a graph of the results of a hemagglutinin inhibition (HAI) assay performed on serum from 75 influenza naive BALB / c mice (n=5 / group) 70 days after first vaccination. Serum collected from 75 influenza naive BALB / c mice (n=5 / group) 70 days after first vaccination was assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated between 2012-2019 (x-axis). Log2 HAI titers are reported as absolute mean 6 standard error of the mean (SEM) (y-axis). The lower dotted line represents an HAI titer of 1:40 and the upper dotted line represents an HAI titer of 1:80. Mice were vaccinated with 3 mg of recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen as follows: J4. HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) with Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). The H3N2 viruses belong to the following clades: Tx / 12 (3c2), Switz / 13 (3c3.a), HK / 14 (3c.2a), Sing / 16 (3c2.a1), Kan / 17 (3c3.a), Tx17 (3c3.a), Switz / 17 (3c3.a2), SA / 19 (3c2.ab / 131K), and HK / 19 (3c2.a1b / 137F). [Figure 13F]Figure 1 shows a graph of the results of a hemagglutinin inhibition (HAI) assay performed on serum from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination. Serum collected from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination was assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated between 2012-2019 (x-axis). Log2 HAI titers are reported as absolute mean 6 standard error of the mean (SEM) (y-axis). The lower dotted line represents an HAI titer of 1:40 and the upper dotted line represents an HAI titer of 1:80. Mice were vaccinated with 3 mg of recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen as follows: NG1. HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) with Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). The H3N2 viruses belong to the following clades: Tx / 12 (3c2), Switz / 13 (3c3.a), HK / 14 (3c.2a), Sing / 16 (3c2.a1), Kan / 17 (3c3.a), Tx17 (3c3.a), Switz / 17 (3c3.a2), SA / 19 (3c2.ab / 131K), and HK / 19 (3c2.a1b / 137F). [Figure 13G]Graph showing results of hemagglutinin inhibition (HAI) assays performed on serum from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination. Serum collected from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination was assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated between 2012-2019 (x-axis). Log2 HAI titers are reported as absolute mean 6 standard error of the mean (SEM) (y-axis). The lower dotted line represents an HAI titer of 1:40 and the upper dotted line represents an HAI titer of 1:80. Mice were vaccinated with 3 mg of recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen as follows: NG2. HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) with Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). The H3N2 viruses belong to the following clades: Tx / 12 (3c2), Switz / 13 (3c3.a), HK / 14 (3c.2a), Sing / 16 (3c2.a1), Kan / 17 (3c3.a), Tx17 (3c3.a), Switz / 17 (3c3.a2), SA / 19 (3c2.ab / 131K), and HK / 19 (3c2.a1b / 137F). [Figure 13H]Graph showing results of hemagglutinin inhibition (HAI) assays performed on serum from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination. Serum collected from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination was assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated between 2012-2019 (x-axis). Log2 HAI titers are reported as absolute mean 6 standard error of the mean (SEM) (y-axis). The lower dotted line represents an HAI titer of 1:40 and the upper dotted line represents an HAI titer of 1:80. Mice were vaccinated with 3 mg of recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen as follows: NG3. HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) with Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). The H3N2 viruses belong to the following clades: Tx / 12 (3c2), Switz / 13 (3c3.a), HK / 14 (3c.2a), Sing / 16 (3c2.a1), Kan / 17 (3c3.a), Tx17 (3c3.a), Switz / 17 (3c3.a2), SA / 19 (3c2.ab / 131K), and HK / 19 (3c2.a1b / 137F). [Figure 13I]Graph showing results of hemagglutinin inhibition (HAI) assays performed on serum from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination. Serum collected from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination was assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated between 2012-2019 (x-axis). Log2 HAI titers are reported as absolute mean 6 standard error of the mean (SEM) (y-axis). The lower dotted line represents an HAI titer of 1:40 and the upper dotted line represents an HAI titer of 1:80. Mice were vaccinated with 3 mg of recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen as follows: Switz / 13. HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) with Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). The H3N2 viruses belong to the following clades: Tx / 12 (3c2), Switz / 13 (3c3.a), HK / 14 (3c.2a), Sing / 16 (3c2.a1), Kan / 17 (3c3.a), Tx17 (3c3.a), Switz / 17 (3c3.a2), SA / 19 (3c2.ab / 131K), and HK / 19 (3c2.a1b / 137F). [Figure 13J]Graph showing results of hemagglutinin inhibition (HAI) assays performed on serum from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination. Serum collected from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination was assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated between 2012-2019 (x-axis). Log2 HAI titers are reported as absolute mean 6 standard error of the mean (SEM) (y-axis). The lower dotted line represents an HAI titer of 1:40 and the upper dotted line represents an HAI titer of 1:80. Mice were vaccinated with 3 mg of recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen as follows: HK / 14. HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) with Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). The H3N2 viruses belong to the following clades: Tx / 12 (3c2), Switz / 13 (3c3.a), HK / 14 (3c.2a), Sing / 16 (3c2.a1), Kan / 17 (3c3.a), Tx17 (3c3.a), Switz / 17 (3c3.a2), SA / 19 (3c2.ab / 131K), and HK / 19 (3c2.a1b / 137F). [Figure 13K]Graph showing results of hemagglutinin inhibition (HAI) assays performed on serum from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination. Serum collected from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination was assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated between 2012-2019 (x-axis). Log2 HAI titers are reported as absolute mean 6 standard error of the mean (SEM) (y-axis). The lower dotted line represents an HAI titer of 1:40 and the upper dotted line represents an HAI titer of 1:80. Mice were vaccinated with 3 mg of recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen as follows: Sing / 16. HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) with Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). The H3N2 viruses belong to the following clades: Tx / 12 (3c2), Switz / 13 (3c3.a), HK / 14 (3c.2a), Sing / 16 (3c2.a1), Kan / 17 (3c3.a), Tx17 (3c3.a), Switz / 17 (3c3.a2), SA / 19 (3c2.ab / 131K), and HK / 19 (3c2.a1b / 137F). [Figure 13L]Graph showing results of hemagglutinin inhibition (HAI) assays performed on serum from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination. Serum collected from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination was assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated between 2012-2019 (x-axis). Log2 HAI titers are reported as absolute mean 6 standard error of the mean (SEM) (y-axis). The lower dotted line represents an HAI titer of 1:40 and the upper dotted line represents an HAI titer of 1:80. Mice were vaccinated with 3 mg of recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen as follows: Kan / 17. HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) with Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). The H3N2 viruses belong to the following clades: Tx / 12 (3c2), Switz / 13 (3c3.a), HK / 14 (3c.2a), Sing / 16 (3c2.a1), Kan / 17 (3c3.a), Tx17 (3c3.a), Switz / 17 (3c3.a2), SA / 19 (3c2.ab / 131K), and HK / 19 (3c2.a1b / 137F). [Figure 13M]Graph showing results of hemagglutinin inhibition (HAI) assays performed on serum from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination. Serum collected from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination was assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated between 2012-2019 (x-axis). Log2 HAI titers are reported as absolute mean 6 standard error of the mean (SEM) (y-axis). The lower dotted line represents an HAI titer of 1:40 and the upper dotted line represents an HAI titer of 1:80. Mice were vaccinated with 3 mg of recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen as follows: Switz / 17. HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) with Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). The H3N2 viruses belong to the following clades: Tx / 12 (3c2), Switz / 13 (3c3.a), HK / 14 (3c.2a), Sing / 16 (3c2.a1), Kan / 17 (3c3.a), Tx17 (3c3.a), Switz / 17 (3c3.a2), SA / 19 (3c2.ab / 131K), and HK / 19 (3c2.a1b / 137F). [Figure 13N]Graph showing results of hemagglutinin inhibition (HAI) assays performed on serum from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination. Serum collected from 75 influenza naïve BALB / c mice (n=5 / group) 70 days after first vaccination was assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated between 2012-2019 (x-axis). Log2 HAI titers are reported as absolute mean 6 standard error of the mean (SEM) (y-axis). The lower dotted line represents an HAI titer of 1:40 and the upper dotted line represents an HAI titer of 1:80. Mice were vaccinated with 3 mg of recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen as follows: SA / 19. HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) with Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). The H3N2 viruses belong to the following clades: Tx / 12 (3c2), Switz / 13 (3c3.a), HK / 14 (3c.2a), Sing / 16 (3c2.a1), Kan / 17 (3c3.a), Tx17 (3c3.a), Switz / 17 (3c3.a2), SA / 19 (3c2.ab / 131K), and HK / 19 (3c2.a1b / 137F). [Figure 14]A graph showing lung viral titers on day 89 is shown. Lungs were harvested from n=3 mice per group on day 89 (3 days after A / Kansas / 14 / 2017 challenge) to assess the viral load present in the lung tissue. Vaccine groups are listed on the x-axis and PFU / g of lung tissue values ​​are reported on the y-axis. Non-parametric one-way ANOVA was used to analyze statistical differences between groups using Prism 9 software (GraphPad Software, Inc., San Diego, CA). P values ​​less than 0.05 were defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). [Figure 15A] A graph showing the results of a nest reduction assay (FRA) against a panel of H3N2 influenza viruses at day 72 performed using sera from pre-immunized mice to assess the presence of antibodies directed against the H3N2 virus panel is presented. Sera collected from 136 H1+H3 pre-immunized DBA / 2J mice (n=8 / group) vaccinated with monovalent and bivalent formulations of H1+H3 recombinant influenza immunogenic polypeptides (Cobra antigens) or WT rHA at day 72 post-primary infection were pooled for each group and assessed for FRA neutralization against a panel of historical H3N2 vaccine strains from 2016-2019. Sera from mice vaccinated with monovalent antigens were tested against the following H1N1: A / Singapore / IFNIMH-16-0019 / 2016. The lower dotted line represents 80% neutralization (Neut80), the middle dotted line represents 50% neutralization (Neut50) and the upper dotted line represents no neutralization of viral infection. [Figure 15B]A graph is presented showing the results of a nest reduction assay (FRA) against a panel of H3N2 influenza viruses at day 72 performed using sera from pre-immunized mice to assess the presence of antibodies directed against the H3N2 virus panel. Sera collected from 136 H1+H3 pre-immunized DBA / 2J mice (n=8 / group) vaccinated with monovalent and bivalent formulations of H1+H3 recombinant influenza immunogenic polypeptides (Cobra antigens) or WT rHA at day 72 post-primary infection were pooled for each group and assessed for FRA neutralization against a panel of historical H3N2 vaccine strains from 2016-2019. Sera from mice vaccinated with monovalent antigens were tested against the following H1N1: A / Kansas / 14 / 2017. The lower dotted line represents 80% neutralization (Neut80), the middle dotted line represents 50% neutralization (Neut50), and the upper dotted line represents no neutralization of viral infection. [Figure 15C] A graph is presented showing the results of a nest reduction assay (FRA) against a panel of H3N2 influenza viruses at day 72 performed using sera from pre-immunized mice to assess the presence of antibodies directed against the H3N2 virus panel. Sera collected from 136 H1+H3 pre-immunized DBA / 2J mice (n=8 / group) vaccinated with monovalent and bivalent formulations of H1+H3 recombinant influenza immunogenic polypeptides (Cobra antigens) or WT rHA at day 72 post-primary infection were pooled for each group and assessed for FRA neutralization against a panel of historical H3N2 vaccine strains from 2016-2019. Sera from mice vaccinated with monovalent antigens were tested against the following H1N1: A / Hong Kong / 2671 / 2019. The lower dotted line represents 80% neutralization (Neut80), the middle dotted line represents 50% neutralization (Neut50), and the upper dotted line represents no neutralization of viral infection. [Figure 15D]A graph showing the results of a nest reduction assay (FRA) against a panel of H3N2 influenza viruses at day 72 performed using sera from pre-immunized mice to assess the presence of antibodies directed against the H3N2 virus panel is presented. Sera collected from 136 H1+H3 pre-immunized DBA / 2J mice (n=8 / group) vaccinated with monovalent and bivalent formulations of H1+H3 recombinant influenza immunogenic polypeptides (Cobra antigens) or WT rHA at day 72 post-primary infection were pooled for each group and assessed for FRA neutralization against a panel of historical H3N2 vaccine strains from 2016-2019. Sera from mice vaccinated with a cocktail of bivalent H1+H3 antigens were tested against the following H1N1 viruses: A / Singapore / IFNIMH-16-0019 / 2016. The lower dotted line represents 80% neutralization (Neut80), the middle dotted line represents 50% neutralization (Neut50) and the upper dotted line represents no neutralization of viral infection. [Figure 15E] A graph showing the results of a nest reduction assay (FRA) against a panel of H3N2 influenza viruses at day 72 performed using sera from pre-immunized mice to assess the presence of antibodies directed against the H3N2 virus panel is presented. Sera collected from 136 H1+H3 pre-immunized DBA / 2J mice (n=8 / group) vaccinated with monovalent and bivalent formulations of H1+H3 recombinant influenza immunogenic polypeptides (Cobra antigens) or WT rHA at day 72 post-primary infection were pooled for each group and assessed for FRA neutralization against a panel of historical H3N2 vaccine strains from 2016-2019. Sera from mice vaccinated with a cocktail of bivalent H1+H3 antigens were tested against the following H1N1 viruses: A / Kansas / 14 / 2017. The lower dotted line represents 80% neutralization (Neut80), the middle dotted line represents 50% neutralization (Neut50) and the upper dotted line represents no neutralization of viral infection. [Figure 15F]A graph showing the results of a nest reduction assay (FRA) against a panel of H3N2 influenza viruses at day 72 performed using sera from pre-immunized mice to assess the presence of antibodies directed against the H3N2 virus panel is presented. Sera collected from 136 H1+H3 pre-immunized DBA / 2J mice (n=8 / group) vaccinated with monovalent and bivalent formulations of H1+H3 recombinant influenza immunogenic polypeptides (Cobra antigens) or WT rHA at day 72 post-primary infection were pooled for each group and assessed for FRA neutralization against a panel of historical H3N2 vaccine strains from 2016-2019. Sera from mice vaccinated with a cocktail of bivalent H1+H3 antigens were tested against the following H1N1 viruses: A / Hong Kong / 2671 / 2019. The lower dotted line represents 80% neutralization (Neut80), the middle dotted line represents 50% neutralization (Neut50) and the upper dotted line represents no neutralization of viral infection. [Figure 16A] A graph is presented showing the results of a nest reduction assay (FRA) against a panel of H1N1 influenza viruses performed using sera from pre-immunized mice at day 72. Sera collected from 136 H1+H3 pre-immunized DBA / 2J mice (n=8 / group) vaccinated with monovalent and bivalent formulations of H1+H3 recombinant influenza immunogenic polypeptides (Cobra antigens) or WT rHA at day 72 post-primary infection were pooled for each group and evaluated for FRA neutralization against a panel of historical H1N1 vaccine strains from 2009-2019. Sera from mice vaccinated with monovalent antigens were tested against the following H1N1: A / California / 07 / 2009. The bottom dotted line represents 80% neutralization (Neut80), the middle dotted line represents 50% neutralization (Neut50), and the top dotted line represents no neutralization of viral infection. [Figure 16B]A graph is presented showing the results of a nest reduction assay (FRA) against a panel of H1N1 influenza viruses performed using sera from pre-immunized mice at day 72. Sera collected from 136 H1+H3 pre-immunized DBA / 2J mice (n=8 / group) vaccinated with monovalent and bivalent formulations of H1+H3 recombinant influenza immunogenic polypeptides (Cobra antigens) or WT rHA at day 72 post-primary infection were pooled for each group and evaluated for FRA neutralization against a panel of historical H1N1 vaccine strains from 2009-2019. Sera from mice vaccinated with monovalent antigens were tested against the following H1N1: A / Brisbane / 2 / 2018. The bottom dotted line represents 80% neutralization (Neut80), the middle dotted line represents 50% neutralization (Neut50), and the top dotted line represents no neutralization of viral infection. [Figure 16C] A graph is presented showing the results of a nest reduction assay (FRA) against a panel of H1N1 influenza viruses performed using sera from pre-immunized mice at day 72. Sera collected from 136 H1+H3 pre-immunized DBA / 2J mice (n=8 / group) vaccinated with monovalent and bivalent formulations of H1+H3 recombinant influenza immunogenic polypeptides (Cobra antigens) or WT rHA at day 72 post-primary infection were pooled for each group and evaluated for FRA neutralization against a panel of historical H1N1 vaccine strains from 2009-2019. Sera from mice vaccinated with monovalent antigens were tested against the following H1N1: A / Guangdong Maonan / SWL1536 / 2019. The bottom dotted line represents 80% neutralization (Neut80), the middle dotted line represents 50% neutralization (Neut50), and the top dotted line represents no neutralization of viral infection. [Figure 16D]A graph is presented showing the results of a nest reduction assay (FRA) against a panel of H1N1 influenza viruses performed using sera from pre-immunized mice at day 72. Sera collected from 136 H1+H3 pre-immunized DBA / 2J mice (n=8 / group) vaccinated with monovalent and bivalent formulations of H1+H3 recombinant influenza immunogenic polypeptides (Cobra antigens) or WT rHA at day 72 post-primary infection were pooled for each group and evaluated for FRA neutralization against a panel of historical H1N1 vaccine strains from 2009-2019. Sera from mice vaccinated with a cocktail of bivalent H1+H3 antigens were tested against the following H1N1 viruses: A / California / 07 / 2009. The bottom dotted line represents 80% neutralization (Neut80), the middle dotted line represents 50% neutralization (Neut50), and the top dotted line represents no neutralization of viral infection. [Figure 16E] Graphs are presented showing the results of a nest reduction assay (FRA) against a panel of H1N1 influenza viruses performed using sera from pre-immunized mice at day 72. Sera collected from 136 H1+H3 pre-immunized DBA / 2J mice (n=8 / group) vaccinated with monovalent and bivalent formulations of H1+H3 recombinant influenza immunogenic polypeptides (Cobra antigens) or WT rHA at day 72 post-primary infection were pooled for each group and evaluated for FRA neutralization against a panel of historical H1N1 vaccine strains from 2009-2019. Sera from mice vaccinated with a cocktail of bivalent H1+H3 antigens were tested against the following H1N1 viruses: A / Brisbane / 2 / 2018. The bottom dotted line represents 80% neutralization (Neut80), the middle dotted line represents 50% neutralization (Neut50), and the top dotted line represents no neutralization of viral infection. [Figure 16F]Graphs are presented showing the results of nest reduction assays (FRA) against a panel of H1N1 influenza viruses performed using sera from pre-immunized mice at day 72. Sera collected from 136 H1+H3 pre-immunized DBA / 2J mice (n=8 / group) vaccinated with monovalent and bivalent formulations of H1+H3 recombinant influenza immunogenic polypeptides (Cobra antigens) or WT rHA at day 72 post-primary infection were pooled for each group and evaluated for FRA neutralization against a panel of historical H1N1 vaccine strains from 2009-2019. Sera from mice vaccinated with a cocktail of bivalent H1+H3 antigens were tested against the following H1N1 viruses: A / Guangdong Maonan / SWL1536 / 2019. The bottom dotted line represents 80% neutralization (Neut80), the middle dotted line represents 50% neutralization (Neut50), and the top dotted line represents no neutralization of viral infection. [Figure 17] Illustrated is an exemplary timeline of a study conducted to evaluate the efficacy of the polypeptide as an immunogen (e.g., vaccine) to protect against and / or reduce the impact of post-immunization viral challenge and severe disease using mice immunized (vaccinated) with a broadly reactive influenza HA polypeptide immunogen described herein (Example 2). In the animal study, 88 BALB / c mice (n=11) were immunized / vaccinated intramuscularly with broadly reactive influenza HA immunogen or wild-type HA in a virus-like particle (VLP) immunogen / vaccine formulated with ADDAVAX™ adjuvant at weeks 0, 4, and 8. Blood was collected from the animals at weeks 6 and 10 after vaccination and serum was separated for analysis. At week 12, all mice were challenged intranasally with 5×104 PFU of A / California / 07 / 2009 H1N1 virus. Lung tissues (n=3 / group) were harvested on days 3 and 6 post-infection (3 dpi and 6 dpi) and assessed for histopathology and virus titer. [Figure 18A]Graphs are presented showing the body weight and survival curves following influenza virus infection of mice immunized / vaccinated with broadly reactive recombinant influenza HA immunogens as described herein or wild-type rHA protein versus PBS control according to the protocol shown in FIG. 17. FIG. 18A shows the percentage of original body weight loss of mice following infection. Mice were observed for clinical signs for 14 days and their body weights were recorded daily following infection. The dotted line indicates 80% of the body weight on day 0 post-infection. Another group of 64 DBA / 2J mice were immunized / vaccinated intramuscularly with broadly reactive recombinant influenza HA immunogens or wild-type rHA immunogens / vaccines formulated with ADDAVAX™ adjuvant using the immunization / vaccination regimen described above. At week 12, all mice were intranasally infected with 8.75×106 PFU of A / Brisbane / 02 / 2018 H1N1 virus as a virus challenge. [Figure 18B] Graphs are presented showing the weight and survival curves following influenza virus infection of mice immunized / vaccinated with broadly reactive recombinant influenza HA immunogens as described herein or wild-type rHA protein versus PBS control according to the protocol shown in FIG. 17. FIG. 18B shows the survival curves following infection with A / California / 07 / 2009 virus. Another group of 64 DBA / 2J mice was immunized / vaccinated intramuscularly with broadly reactive recombinant influenza HA immunogens or wild-type rHA immunogens / vaccines formulated with ADDAVAX™ adjuvant using the immunization / vaccination regimen described above. At week 12, all mice were infected intranasally with 8.75×106 PFU of A / Brisbane / 02 / 2018 H1N1 virus as a virus challenge. [Figure 18C]Graphs are presented showing the weight and survival curves of mice immunized / vaccinated with broadly reactive recombinant influenza HA immunogens as described herein or wild-type rHA protein following influenza virus infection versus PBS control according to the protocol shown in FIG. 17. Another group of 64 DBA / 2J mice was immunized / vaccinated intramuscularly with broadly reactive recombinant influenza HA immunogens or wild-type rHA immunogens / vaccines formulated with ADDAVAX™ adjuvant using the immunization / vaccination regimen described above. At week 12, all mice were infected intranasally with 8.75×106 PFU of A / Brisbane / 02 / 2018 H1N1 virus as a virus challenge. FIG. 18C shows the weight loss curves of DBA / 2J mice following challenge with A / Brisbane / 02 / 2018 H1N1 virus. [Figure 18D] Graphs are presented showing the weight and survival curves following influenza virus infection of mice immunized / vaccinated with broadly reactive recombinant influenza HA immunogens as described herein or wild-type rHA protein versus PBS control according to the protocol shown in FIG. 17. Another group of 64 DBA / 2J mice was immunized / vaccinated intramuscularly with broadly reactive recombinant influenza HA immunogens or wild-type rHA immunogens / vaccines formulated with ADDAVAX™ adjuvant using the immunization / vaccination regimen described above. At week 12, all mice were infected intranasally with 8.75×106 PFU of A / Brisbane / 02 / 2018 H1N1 virus as a virus challenge. FIG. 18D shows the survival curves following challenge with A / Brisbane / 02 / 2018 virus. [Figure 19A]A graph is presented showing serum HAI antibody titers in mice after immunization / vaccination against a panel of H1N1 viruses. Immunologically naive BALB / c mice were immunized / vaccinated three times at 4-week intervals with a VLP immunogen / vaccine containing a polynucleotide encoding broadly reactive recombinant influenza H1N1 HA immunogen Y2 (e.g., SEQ ID NO: 15), or with H1N1 wild-type Bris / 07, CA / 09, or Bris / 18 VLP immunogen / vaccine. Serum was collected from animals 10 weeks after the first immunization / vaccination, and HAI assays were performed with sear against a panel of seven H1N1 influenza viruses: broadly reactive recombinant influenza H1 HA immunogen. The y-axis shows the log2 HAI titers for each immunization / vaccination group of animals, which are presented as absolute mean ± SEM. The dotted lines indicate HAI titers ranging from 1:40 (lower line) and 1:80 (upper line). HAI titers were statistically analyzed using nonparametric one-way ANOVA with GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 19B]A graph is presented showing serum HAI antibody titers in mice after immunization / vaccination against a panel of H1N1 viruses. Immunologically naive BALB / c mice were immunized / vaccinated three times at 4-week intervals with a VLP immunogen / vaccine containing a polynucleotide encoding broadly reactive recombinant influenza H1N1 HA immunogen Y2 (e.g., SEQ ID NO: 15), or with H1N1 wild-type Bris / 07, CA / 09, or Bris / 18 VLP immunogen / vaccine. Serum was collected from animals 10 weeks after the first immunization / vaccination, and HAI assays were performed with sear against a panel of seven H1N1 influenza viruses: Bris / 18. The y-axis shows the log2 HAI titers for each immunization / vaccination group of animals, which are presented as absolute mean ± SEM. The dotted lines indicate HAI titers ranging from 1:40 (lower line) and 1:80 (upper line). HAI titers were statistically analyzed using nonparametric one-way ANOVA with GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 19C]A graph showing serum HAI antibody titers in mice after immunization / vaccination against a panel of H1N1 viruses is presented. Immunologically naive BALB / c mice were immunized / vaccinated three times at 4-week intervals with a VLP immunogen / vaccine containing a polynucleotide encoding broadly reactive recombinant influenza H1N1 HA immunogen Y2 (e.g., SEQ ID NO: 15), or with H1N1 wild-type Bris / 07, CA / 09, or Bris / 18 VLP immunogen / vaccine. Serum was collected from animals 10 weeks after the first immunization / vaccination, and HAI assays were performed with sear against a panel of seven H1N1 influenza viruses: CA / 09. The y-axis shows the log2 HAI titers for each immunization / vaccination group of animals, which are presented as absolute mean ± SEM. The dotted lines indicate HAI titers ranging from 1:40 (lower line) and 1:80 (upper line). HAI titers were statistically analyzed using nonparametric one-way ANOVA with GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 19D]A graph is presented showing serum HAI antibody titers in mice after immunization / vaccination against a panel of H1N1 viruses. Immunologically naive BALB / c mice were immunized / vaccinated three times at 4-week intervals with a VLP immunogen / vaccine containing a polynucleotide encoding broadly reactive recombinant influenza H1N1 HA immunogen Y2 (e.g., SEQ ID NO: 15), or with H1N1 wild-type Bris / 07, CA / 09, or Bris / 18 VLP immunogen / vaccine. Serum was collected from animals 10 weeks after the first immunization / vaccination, and HAI assays were performed with sear against a panel of seven H1N1 influenza viruses: Bris / 07. The y-axis shows the log2 HAI titers for each immunization / vaccination group of animals, which are presented as absolute mean ± SEM. The dotted lines indicate HAI titers ranging from 1:40 (lower line) and 1:80 (upper line). HAI titers were statistically analyzed using nonparametric one-way ANOVA with GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 19E]A graph is presented showing serum HAI antibody titers in mice after immunization / vaccination against a panel of H1N1 viruses. Immunologically naive BALB / c mice were immunized / vaccinated three times at 4-week intervals with a VLP immunogen / vaccine containing a polynucleotide encoding broadly reactive recombinant influenza H1N1 HA immunogen Y2 (e.g., SEQ ID NO: 15), or with H1N1 wild-type Bris / 07, CA / 09, or Bris / 18 VLP immunogen / vaccine. Serum was collected from animals 10 weeks after the first immunization / vaccination, and HAI assays using sear were performed against a panel of seven H1N1 influenza viruses: PBS. The y-axis shows the log2 HAI titers for each immunization / vaccination group of animals, which are presented as absolute mean ± SEM. The dotted lines indicate HAI titers ranging from 1:40 (lower line) and 1:80 (upper line). HAI titers were statistically analyzed using nonparametric one-way ANOVA with GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 20A] A graph showing neutralizing antibody titers in mouse serum after vaccination is presented. Immunologically naive BALB / c mice (n=11 / group) were vaccinated three times at 4-week intervals with broadly reactive HA immunogenic polypeptide (Y2 COBRA H1N1) VLP vaccine or H1N1 wild-type Bris / 07, CA / 09, or Bris / 18 VLP vaccine. Ten weeks after vaccination, serum was collected and used to perform FRA assays against A / California / 07 / 2009 virus. For each virus, virus concentration was normalized to 1.2×104 FFU / mL, and virus-only infected wells were normalized as 100% infection. The x-axis shows the log2 serum dilution, and the y-axis represents the percentage of infected cells compared to control wells infected with virus alone. The dotted lines represent the 50% inhibitory (upper line) and 80% inhibitory (lower line) activity of antibodies present in the antisera. [Figure 20B]A graph showing neutralizing antibody titers in mouse serum after vaccination is presented. Immunologically naive BALB / c mice (n=11 / group) were vaccinated three times at 4-week intervals with broadly reactive HA immunogenic polypeptide (Y2 COBRA H1N1) VLP vaccine or H1N1 wild-type Bris / 07, CA / 09, or Bris / 18 VLP vaccine. Ten weeks after vaccination, serum was collected and used to perform FRA assays against A / Brisbane / 02 / 2018 virus. For each virus, virus concentration was normalized to 1.2×104 FFU / mL, and virus-only infected wells were normalized as 100% infection. The x-axis shows the log2 serum dilution, and the y-axis represents the percentage of infected cells compared to control wells infected with virus alone. The dotted lines represent the 50% inhibitory (upper line) and 80% inhibitory (lower line) activity of antibodies present in the antisera. [Figure 21A] A graph showing total IgG antibody responses in mice is presented. Vaccine responses in BALB / c mice were evaluated 10 weeks after vaccination with broadly reactive HA immunogenic polypeptide vaccine (H1 Cobra HA), wild-type HA VLP vaccine (e.g., Bris / 18HA, CA / 09HA, Bris / 07HA), or PBS (x-axis) formulated with ADDAVAX™ adjuvant. IgG antibody titers were determined against A / California / 07 / 2009 HA protein. Data are presented as area under the curve (AUC) OD141 values ​​obtained from 3-fold serially diluted sera + SEM. For each independent experiment, mouse sera were assayed in duplicate. One-way ANOVA was used to analyze statistical differences between groups by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p-value less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 21B]A graph showing total IgG antibody responses in mice is presented. Vaccine responses in BALB / c mice were evaluated 10 weeks after vaccination with broadly reactive HA immunogenic polypeptide vaccine (H1 Cobra HA), wild-type HA VLP vaccine (e.g., Bris / 18HA, CA / 09HA, Bris / 07HA), or PBS (x-axis) formulated with ADDAVAX™ adjuvant. IgG antibody titers were determined against A / Brisbane / 02 / 2018 HA protein. Data are presented as area under the curve (AUC) OD141 values ​​obtained from 3-fold serially diluted sera + SEM. For each independent experiment, mouse sera were assayed in duplicate. One-way ANOVA was used to analyze statistical differences between groups by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p-value less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 21C] A graph showing total IgG antibody responses in mice is presented. Vaccine responses in BALB / c mice were evaluated 10 weeks after vaccination with broadly reactive HA immunogenic polypeptide vaccine (H1 Cobra HA), wild-type HA VLP vaccine (e.g., Bris / 18HA, CA / 09HA, Bris / 07HA), or PBS (x-axis) formulated with ADDAVAX™ adjuvant. IgG antibody titers were determined against cH6 / 1HA protein (chimeric rHA with globular head and stalk morphology subtype H6 influenza virus HA from A / California / 07 / 2009HA). Data are presented as area under the curve (AUC) OD141 values ​​obtained from 3-fold serially diluted sera + SEM. For each independent experiment, mouse sera were assayed in duplicate. One-way ANOVA was used to analyze statistical differences between groups by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p-value less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 22A] Graphs showing virus titers in lung tissues of BALB / c and DBA / 2J mice are presented. BALB / c mice were vaccinated intramuscularly with broadly reactive HA immunogenic polypeptide (H1 Cobra HA) or wild-type HA VLP vaccines (e.g., Bris / 18HA, CA / 09HA, Bris / 07HA) (x-axis) and then challenged with H1N1 A / California / 07 / 2009 virus 12 weeks after vaccination. Lung samples (n=3) were taken 3 days after infection and lung virus titers were measured. Viral titers in lung tissues are presented as PFU / mL, shown on the y-axis. The x-axis indicates the different vaccines used in this study. Non-parametric one-way ANOVA was used to analyze statistical differences between groups using GraphPad Prism 9 software. A p-value less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). [Figure 22B] A graph showing virus titers in lung tissue of BALB / c and DBA / 2J mice is presented. Lung samples (n=3 mice / group) were collected 3 days post-infection to determine lung virus titers. Another set of DBA / 2J mice immunized / vaccinated with the same vaccines as above, delivered in rHA format, was challenged with A / Brisbane / 02 / 2018 12 weeks post-vaccination. Lung samples (n=3) were collected 3 days post-infection to determine lung virus titers. Viral titers in lung tissue are presented as PFU / mL, shown on the y-axis. The x-axis indicates the different vaccines used in this study. Non-parametric one-way ANOVA was used to analyze statistical differences between groups using GraphPad Prism 9 software. A p-value less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0107] Detailed Description of the Disclosure Featured herein are synthetic (non-naturally occurring) immunogenic antigens, e.g., protein (polypeptide) and glycoprotein antigens, derived from influenza ("flu") hemagglutinin (HA) protein of influenza virus strains, e.g., H1 or H3, that elicit a strong, broadly reactive and long-lasting immune response in a subject. In some embodiments, the subject is a mammalian subject. In some embodiments, the subject is a human subject. In some embodiments, the subject is an avian subject. In some embodiments, the HA protein is a full-length HA polypeptide or a soluble HA (sHA) polypeptide. In some embodiments, the sHA polypeptide does not include a transmembrane (TM) or tail domain. Such immunogenic antigens are also referred to herein as immunogens, immunogenic polypeptides, proteins, or peptides, or vaccines.

[0108] Provided are broadly reactive immunogens that protect against disease caused by influenza strains such as H1 or H3 (e.g., H1N1 or H3N2). In certain embodiments, fully synthetic protein antigens, such as influenza virus HA protein antigens, are featured. Such HA antigens are synthetic proteins not found in nature, but retain all the functions of natural influenza virus HA proteins, and are immunogenic, i.e., capable of eliciting an immune response, particularly a broadly active immune response in the form of neutralizing antibodies and / or reactive T lymphocytes, particularly after administration or delivery or introduction to a subject against the influenza virus antigen immunogen. In some embodiments, the HA protein antigen is a full-length HA polypeptide or a soluble HA (sHA) polypeptide. Also provided are immunogenic compositions, such as vaccines comprising the synthetic viral protein antigens, or nucleic acids (e.g., DNA or RNA) encoding the antigens.

[0109] The HA amino acid sequence and protein antigens having such sequences are intended for use as immunogens or in immunogenic compositions, e.g., vaccines, to elicit broadly reactive immune responses, particularly in subjects to which the composition or vaccine is administered, particularly human subjects. The synthetic antigens are designed to generate a broadly active immune response, particularly in the form of neutralizing antibodies, in the subject, in some cases together with a cellular immune response. In certain embodiments, the subject is a mammalian subject, particularly a human subject. Such antigens are useful as immunogens to elicit an immune response (e.g., production of neutralizing antibodies and / or a cellular immune response) against viruses, particularly when more than one viral strain is co-circulating at a given time. By way of example, broadly reactive influenza immunogenic antigens may be derived from influenza viruses that frequently mutate parts of their genome to escape immune pressure, thus avoiding immune surveillance of subjects whose immune systems have not been primed or stimulated to generate antibodies against antigenic epitopes (determinants) on viral antigens following infection. Thus, a synthetic influenza virus antigen, e.g., an H1 or H3 HA antigen, comprises an amino acid (or polynucleotide) sequence that elicits a greater number of neutralizing antibodies (and / or an improved cellular immune response) against potential influenza virus variants that exhibit antigenic drift compared to the wild-type antigen sequence.

[0110] The H1 or H3 influenza virus HA immunogenic proteins or immunogens described herein can be used in immunogenic compositions or as vaccines that can provide protection against many virus strains over time. In embodiments, the H1 or H3 influenza virus HA immunogenic proteins or immunogens comprise the sequences set forth in SEQ ID NOs: 1-17 herein. The broadly reactive viral antigen immunogens and vaccines described herein are advantageous in that they are designed to provide broader and longer lasting protection against several different virus (e.g., influenza virus) strains (or clades), such as those occurring in different regions.

[0111] Immunogenic influenza virus HA antigens, including full length and soluble forms of HA described herein, may be used in immunogenic compositions (e.g., vaccines) that can provide protective immunity against influenza virus infection and disease in a subject. Protective immunity is provided in the subject through the induction of broadly reactive anti-HA specific antibody or cellular immune responses that protect the subject against viral strains that may have mutated or undergo antigenic drift.

[0112] Influenza virus Influenza viruses are segmented negative-stranded RNA viruses that belong to the family Orthomyxoviridae. There are three types of influenza viruses: types A, B, and C. Influenza A viruses infect a wide variety of birds and mammals, including 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 H1N1 ("H1"), H3N2 ("H3"), or H5N1 ("H5'"), or H7, or H9 strains, as non-limiting examples, can cause systemic infections in poultry with mortality rates approaching 100%. Animals infected with influenza A often act as reservoirs for influenza viruses, and certain subtypes have been shown to cross the species barrier to humans, where they can cause severe disease and devastating influenza outbreaks that can lead to the death of infected human subjects.

[0113] Influenza A viruses can be classified into subtypes based on allelic variations in the antigenic regions of two genes encoding surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), required for virus attachment and cell release, respectively. Currently, 16 subtypes of HA (H1-H16) and 9 NA (N1-N9) antigenic variants are known for influenza A viruses. Previously, only three subtypes were known to circulate in humans (H1N1 or H1N2). However, in recent years, it has been reported that the pathogenic H5N1 subtype of avian influenza A can cross the species barrier to infect humans and cause the deaths of several patients, for example, as reported in Hong Kong in 1997 and 2003.

[0114] In humans, avian influenza viruses infect cells of the respiratory tract as well as the intestinal tract, liver, spleen, kidneys, and other organs. Symptoms of avian influenza infection include fever, respiratory distress such as shortness of breath and cough, lymphopenia, diarrhea, and difficulty regulating blood sugar levels. In contrast to seasonal influenza, the group most at risk is healthy adults, who make up the majority of the population. Due to the high virulence of certain avian influenza A subtypes and their demonstrated ability to cross-transfect to infect humans, there are significant economic and public health risks associated with these virus strains, including the threat of actual epidemics and pandemics.

[0115] The influenza A virus genome encodes nine structural proteins and one nonstructural (NS1) protein with regulatory functions. The segmented genome of influenza viruses contains eight negative-sense RNA (nsRNA) gene segments (PB2, PB1, PA, NP, M, NS, HA, and NA) that encode at least ten polypeptides, including RNA-directed RNA polymerase proteins (PB2, PB1, and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin, e.g., subunits HA1, often referred to as the "head" subunit, and HA2, often referred to as the "tail" or "stalk" subunit, matrix proteins (M1 and M2), and nonstructural proteins (NS1 and NS2) (see, e.g., Krug et al., 1989, In: The Influenza Viruses, RMKrug ed., Plenum Press, NY, pp. 89 152).

[0116] The ability of influenza viruses to cause a wide range of disease is due to their ability to evade the immune system by undergoing antigenic changes that likely occur when a host is simultaneously infected with both animal and human influenza viruses. During mutation and reassortment in the host, a virus may incorporate HA and / or NA surface protein genes from another virus into its genome, thereby producing new influenza subtypes and evading the immune system.

[0117] Due to antigenic drift (drifting) in circulating strains of influenza virus, particularly in the HA and NA proteins of the virus, the efficacy of immunogenic compositions, e.g., vaccines, against influenza virus is often suboptimal. The immunogens, compositions and methods described herein provide broadly reactive HA antigens that generate a broadly reactive immune response, particularly a form of neutralizing antibody that binds to viral antigens and neutralizes the activity of the virus (e.g., its ability to infect cells) to more effectively treat influenza and its symptoms.

[0118] Influenza virus hemagglutinin (HA) and neuraminidase (NA) proteins HA is a viral surface glycoprotein that generally contains approximately 560 amino acids (e.g., 566 amino acids) and accounts for 25% of all viral proteins. As described herein, HA is a protein antigen that is highly useful as an immunogen because it contains a diverse repertoire of epitopes against which antibodies are generated in subjects or hosts that encounter the HA antigen of influenza viruses during infection.

[0119] HA is involved in the attachment of virus particles to and penetration of host cells, especially respiratory epithelia, early in infection. Cleavage of the viral HA0 precursor into HA1 and HA2 subfragments is a necessary step for the virus to infect cells. Thus, cleavage is necessary to convert new virus particles in the host cell into virions capable of infecting new cells. Cleavage is known to occur during transport of the integral HA0 membrane protein from the endoplasmic reticulum to the plasma membrane of infected cells. During transport, HA undergoes a series of co- and post-translational modifications, including proteolytic cleavage of the precursor HA into the amino-terminal fragment HA1 ("head") and the carboxy-terminal HA2 ("tail" or "stalk"). One of the major difficulties in growing influenza strains in primary tissue cultures or established cell lines arises from the need for proteolytic cleavage activation of influenza hemagglutinin in host cells.

[0120] Although it is known that uncleaved HA can mediate the attachment of the virus to its neuraminic acid-containing receptors on the cell surface, it cannot enable the next step of the infection cycle, i.e., fusion. It has been reported that the exposure of the hydrophobic amino terminus of HA2 is required so that it can be cleaved and inserted into the target cell, thereby forming a bridge between the virus and the target cell membrane. After this process, the two membranes fuse and the virus enters the target cell.

[0121] Proteolytic activation of HA involves cleavage at arginine residues by trypsin-like endoproteases, which are intracellular enzymes that are often calcium-dependent and have a neutral pH optimum. Since the activating proteases are cellular enzymes, the infected cell type determines whether HA is cleaved. The HA of mammalian influenza viruses and nonpathogenic avian influenza viruses is susceptible to proteolytic cleavage only in a limited number of cell types. On the other hand, the HA of pathogenic avian viruses, for example among the H5 and H7 subtypes, is cleaved by proteases present in a wide range of different host cells. Thus, there are host range differences resulting from differences in hemagglutinin cleavability that correlate with the pathogenicity of the virus.

[0122] Neuraminidase (NA) is the second membrane glycoprotein of influenza viruses. The presence of viral NA has been shown to be important for generating a multifaceted protective immune response against infecting viruses. For most influenza A viruses, NA is 413 amino acids long and is encoded by a gene of 1413 nucleotides. Nine different NA subtypes (N1, N2, N3, N4, N5, N6, N7, N8 and N9) have been identified in influenza viruses, all of which have been found in wild birds. NA is involved in the destruction of the cellular receptor for viral HA by cleaving terminal neuraminic acid (also called sialic acid) residues from carbohydrate moieties on the surface of infected cells. NA also cleaves sialic acid residues from viral proteins, preventing virus aggregation. Using this mechanism, NA is hypothesized to promote the release of viral progeny by preventing newly formed viral particles from accumulating along cell membranes and by facilitating the transport of the virus through mucus present at mucosal surfaces. NA is an important antigenic determinant that is subject to antigenic variation.

[0123] In addition to the surface proteins HA and NA, influenza viruses contain six additional internal genes that give rise to eight different proteins, including the polymerase genes PB1, PB2, and PA, the matrix proteins M1 and M2, the nucleoprotein (NP), and the nonstructural proteins NS1 and NS2 (see, e.g., Horimoto et al., 2001, Clin Microbiol Rev. 14(1):129-149).

[0124] For packaging into progeny virions, viral RNA is transported from the nucleus as a ribonucleoprotein (RNP) complex composed of three influenza virus polymerase proteins, nucleoprotein (NP), and viral RNA in association with influenza virus matrix 1 (M1) protein and nuclear export protein (Marsh et al., 2008, J Virol, 82:2295-2304). The M1 protein, located within the envelope, is thought to function in assembly and budding. A limited number of M2 proteins are incorporated into virions (Zebedee, 1988, J. Virol. 62:2762-2772). These M2 proteins form tetramers with H+ ion channel activity, which, when activated by the low pH in the endosome, acidify the interior of the virion, thus facilitating its uncoating (Pinto et al., 1992, Cell 69:517-528). Amantadine is an anti-influenza drug that prevents viral infection by blocking M2 ion channel activity and inhibits viral uncoating.

[0125] The nonstructural protein NS1 has multiple functions, including regulating splicing and nuclear export of cellular mRNAs, and stimulating translation. The primary function of NS1 appears to be to counteract host interferon activity, since NS1 knockout viruses were viable but did not grow as efficiently as the parent virus in interferon-uninfected cells (Garcia-Sastre, 1998, Virology 252:324-330).

[0126] The NS2 nonstructural protein has been detected in virus 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 a virus particle was estimated to be 130-200 molecules. In vitro binding assays have demonstrated direct protein-protein contacts between M1 and NS2. NS2-M1 complexes have also been detected by immunoprecipitation in virus-infected cell lysates. The NS2 protein is thought to play a role in export of RNPs from the nucleus via interaction with the M1 protein (Ward et al., 1995, Arch. Virol. 140:2067-2073).

[0127] Viral proteins and virus-like particles (VLPs) Provided are virus-like particles (VLPs) that contain non-naturally occurring broadly reactive influenza (e.g., H1 or H3) HA immunogenic polypeptides (immunogens) and influenza virus HA immunogens that contain diverse epitopes (antigenic determinants) that confer upon the HA antigen the ability to generate a broadly active immune response against influenza and its symptoms, either prophylactically or therapeutically, following administration and delivery to a susceptible subject. By way of example, representative influenza virus HA immunogenic antigen sequences are set forth herein (Example 1) in SEQ ID NOs: 1-17. In particular embodiments, the broadly reactive HA polypeptides are administered as part of a VLP. In some embodiments, the VLP contains one or more polynucleotides encoding one or more broadly reactive influenza HA immunogenic antigens described herein.

[0128] It will be understood that the influenza virus immunogens and sequences described and provided herein are non-naturally occurring and broadly reactive, whether or not these properties and characteristics are explicitly described. It will be further understood that the antigenic proteins described herein and used as immunogens are non-naturally occurring or synthetic antigens that elicit an immune response in a subject, e.g., a neutralizing antibody and / or a cellular immune response.

[0129] Influenza VLPs contain viral HA, NA and M1 proteins. The production of influenza VLPs has been described in the art and is within the skill and expertise of one of ordinary skill in the art. Briefly and as described, influenza VLPs can be produced by transfection of host cells with one or more plasmids containing polynucleotide sequences encoding HA, NA and M1 proteins. After incubating the transfected cells for an appropriate time to allow protein expression (e.g., approximately 72 hours), the VLPs can be isolated from the cell culture supernatant. Influenza VLPs can be purified from the cell supernatant using procedures practiced in the art, for example, VLPs can be isolated by low speed centrifugation (to remove cell debris), high vacuum filtration, and ultracentrifugation through 20% glycerol. In certain embodiments, VLPs containing a broad range of antigens from other pathogens can also be produced, isolated and used as immunogens or in immunogenic compositions.

[0130] Influenza VLPs can be used as influenza vaccines to induce an immune response against H1 or H3 influenza viruses. In particular, the broadly reactive influenza HA polypeptides that are components of the vaccine (or VLP) are broadly reactive and contain antigenic determinants useful for eliciting an immune response in a subject (e.g., production of neutralizing antibodies and / or activated T cells) that can treat a subject infected with the virus (e.g., neutralize the infectious virus) and / or protect the subject from full-blown viral infection or the signs and symptoms thereof.

[0131] In some embodiments, the antigenic sequences of the broadly reactive and immunogenic influenza antigens described herein, such as H1 or H3 HA antigens, contain a diverse repertoire of epitopic determinants that can reflect drift and sequence variability in the antigenic proteins of the virus. In particular, the influenza virus HA antigens described herein can include amino acid sequences that contain antigenic determinants (epitopes) derived from sequence-diverse influenza virus strains, including drift variants that can raise broadly reactive neutralizing antibodies, particularly when the antigen is used as an immunogenic product, (immunogen), such as an anti-viral vaccine, introduced into a subject. In some embodiments, the H1 or H3 immunogenic antigen sequences are as set forth in SEQ ID NOs: 1-17. In some embodiments, the HA immunogenic antigen is a full-length HA polypeptide. In some embodiments, the HA immunogenic antigen is a soluble HA polypeptide (sHA) that lacks the transmembrane and tail domains.

[0132] Because the broadly reactive influenza HA antigens and sequences thereof described herein and used as immunogens or immunogenic compositions, e.g., vaccines, elicit broadly reactive immune responses in immunocompetent subjects, they provide superior immunogenic products (e.g., vaccines) that capture antigenic determinants of different influenza isolates (subtypes or strains) against which a broadly active immune response (e.g., a broadly active neutralizing antibody and / or cellular immune response) is generated. Note that the terms "broadly active" and "broadly reactive" are used interchangeably herein.

[0133] In one embodiment, the influenza virus antigens described herein are polypeptide or peptide antigens of viruses currently causing disease or infection, such as influenza, influenza, or infectious bronchitis, and symptoms thereof. In another embodiment, the influenza virus antigens are polypeptide or peptide antigens that may cause future disease and infection. In one embodiment, the influenza virus antigen is a polynucleotide sequence. In one embodiment, the influenza virus antigen is a polynucleotide sequence that encodes a polypeptide or peptide antigen described herein. By way of example, representative broadly reactive influenza virus HA immunogenic antigen sequences are provided in Example 1 below as SEQ ID NOs: 1-17.

[0134] In another embodiment, the influenza immunogen sequences described herein are expressed intracellularly as a polypeptide, protein, or peptide. In an embodiment, the influenza immunogen is isolated and / or purified. In an embodiment, the immunogen is formulated for administration to a subject in need thereof. In an embodiment, the immunogen is administered to a subject in need thereof in an effective amount to elicit an immune response in the subject. In an embodiment, the immune response elicits neutralizing antibodies. In an embodiment, a cellular immune response is elicited. In an embodiment, the immune response is prophylactic or therapeutic.

[0135] In certain embodiments, non-natural influenza virus immunogens (immunogenic sequences), e.g., vaccines, are provided that elicit a broadly reactive immune response in a subject following introduction, administration, or delivery of the immunogen to the subject. The route of introduction, administration, or delivery is not limited and may include, for example, intravenous, subcutaneous, intramuscular, oral, etc. Vaccines may be therapeutic (e.g., administered to a subject after symptoms of a disease caused by an influenza virus (influenza or bronchitis)) or prophylactic (protective) (e.g., administered to a subject before the subject has or develops symptoms of a disease caused by a virus (influenza or bronchitis) or full-blown disease).

[0136] In one embodiment, the final amino acid sequence of a viral antigen (e.g., HA) is reverse translated and optimized for expression in mammalian cells. As will be appreciated by those skilled in the art, optimization of a nucleic acid sequence includes codon optimization and RNA optimization (such as RNA stability) for expression of the sequence in mammalian cells.

[0137] In certain embodiments, an isolated nucleic acid molecule (polynucleotide) is provided that comprises a nucleotide sequence encoding a polypeptide or peptide antigen, such as an influenza virus HA polypeptide. In certain embodiments, the nucleotide sequence encoding the HA polypeptide is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polynucleotide encoding the HA polypeptide sequence of SEQ ID NOs: 1-17 herein.

[0138] In other embodiments, a nucleotide sequence encoding an influenza virus HA 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 a polynucleotide encoding an influenza virus HA polypeptide sequence of SEQ ID NOs: 1-17 herein lacks a start codon encoding an N-terminal methionine. In some embodiments, the nucleotide sequence encodes a start codon encoding an N-terminal methionine.

[0139] Vectors are provided that contain a nucleotide sequence encoding a non-naturally occurring broadly reactive polypeptide or peptide antigen, e.g., an influenza HA polypeptide. In some embodiments, the vector comprises a nucleotide sequence encoding a polypeptide or peptide antigen, e.g., an influenza H1 or H3 HA polypeptide antigen, that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polynucleotide encoding an HA polypeptide sequence of SEQ ID NOs: 1-17 herein. In some embodiments, the vector further comprises a promoter operably linked to the nucleotide sequence encoding the HA polypeptide. In particular 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 a polynucleotide encoding an HA polypeptide sequence of SEQ ID NOs: 1-17 herein. In particular embodiments, the nucleotide sequence of the vector comprises a polynucleotide encoding an HA polypeptide sequence of SEQ ID NOs: 1-17 herein. In some embodiments, the vector is a prokaryotic or eukaryotic vector. In one embodiment, the vector is an expression vector, such as a eukaryotic (e.g., mammalian) expression vector. In another embodiment, the vector is a plasmid (prokaryotic or bacterial) vector. In another embodiment, the vector is a viral vector.

[0140] The vector used to express influenza virus antigens, e.g., H1 or H3 viral proteins, such as the HA protein described herein, may be any suitable expression vector known and used in the art. The vector may be, for example, a mammalian expression vector or a viral 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, which are incorporated herein by reference).

[0141] Provided are non-naturally occurring polypeptide immunogens derived from influenza virus, e.g., H1 or H3 influenza HA polypeptide antigens, produced by transfecting a host cell with an expression vector known and used in the art under conditions sufficient to allow expression of the HA polypeptide in the cell. Also provided are isolated cells containing the vector.

[0142] Also provided are non-naturally occurring broadly reactive influenza virus antigen polypeptides as described herein, e.g., broadly reactive H1 or H3 influenza HA polypeptides. In certain embodiments, the amino acid sequence of the polypeptide is at least 95%-99% (including the boundaries) identical to the amino acid sequence of the HA polypeptide set forth in SEQ ID NOs: 1-17 herein (Example 1). In certain embodiments, the amino acid sequence of the influenza HA polypeptide that is at least 95%-99% (including the boundaries) identical to the amino acid sequence of the HA polypeptide of SEQ ID NOs: 1-17 lacks an N-terminal methionine residue. In certain embodiments, the amino acid sequence of the influenza HA polypeptide is at least 95%-99% (including the boundaries) identical to the amino acid sequence of the HA polypeptide of SEQ ID NOs: 1-17.

[0143] In some embodiments, fusion proteins are also provided that include a broadly reactive influenza virus antigen polypeptide as described herein, including, but not limited to, an HA polypeptide as disclosed herein. In some embodiments, an influenza HA polypeptide can be fused to any heterologous amino acid sequence to form a fusion protein. By way of example, HA1 and HA2 polypeptides may be produced independently and then fused together to produce an influenza HA polypeptide antigen.

[0144] Also provided are virus-like particles (VLPs) as described herein, particularly H1 or H3 influenza VLPs that contain a broadly reactive protein antigen, such as an HA protein. In certain embodiments, the HA protein of the VLP is at least 94% or equal, at least 95% or equal, at least 96% or equal, at least 97% or equal, at least 98% or equal, at least 99% or equal, or 100% identical to the influenza virus HA protein set forth in SEQ ID NOs: 1-17 herein. The virus or influenza VLP may further comprise any additional viral or influenza proteins necessary to form a viral particle. In certain embodiments, the virus or influenza VLP further comprises an influenza neuraminidase (NA) protein, an influenza matrix (M1) protein, or both.

[0145] Also provided is an influenza VLP containing an H1 or H3 influenza virus HA polypeptide as described herein, produced by transfecting a host cell with a vector containing a polynucleotide encoding the HA polypeptide. In some embodiments, the polynucleotide is DNA or RNA, e.g., mRNA. In certain embodiments, also provided is an influenza VLP containing an influenza HA polypeptide as described herein, produced by transfecting a host cell with a vector encoding an influenza virus HA polypeptide, a vector encoding an influenza NA protein, and a vector encoding an influenza M1 protein, under conditions sufficient to allow expression of the influenza virus HA, NA, and M1 proteins. Such a VLP comprises a sequence as set forth in SEQ ID NOs: 1-17 and is used as an immunogen to generate antibodies with high hemagglutinin inhibition (HAI) titers against different strains of influenza virus types as described herein.

[0146] A collection of plasmids (vectors) is also contemplated. In certain embodiments, the collection of plasmids includes a plasmid encoding an influenza virus NA, a plasmid encoding an influenza MA, and a plasmid encoding a broadly reactive influenza virus HA protein as described herein. In some embodiments, the nucleotide sequence encoding the influenza 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 a polynucleotide encoding the HA amino acid sequence set forth in SEQ ID NOs: 1-17. In some embodiments, the nucleotide sequence encoding the codon-optimized influenza 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 a polynucleotide encoding the influenza HA amino acid sequence set forth in SEQ ID NOs: 1-17. In another embodiment, the collection of plasmids contains a plasmid encoding a broadly reactive HA protein as described herein, comprising a polynucleotide encoding the HA amino acid sequence set forth in SEQ ID NOs: 1-17.

[0147] In the context of the present disclosure, "broadly reactive" or "broadly active" refers to an influenza virus protein (e.g., an H1 or H3 HA protein sequence) that is immunogenic and contains a diversity of epitopes (antigenic determinants) that elicit a sufficient immune response (e.g., neutralizing antibodies directed against epitopes contained in the broadly reactive protein immunogen, frequently accompanied by a T cell response) in a subject to treat disease or infection and / or inhibit, neutralize, or prevent infection caused by most or all of the influenza viruses within a particular subtype or related virus strains. In embodiments, an HA antigen protein from a broadly reactive H1 or H3 influenza virus can elicit a protective immune response against most or all of the known H1 or H3 influenza virus isolates, such as about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 96%-99% of the known H1 or H3 influenza virus isolates. In certain embodiments, an antigenic protein, e.g., HA protein, from a broadly reactive H1 or H3 influenza virus is capable of eliciting a protective immune response against most or all of the known H1 or H3 influenza virus isolates, e.g., about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 96%-99% of the known H1 or H3 influenza virus isolates.

[0148] Compositions for Administration and Pharmaceutical Compositions Compositions are provided that include broadly reactive influenza HA proteins, or fusion proteins or VLPs that include such broadly reactive influenza or HA proteins described herein. In some embodiments, the compositions further include a pharma- ceutically acceptable carrier, excipient, or vehicle. In some embodiments, adjuvants (pharmacologic or immunologic agents that modify or enhance immune responses, e.g., produce more and longer-lasting antibodies) are also used. For example, but not limited to, adjuvants can be inorganic compounds, e.g., alum, aluminum hydroxide, or aluminum phosphate; mineral or paraffin oil; squalene; detergents, e.g., Quil A; plant saponins; Freund's complete or incomplete adjuvant, biological adjuvants (e.g., cytokines, e.g., IL-1, IL-2, or IL-12); bacterial products, e.g., killed Bordetella pertussis, or toxoids; or immunostimulatory oligonucleotides (e.g., CpG oligonucleotides).

[0149] Compositions and preparations (e.g., physiologically or pharma- ceutically acceptable compositions) containing non-native broadly reactive influenza virus HA polypeptides and influenza virus-like particles (VLPs) for parenteral administration include, but are not limited to, sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Non-limiting examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and canola oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives, such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present in such compositions and preparations.

[0150] Some of the present compositions may potentially be administered as pharma- ceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric, hydrobromic, perchloric, nitric, thiocyanic, sulfuric, and phosphoric acid, and organic acids such as formic, acetic, propionic, glycolic, lactic, pyruvic, oxalic, malonic, succinic, maleic, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl, and aryl amines, and substituted ethanol amines.

[0151] Provided herein is a pharmaceutical composition comprising a therapeutically effective amount of a non-native broadly reactive influenza virus protein HA antigen, or influenza VLP, alone or in combination with a pharma- ceutically acceptable carrier. In some embodiments, the influenza virus HA antigen comprises, for example, that of an H1 or H3 influenza virus having a sequence as set forth in SEQ ID NOs: 1-17 herein. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile, and the formulation is suitable for the mode of administration. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid or aqueous solution, suspension, emulsion, dispersion, tablet, pill, capsule, powder, or sustained-release formulation. Liquid or aqueous compositions can be lyophilized and reconstituted with a solution or buffer prior to use. The composition can be formulated as a suppository, with traditional binders and carriers, such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Any commonly known pharmaceutical carriers such as sterile saline or sesame oil may be used. The vehicle may also contain conventional pharmaceutical auxiliary materials such as pharma- ceutically acceptable salts for adjusting osmotic pressure, buffers, preservatives, etc. Other vehicles that may be used in the compositions and administration methods as described are saline and sesame oil.

[0152] Methods of Treatment, Administration and Delivery Methods are provided for treating a disease or infection caused by an influenza virus (e.g., an H1 or H3 influenza virus), or a symptom thereof. The methods include administering a therapeutically effective amount of a broadly reactive immunogen as described herein, or a pharmaceutical composition comprising an immunogen as described herein, or a vaccine (e.g., a VLP vaccine) to a subject (e.g., a mammal), particularly a human subject, a non-human animal or veterinary subject, or an avian subject. It is to be understood that the term administering encompasses inoculation, immunization, or vaccination of a subject. The terms immunization and vaccination ("immunization / vaccination") may be used interchangeably herein.

[0153] One embodiment includes a method of treating a subject suffering from, at risk of suffering from, or susceptible to a disease or infection caused by an influenza virus, or a symptom thereof, comprising administering to the subject (e.g., a mammalian subject) an immunogenic composition or vaccine comprising a non-native broadly reactive influenza virus antigen polypeptide, such as an HA polypeptide or VLP, in an amount sufficient or therapeutic to treat the disease, infection, or symptom thereof caused by an influenza virus, under conditions such that the disease, infection, and / or symptom thereof is treated.

[0154] In certain embodiments, the methods herein comprise administering to a subject (including a human or non-human subject identified as in need of such treatment) an effective amount of a non-natural broadly reactive influenza virus antigen polypeptide, such as an H1 or H3 influenza virus HA polypeptide described herein, or a vaccine, or a composition described herein, to produce an immune response. The therapeutic methods are suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk of having a disease, disorder, infection, or symptoms thereof, such as influenza or influenza, or infectious bronchitis. In some embodiments, the therapeutic methods are also suitably administered to non-human subjects, such as non-human animal subjects, veterinary subjects, or avian subjects. Identifying a subject in need of such treatment can be based on the judgment of the subject or a medical or veterinary medical professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method). Briefly, the determination of a subject in need of treatment, or "at risk" or "susceptible" can be made by any objective or subjective determination, including the opinion of the subject or health care provider, by diagnostic tests (e.g., genetic testing, enzyme or protein marker assays), marker analysis, family medical history, etc. The non-natural broadly reactive viral immunogens, such as the H1 or H3 influenza virus HA polypeptides, immunogens, and vaccines described herein, can also be used to treat any other disorder that may involve infection or disease caused by H1 or H3 influenza viruses. The subject to be treated can be a non-human mammal, such as a veterinary subject, an avian subject, or a human subject (also referred to as a "patient").

[0155] Additionally, prophylactic methods are provided for preventing or protecting against disease or infection caused by influenza viruses, e.g., H1 or H3 influenza viruses, or symptoms thereof, comprising administering to a subject (e.g., a mammal, such as a human) in need thereof a pharmaceutical composition comprising a therapeutically effective amount of an H1 or H3 influenza virus HA polypeptide immunogenic composition or vaccine (e.g., an H1 or H3 influenza virus VLP vaccine) described herein, particularly prior to infection of the subject or prior to the onset of disease, such as an H1 or H3 virus-associated disease.

[0156] In another embodiment, a method is provided for monitoring the progression of influenza virus infection or disease, or for monitoring the treatment of influenza virus infection or disease, e.g., caused by H1 or H3 influenza virus. The method comprises determining the level of a diagnostic marker or biomarker (e.g., an influenza virus protein such as H1 or H3 HA) or diagnostic measurement (e.g., a screening assay or detection assay) in a subject suffering from or susceptible to an infection, disease, or symptom thereof associated with an influenza virus, wherein the subject has been administered a sufficient amount (e.g., a therapeutic amount) of a non-native, broadly reactive influenza virus HA protein immunogen as described herein, or a vaccine as described herein, to treat the infection, disease, or symptom thereof. The level or amount of the marker or biomarker (e.g., a viral protein) determined by the method can be compared to known levels of the marker or biomarker in samples from healthy (uninfected) normal controls, in pre-infection or pre-disease samples from the subject, or in other diseased / infected / disease patients to establish the disease state of the subject being treated. For monitoring, a second level or amount of the marker or biomarker in a sample obtained from the subject is determined at a time later than the determination of the first level or amount, and the levels or amounts of the two markers or biomarkers can be compared to monitor the course of a disease or infection, or the effectiveness of a therapy / treatment. In certain embodiments, a pre-treatment level of the marker or biomarker in the subject (e.g., in a sample obtained from the subject) is determined before starting a treatment as described. This pre-treatment level of the marker or biomarker can then be compared to the level of the marker or biomarker in the subject after the start of treatment and / or during the course of treatment to determine (monitor) the effectiveness of the disease treatment. In the methods described, the subject can be a human subject or patient, or a non-human animal or veterinary subject.

[0157] Non-native broadly reactive influenza virus polypeptides, such as H1 or H3 influenza virus HA polypeptides as described, as well as VLPs comprising such HA polypeptides, or compositions thereof, can be administered to a subject by any of the routes typically 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, intranasal, inhalation, intraocular, intracranial, or oral. Parenteral administration, such as subcutaneous, intravenous, or intramuscular administration, is generally accomplished by injection (immunization). Injectables can be prepared in conventional forms and formulations, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection (e.g., lyophilized forms), or emulsions. Injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets. Administration can be systemic or local.

[0158] The non-natural broadly reactive influenza virus polypeptides, such as the described H1 or H3 influenza virus HA polypeptides, and VLPs comprising such HA polypeptides, or compositions thereof, can be administered in any suitable manner, such as with a pharma- ceutically acceptable carrier, as described above. Pharmaceutically acceptable carriers are determined in part by the particular immunogen or composition being administered, as well as by the particular method used to administer the composition. Thus, the immunogenic non-natural influenza virus antigen polypeptides, such as the described H1 or H3 influenza virus HA polypeptides, and pharmaceutical compositions comprising such HA polypeptides, or compositions thereof, can be prepared using a wide variety of suitable physiologically and pharma- ceutical acceptable formulations.

[0159] Administration of broadly reactive immunogenic viral antigen polypeptides, such as the described H1 or H3 influenza virus HA polypeptides, and VLPs comprising such HA polypeptides, or compositions thereof, can be accomplished by a single dose or multiple doses. The dose administered to a subject should be sufficient to induce a beneficial therapeutic response in the subject over time, for example, to inhibit, block, reduce, ameliorate, protect against, or prevent disease or infection by influenza viruses (e.g., H1 or H3 influenza viruses). The required dose will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, depending on the severity of the infection being treated, depending on the specific composition being used, and depending on the method of administration. Appropriate doses can be determined by those skilled in the art, such as clinicians or medical professionals, using only routine experimentation.

[0160] Further provided is a method of eliciting an immune response against influenza virus in a subject by administering to the subject a non-natural broadly reactive H1 or H3 influenza virus HA polypeptide as described, or a VLP comprising such an HA polypeptide, a composition thereof, or a fusion protein containing an H1 or H3 influenza virus HA polypeptide as described herein. In some embodiments, the influenza virus HA protein, fusion protein, or VLP can be administered using any suitable route of administration, such as, for example, intramuscular injection. In some embodiments, the influenza virus HA protein, fusion protein, or VLP is administered as a composition comprising a pharma- ceutically acceptable carrier. In some embodiments, the composition comprises an adjuvant selected from, for example, alum, Freund's complete or incomplete adjuvant, a biological adjuvant, or an immunostimulatory oligonucleotide (e.g., a CpG oligonucleotide). In other embodiments, the composition can be administered in combination with another therapeutic agent or molecule, such as an antiviral agent, or a combination thereof, as used by those skilled in the art.

[0161] Also provided are methods of immunizing a subject against infection or disease caused by an H1 or H3 influenza virus or symptoms thereof, comprising administering to the subject a VLP containing a non-native broadly reactive H1 or H3 influenza HA protein as described herein, or administering an immunogenic composition thereof. In some embodiments of the methods, the composition further comprises a pharma- ceutically acceptable carrier and / or an adjuvant. For example, the adjuvant can be alum, Freund's complete or incomplete adjuvant, a biological adjuvant, or an immunostimulatory oligonucleotide (e.g., a CpG oligonucleotide). In certain embodiments, the VLP (or composition thereof) is administered intramuscularly.

[0162] In some embodiments of the method of inducing an immune response or immunizing a subject against a viral infection or disease caused by or associated with influenza virus (e.g., H1 or H3 influenza), the subject is administered VLPs containing at least 1 μg of a non-native, broadly reactive influenza virus (e.g., H1 or H3 influenza) HA protein, 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 a non-native, broadly reactive influenza virus HA protein, e.g., about 1 to about 50 μg, or about 1 to about 25 μg of a VLP. In a specific but non-limiting example, the subject is administered about 5 to about 20 μg of VLPs, or about 10 to about 15 μg of VLPs. In a specific but non-limiting example, the subject is administered about 15 μg of VLPs. However, one of skill in the art can determine a suitable therapeutically effective amount of VLPs (e.g., an amount that provides a therapeutic effect or protection against influenza virus (e.g., H1 or H3 influenza) infection) to administer to a subject in need of treatment or protection from viral infection.

[0163] Administration of VLPs comprising a non-native, broadly reactive influenza virus HA protein as described herein is expected to elicit high titers of neutralizing antibodies directed against a diverse repertoire of epitopic determinants on the HA protein immunogen, as well as therapeutic or protective levels of HA inhibitory (HAI) antibodies directed against several representative influenza isolates and providing complete protection against lethal challenge with influenza virus (e.g., H1 or H3 influenza) and / or related influenza virus types. VLPs containing a non-native, broadly reactive influenza HA protein (e.g., H1 or H3 influenza HA protein) as described herein elicit a broader immune response (e.g., elicit neutralizing antibodies directed against a broader range of influenza virus isolates compared to the immune response elicited by a multivalent influenza virus (e.g., multivalent H1 or H3 influenza virus) vaccine.

[0164] Adjuvant and Combination Therapies Influenza virus immunogens or immunogenic compositions containing influenza protein antigens (e.g., H1 or H3 influenza HA antigens) or containing influenza virus (e.g., H1 or H3 influenza 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 an immune response, such as elicitation of specific antibodies. By way of example, H1 or H3 influenza virus VLPs can be administered with an adjuvant, such as alum, Freund's incomplete adjuvant, Freund's complete adjuvant, ADDAVAX™ adjuvant, biological adjuvant, or immunostimulatory oligonucleotides (e.g., CpG oligonucleotides). ADDAVAX™ adjuvant (InvivoGen, San Diego, CA; ThermoFisher) is a squalene-based oil-in-water nanoemulsion with a formulation similar to MF59®. Such squalene oil-in-water emulsion adjuvants induce both cellular (Th1) and humoral (Th2) immune responses and are believed to act through the recruitment and activation of antigen-presenting cells (APCs) and the stimulation of cytokine and chemokine production by macrophages and granulocytes.

[0165] One or more cytokines, such as interleukin-1 (IL-2), interleukin-6 (IL-6), interleukin-12 (IL-12), 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), 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 membrane glycoprotein receptor belonging to the TNF superfamily (4-1BBL), or combinations of these molecules, can be used as a biological adjuvant if desired or warranted (see, e.g., Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze et al., 1999, J. Surg. Oncol. 68(2):122-38; (see, e.g., E. 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 locally) to the subject. In another embodiment, one or more antiviral agents used by those skilled in the art (such as, but not limited to, influenza drugs Rapivab (peramivir), Relenza (zanamivir), Tamiflu (oseltamivir phosphate), or Xofluza (baloxavir marboxil)) can be co-administered to the subject.

[0166] Several methods of inducing cellular responses both in vitro and in vivo are known and practiced in the art. Lipids have been identified as agents that can help prime 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 such as glycine, glycine-glycine, serine, serine-serine, etc.) (U.S. Patent No. 5,662,907). The lipidated peptides can then be directly injected in micellar form, incorporated into liposomes, or emulsified in adjuvants. As another example, E. coli lipoproteins such as tripalmitoyl-S-glycerylcysteine ​​lyseryl-serine can be used to prime tumor-specific CTLs when covalently attached to the appropriate peptide (see, e.g., Deres et al., 1989, Nature 342:561). Furthermore, the induction of neutralizing antibodies can also be primed with the same molecule conjugated to a peptide displaying the appropriate epitope, and the two compositions can be combined to elicit both humoral and cell-mediated responses if such a combination is deemed desirable.

[0167] Although the methods of treatment may involve administration of VLPs containing the non-native broadly reactive HA immunogenic proteins described herein, one of skill in the art will appreciate that the non-native broadly reactive HA protein itself (in the absence of viral particles), either as a component of a pharma- ceutically acceptable composition or as a fusion protein, can be administered to a subject in need thereof to elicit an immune response in the subject.

[0168] kit Also provided are kits containing the non-natural broadly reactive influenza virus immunogens as described, or vaccines, or pharma- ceutically acceptable compositions containing the immunogens and pharma- ceutically acceptable carriers, diluents, or excipients, for example, for administration to a subject in need thereof. The immunogens may be in the form of influenza virus (e.g., H1 or H3 influenza virus) proteins (polypeptides) or polynucleotides (polynucleotides encoding influenza virus proteins), e.g., H1 or H3 influenza virus HA as described herein. Also provided are kits containing one or more of the plasmids, or collections of plasmids, described herein. As will be appreciated by those of skill in the art, such kits may contain one or more containers housing the immunogens, vaccines, or compositions, diluents, or excipients, as appropriate, and instructions for use.

[0169] The practice of the aspects and embodiments described herein employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology that are well within the skill of the art. Such techniques are fully explained in such publications as "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 polynucleotides and polypeptides of the aspects and embodiments described and / or exemplified herein, and therefore may be considered in making and practicing such aspects and embodiments. Techniques useful for particular embodiments are described in the following sections. EXAMPLES

[0170] The following examples are provided to illustrate certain specific features and / or embodiments and should not be construed as limiting the disclosure to the specific features or embodiments described.

[0171] Example 1: Influenza virus hemagglutinin (HA) sequence This example presents the amino acid sequence of a full-length non-natural broadly reactive influenza virus hemagglutinin (HA) immunogenic polypeptide antigen, as well as a soluble influenza virus HA (sHA) antigen amino acid sequence derived from influenza H1 and H3 types, such as H1N1 and H3N2. In certain embodiments, the HA antigen provides epitopes of HA antigens derived from influenza H1 or H3 in a particular time frame (e.g., a given span of years or flu seasons) that provide a broadly reactive immune response against current and future H1 or H3 HA antigens (e.g., H1 or H3 antigens of influenza viruses in epidemics during future flu seasons) when the HA antigen is administered to a subject or host as an immunogen. As an immunogen, the non-natural HA immunogenic polypeptide antigen generates a broadly reactive immune response (antibody and / or cellular immune response) against influenza viruses (e.g., viral antigens) in the recipient subject or host. In some embodiments, the HA antigen used as an immunogen generates a therapeutic and / or protective immune response (e.g., an antibody response and / or a cellular immune response) in a subject against influenza virus strains or types that may occur in different (e.g., subsequent or future) influenza seasons. By way of example, non-natural broadly reactive influenza virus hemagglutinin (HA) or neuraminidase (NA) polypeptide antigen sequences for use as immunogens may be generated by methods such as those described in published PCT application numbers WO2020 / 014673 or WO2020 / 014675, the contents of which are incorporated herein by reference in their entireties. In some instances herein, the methods for generating broadly reactive influenza HA or NA immunogenic polypeptides (or peptides) are referred to as "computationally optimized broadly reactive antigens" (Cobra) methods, and non-natural broadly reactive influenza virus HA or NA immunogenic polypeptides (or peptides), including soluble forms thereof, are referred to as "Cobra" antigens or immunogens.The amino acid sequences of the H1 or H3 viral HA polypeptides provided herein are as follows: TIFF2024522193000002.tif163163TIFF2024522193000003.tif241163TIFF2024522193000004.tif82163Soluble HA sequence TIFF2024522193000005.tif143163TIFF2024522193000006.tif163163H1N1 soluble HA sequence TIFF2024522193000007.tif246163

[0172] Example 2: Ferret and mouse animal studies using recombinant influenza virus HA immunogenic peptides (influenza A(H3)) as immunogens (vaccine) Recombinant influenza virus HA immunogenic peptides (such as influenza A(H3N2)) were used as immunogens (vaccines) in vivo in pre-immunized and naive mice and pre-immunized and naive ferrets to determine the immunogenicity and efficacy of recombinant influenza HA immunogens in eliciting broad-spectrum protective immune responses against seasonal and pandemic influenza viruses using in vivo animal models. Pre-immunized mice and ferret animals do not typically exhibit pre-existing antibodies to seasonal influenza virus antigens (e.g., HA), and therefore provide a relevant animal model for evaluating the efficacy of influenza HA immunogens described herein as therapeutics in preventing and / or treating disease. In contrast, most human subjects have pre-existing antibodies to seasonal influenza virus antigens. As described in Example 1, the HA polypeptide immunogen(s) (and recombinant HA polypeptide immunogen(s)) comprise influenza virus hemagglutinin (HA) antigenic amino acid sequences derived from influenza H1 and H3 types, such as H1N1 and H3N2, containing HA sequences representative of seasonal or pandemic influenza viruses. In the following examples, the terms "immunization" and "vaccination" are used interchangeably.

[0173] Ferret Test Viral infection and immunization (vaccination) of ferrets with a broadly reactive recombinant HA polypeptide immunogen. Fitch ferrets (Mustela putorius furo, females, 6-12 months of age) that were antibody negative to pandemic influenza A (H1N1, H3N2) and influenza B viruses were descended and purchased from Triple F Farms (Sayre, PA). Ferrets were housed in pairs in stainless steel cages (Shor-line, Kansas City, KS) containing Sani-Chips laboratory animal bedding (PJ Murphy Forest Products, Montville, NJ). Ferrets were provided with Teklad Global Ferret Diet (Harlan Teklad, Madison, WI) and fresh water available ad libitum. In the pre-immune vaccine group, ferrets were immunized with CA / 09, Pan / 99, and B / HK / 01 virus strains (10 mg each) 60 days prior to vaccination. 6PFU) (FIG. 1). Ferrets were infected with recombinant influenza virus hemagglutinin (HA) and neuraminidase (NA) immunogenic polypeptide antigens (Y4, Z1, NG3, IAN8, Q6, BC2, N1I (also referred to as NA-A), and N2A), such as the octavalent formulations of non-natural broadly reactive influenza immunogenic polypeptides described herein (e.g., Y4 (SEQ ID NO: 17); NG3 (SEQ ID NO: 13), as well as the octavalent formulations of non-natural broadly reactive influenza immunogenic polypeptides described in WO 2020 / 014673 A1 (e.g., Z1, IAN8), in WO 2021 / 142256 A2 (e.g., Q6 (H7 HA) and neuraminidase antigens, e.g., N1, N2, N1I (NA-A and NA-D)), in WO 2020 / 014656 A1, by Y. Huang et al. Mice were vaccinated intranasally (days 0 and 28) with either J.D. Allen and TM Ross, 2022, J. Virology, Vol. 96, No. 7: doi.org / 10.1128 / jvi.01652-21, the entire contents of which are incorporated herein by reference in their entirety. Vaccine immunogens contained 15 μg of each antigen formulated with 50 μg of cyclic-di-AMP as an adjuvant (InvivoGen, San Diego, CA). Diego, CA). Ferrets were boosted 28 days after the first vaccination. Blood was collected from all anesthetized ferrets via the anterior vena cava prior to vaccination and on days 28 and 56 after the first vaccination. Serum collected from the animals was transferred to centrifuge tubes and centrifuged at 2500 rpm. Clarified serum was removed and frozen at -20±5°C. Control (mock) ferrets were immunized with a placebo containing phosphate buffered saline (PBS), pH 7.4, formulated with 50 μg of c-di-AMP. All vaccine immunogens and placebos were stored in a refrigerator at a temperature of 2°C to 8°C until use.

[0174] On day 56 after vaccination, ferrets were administered 10 8 PFU Bris / 18 (H1N1), 10 7PFU's B / WA / 19(IBV) or 10 5Ferrets were challenged intranasally with PFU of Vn / 04 (H5N1) virus (n=5 per vaccine group per challenge). Ferrets were monitored daily for weight loss, signs of disease, and mortality for 14 days post-infection. Individual body weights and mortality were recorded for each group on each day post-virus challenge. Experimental endpoints were defined as weight loss of more than 20%. Nasal washes were performed by instilling 3 ml of PBS into the nose of anesthetized ferrets on days 1, 3, 5, and 7 post-infection (Figure 1). Washes were collected and stored at -80°C until use. The University of Georgia Institutional Animal Care and Use Committee approved all experiments under the Animal Use Protocol (number A2020 11-016) and were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, and the CDC / NIH's Biosafety in Microbiological and Biomedical Laboratories guide. Figures 2A-2D present graphs showing the results of ELISA analysis performed on sera obtained from ferrets on day 56 of the study. The results demonstrated that immunization with the octavalent HA immunogenic polypeptide antigen (Cobra antigen) described herein elicited an immune response and the production of antibodies that bound to all components of the HA immunogenic polypeptide / vaccine (Figures 2A and 2C). Antibodies from pre-immunized mock animals bound to H1, N1 and N2 HA antigens (Figure 2B). Antibodies from naive mock animals showed no binding to HA antigens (Figure 2D). Figures 3A and 3B present graphs showing the results of ELISA analysis performed to compare the total IgG antibody responses before and after vaccination in pre-immunized groups of ferrets on days 0 and 56 of the study using sera obtained from pre-immunized ferrets immunized with HA immunogenic polypeptide antigen or from pre-immunized mock immunized ferrets.Sera were collected prior to immunization / vaccination (d0) and after the final immunization / vaccination (d56) for individual pre-immunized ferrets immunized with the HA polypeptide immunogen (Cobra) (Figure 3A) and mock-vaccinated pre-immunized ferrets (Figure 3B). Results demonstrated that pre-immunized ferrets immunized with the HA immunogenic polypeptide antigen (Cobra antigen) described herein had an increase in antibodies against all HA components of the immunogenic polypeptide antigen / vaccine, and a statistically significant increase in antibodies generated against the Z1, NG3, IAN8, Q6, and BC2 immunogens.

[0175] Figures 4A and 4B show serum HAI antibody titers for H1N1 virus in sera obtained from pre-immunized ferrets immunized with HA immunogenic polypeptide antigen (Cobra antigen), naive ferrets immunized with HA immunogenic polypeptide antigen (Cobra antigen), and pre-immune mock immunized ferrets before and after vaccination. Ferrets were vaccinated intranasally twice, 4 weeks apart, with c-di-AMP as the adjuvant. Sera were collected prior to vaccination (H1N1, day 0, Figure 4A) and 4 weeks after the second vaccination (H1N1, day 56, Figure 4B) to perform HAI assays against a panel of six H1N1 influenza viruses. The results showed that the pre-immunized mock group had statistically significantly lower HAI titers for Mich / 15 and Bris / 18 strains of virus, the pre-immunized group immunized with HA immunogenic polypeptide antigen (Cobra antigen) maintained high HAI titers, and the naive group with HA immunogenic polypeptide antigen (Cobra antigen) reached HAI titers of 1:40 for pandemic-like strains, except for Guangdong / 19.

[0176] Figures 5A and 5B show serum HAI titers for H3N2 viruses in serum obtained from test ferrets before and after vaccination. Ferrets were vaccinated intranasally twice, 4 weeks apart, with c-di-AMP as the adjuvant. Vaccine groups included pre-immunized ferrets receiving octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black bars), naive ferrets receiving octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (white bars), or pre-immunized ferrets receiving mock vaccination (gray bars). Serum was collected prior to vaccination (Figure 5A) and 4 weeks after the second vaccination (Figure 5B) for HAI assays against a panel of six H3N2 influenza viruses. The results showed that pre-immunized ferrets that received the octavalent recombinant influenza immunogenic polypeptides as an immunogen had a statistically significant increase in HAI titers, with the exception of KS / 17 and HK / 19, and naive ferrets that received the octavalent recombinant influenza immunogenic polypeptides as an immunogen had titers that were increased but not statistically significant.

[0177] Figures 6A and 6B show serum HAI antibody titers in test ferrets against influenza B (IBV) viruses before and after vaccination. Vaccine groups were pre-immunized ferrets receiving octavalent recombinant influenza immunogenic polypeptides (Cobra antigens), naive ferrets receiving octavalent recombinant influenza immunogenic polypeptides (Cobra antigens), or pre-immunized ferrets receiving mock vaccinations. Serum was collected prior to vaccination and 4 weeks after the second vaccination for HAI assays against a panel of six IBV influenza viruses. Results showed that ferrets in the pre-immunized group receiving octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) had statistically significantly increased HAI titers for B / Bris / 08 and B / CO / 17, whereas naive ferrets receiving octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) and pre-immunized mock immunized ferrets had no statistically significant changes.

[0178] Figures 7A and 7B show serum HAI antibody titers in test ferrets against H5 viruses before and after immunization / vaccination. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as an adjuvant. Vaccine groups were pre-immunized ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens), naive ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens), or pre-immunized ferrets that received a mock vaccination. Sera were collected before immunization / vaccination and 4 weeks after the second immunization / vaccination for HAI assays against a panel of six H5 influenza viruses. A / Vietnam / 1203 / 2004 (H5N1, Vn / 04), A / Wu / Mongolia / 244 / 2005 (H5N1, ws / Mo / 05), A / Egypt / 321 / 2007 (H5N1, Eg / 07), A / Hupei / 01 / 2010 (H5N1, Hu / 10), A / Guizhou / 01 / 2013 (H5N1, Gu / 13), and A / Sichuan / 26221 / 2014 (H5N6, Si / 14). The results showed that none of the herds had HAI titers to the H5N1 viruses.

[0179] Figures 8A and 8B show the weight and survival curves of ferrets in the study after challenge with influenza H5N1 virus. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as adjuvant. Vaccine groups were pre-immunized ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black line), naive ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens) (black line with circles), or pre-immunized ferrets that received a mock immunization / vaccination (gray line). Four weeks after the final immunization / vaccination, ferrets were vaccinated with a lethal dose of A / Vietnam / 1203 / 2004 (1000 mg / kg) in a volume of 1 mL. 5Animals were intranasally infected with 100 mg / kg of H5N1 virus (PFU). Animals were observed for clinical signs and their body weights were recorded daily post-infection (Figure 8A). Post-infection survival curve data show that all animals survived the lethal virus challenge (Figure 8B). The results show that immunization / vaccination of ferrets effectively protects against lethal H5N1 virus challenge (10 5 These results show that H1 pre-immunization protected animals from H5N1 infection (PFU).

[0180] Figures 9A and 9B show the weight curves of ferrets after challenge with influenza virus. Ferrets were vaccinated intranasally twice at 4-week intervals with c-di-AMP as adjuvant. Vaccine groups were pre-immunized ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens), naïve ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens), pre-immunized ferrets mock immunized / vaccinated, or naïve ferrets mock immunized / vaccinated. Four weeks after the final vaccination, ferrets were vaccinated with influenza virus A / Brisbane / 02 / 2018 (100 mg / kg) in a volume of 1 mL. 8 PFU) (Figure 9A), or influenza virus (b) B / Washington / 02 / 2019 (10 7 Animals were intranasally infected with 1000 ng / mL IgG 1000-fold increase in ...

[0181] Figures 11-11C show graphs of virus titers in the upper respiratory tract of ferrets following infection with influenza virus A / Brisbane / 02 / 2018 (H1N1), and Figures 12A-12C show graphs of nasal wash titers in ferrets following infection with influenza virus B / Washington / 02 / 2019 (IBV). Prior to these analyses, ferrets were intranasally vaccinated twice at 4-week intervals with c-di-AMP as an adjuvant. Groups of immunized animals included pre-immunized and naive ferrets immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens), as well as mock-immunized pre-immunized and naive ferrets. Four weeks after the second vaccination, groups of animals were challenged with H1N1 A / Brisbane / 02 / 2018 virus (Figures 11A-11C), or IBV B / Washington / 02 / 2019 virus (Figures 12A-12C). Nasal washes were taken from animals on days 1, 3, and 5 post-infection and viral titers were determined. Results demonstrated that by day 5 post-infection, no virus was detectable in nasal fluids obtained from pre-immunized and naive animals immunized with octavalent recombinant influenza immunogenic polypeptides (Cobra antigens), as well as mock-immunized pre-immunized animals.

[0182] In summary, the results of the ferret studies demonstrated that the multivalent recombinant influenza immunogenic polypeptides (Cobra antigens) described herein elicited broad-spectrum protective immune responses against influenza viruses of multiple subtypes. In addition, it was observed that pre-immunization improved the immune response of animals to virus challenge, and improved the immune response of animals immunized with the recombinant influenza immunogenic polypeptides (Cobra antigens) and ameliorated disease caused by viruses of multiple subtypes.

[0183] Mouse studies Viral infection and immunization (vaccination) of mice with broadly reactive recombinant HA polypeptide immunogens BALB / c and DBA / 2J mice (female, 6-8 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed in microisolator units and allowed free access to food and water. All animals were cared for under USDA guidelines for laboratory animals, and all procedures were approved by the Georgia Institutional Animal Care and Use Committee (IACUC) (no. A2018 06-018-Y3-A16). Seventy-five influenza naïve BALB / c mice were randomized into 15 groups (5 animals / group) and challenged with 3 μg of either broadly reactive recombinant HA (rHA) immunogenic polypeptides (Cobra antigens) described herein, J1, J2, J3, J4, NG1, NG2, or NG3 rHA (e.g., J1: SEQ ID NOs: 3, 7, 9; J2: SEQ ID NO: 4); J3 (SEQ ID NO: 5); J4 (SEQ ID NO: 6, 8); NG1 (SEQ ID NO: 11); NG2 (SEQ ID NOs: 2, 12); NG3 (SEQ ID NO: 13)) or 3 μg of WT H3N2 vaccine strains from the following historical vaccine strains: rHA (wild-type recombinant HA antigen): A / Switzerland / 9715293 / 2013 (Switz / 13) (EPI_ISL_162149, MDCK-SP2), A / Hong Kong / 4801 / 2014 (HK / 14) (EPI_ISL_259080, MDCK-SP3), A / Singapore / IFNIMH-16-0019 / 2016 (Sing / 16) (EPI_ISL_285898, MDCK-SP3), A / Kansas / 14 / 2017 (Kan / 17) (EPI_ISL_ Mice were vaccinated intramuscularly with either A / Switzerland / 8020 / 2017 (EPI_ISL_303951, MDCK-SP1), A / South Australia / 34 / 2019 (SA / 19) (EPI_ISL_395032, MDCK-SP2), or 25 μL of phosphate-buffered saline (PBS, Corning, Tewksbury, MA, USA) alone as a mock-vaccinated control.

[0184] All immunogens / vaccines (broadly reactive influenza immunogenic polypeptide antigens (Cobra antigens), wild-type (WT) rHA antigens, and mock) were formulated in ADDAVAX™ (InvivoGen, San Diego, CA, USA), an emulsified squalene-based oil-in-water emulsion adjuvant, with a final concentration of 2.5% squalene after mixing 1:1 with rHA. Immunogens / vaccines were administered into the hind paws of animals on days 0, 28, and 56 in a homologous prime-boost-boost regimen. Blood was collected from the facial vein 14 days after each vaccination, on days 14, 42, and 70. Serum was isolated from the animals' blood by centrifugation at 2,500 rpm for 10 min. Clarified serum was removed and frozen at -20±5°C.

[0185] Figures 13A-N show graphs of the results of hemagglutinin inhibition (HAI) assays performed on sera from 75 influenza-naive BALB / c mice 70 days after initial vaccination with recombinant influenza hemagglutinin (rHA) immunogenic polypeptide antigen. The results demonstrated that mock-vaccinated mice had no detectable titers against any of the H3N2 viruses in the panel (Figure 13A). Mice vaccinated with broadly reactive recombinant influenza immunogenic polypeptide antigen (Cobra antigen (rHA)) J1 (e.g., SEQ ID NOs: 3, 7) had antibodies with HAI activity against H3N2 influenza viruses isolated in 2012-2016, as well as the Switz / 17 and SA / 19 strains, but these antibodies failed to recognize the Kan / 17, Tx / 17, and HK / 19 viruses (Figure 13B). J2 vaccinated mice had antibodies with HAI activity against Tx / 12, HK / 14, Sing / 16, and Switz / 17 (Figure 13C). Mice vaccinated with J3 rHA had antibodies with HAI activity against all H3N2 influenza virus strains isolated in 2012-2016, as well as Switz / 17 and SA / 19, with the highest antibody titers directed against the Tx / 12 isolate (Figure 13D). J4 rHA induced antibodies with HAI activity against Tx / 12, HK / 14, Sing / 16, Switz / 17, and SA / 19 in vaccinated mice, with the highest antibody titers detected against the HK / 14 and Sing / 16 strains (Figure 13E). Mice vaccinated with NG1 rHA seroconverted to all H3N2 strains isolated between 2012 and 2019, except Kan / 17 and HK / 19 (Figure 13F). Mice vaccinated with NG2 rHA had seroprotective antibody titers against all of the H3N2 strains in the panel, with the highest titers directed against the SA / 19 virus (Figure 13G). The NG3 rHA vaccine induced seroconversion to Tx / 12, Switz / 13, Switz / 17, and SA / 19 (Figure 13H).Animals vaccinated with Switz / 13 seroconverted only to the homologously matched virus, Switz / 13, and not to any other viruses in the H3N2 panel (Figure 13I). The HK / 14 rHA vaccine induced antibodies with HAI activity against all viruses from 2012-2016, with the largest antibody titers directed against the homologously matched HK / 14 virus. The HK / 14 rHA vaccine did not induce antibodies with HAI activity against any of the strains isolated between 2017-2019 (Figure 13J). Mice vaccinated with Sing / 16 rHA had antibodies with HAI activity against all strains isolated between 2012-2019, with the exception of Kan / 17 and HK / 19 viruses. The largest antibody response in these mice was directed against the homologously matched Sing / 16 virus (Figure 13K). The Kan / 17 rHA vaccine induced antibodies with HAI activity against all H3N2 isolates from 2012 to 2017, except for the Sing / 16 virus. These mice also did not seroconvert to any of the 2019 isolates SA / 19 or HK / 19, and the largest antibody response from this group was directed against the homologously matched Kan / 17 virus (Figure 13L). Mice vaccinated with Switz / 17 rHA had antibodies with HAI activity against Tx / 12, Tx / 17, Switz / 17, and SA / 19, with the largest antibody response directed against the homologously matched Switz / 17 virus (Figure 13M). The SA / 19 rHA vaccine induced antibodies with HAI activity against Tx / 12, HK / 14, Tx / 17, Switz / 17, and SA / 19 viruses. The highest titers of HAI antibodies in this group were directed against the homologously matched SA / 19 virus (Fig. 13N).

[0186] Figure 14 shows virus titers from mouse lungs harvested from mice (n=3 per group) on day 89 (3 days after A / Kansas / 14 / 2017 challenge) to assess the viral load present in lung tissue. Influenza-naïve mock-vaccinated animals had the most virus present in their lungs, which was above the limit of detection (approximately 1 × 10) at 3 days post-infection. 2 PFU / g of lung tissue) was significantly higher than pre-immunized animals vaccinated with either the monovalent J4 HA immunogenic polypeptide antigen or the bivalent formulations of NG2 rHA and Y2+J4 or Y2+NG2 rHA described herein. In contrast, mice vaccinated with monovalent H1 rHA, Y2, Bris / 07, or Cal / 09 had lung titers (approximately 1×10) higher than those of mock-vaccinated pre-immunized animals. 5 The lung titers were similar to those of the control group (PFU / g of lung tissue).

[0187] 138 influenza-naïve DBA / 2J mice were randomly divided into 17 groups (8 mice / group) for use in the pre-immune mouse study. On day 0, 5 × 10 5Sixteen groups of mice were pre-immunized against both H1N1 and H3N2 influenza viruses by administering a mixture containing equal concentrations of H1N1 virus (A / Singapore / 6 / 1986 (Sing / 86)) and H3N2 virus (A / Panama / 2007 / 1999 (Pan / 99)) at a final concentration of PFU / 50 μL, with 50 μL administered intranasally to each mouse. Mock-pre-immunized animals were inoculated intranasally with 50 μL of PBS. After pre-immunization infection, animals were monitored twice daily in the morning and evening for weight loss and clinical signs (labored breathing, lethargy, hunched back, disheveled fur, unresponsiveness to stimuli, and severe respiratory distress) for 14 days post-infection. During this time, none of the mice lost more than 5% of their original body weight or showed any clinical signs. The animals were then allowed to rest for 30 days, at which point they were administered 3 μg total of a monovalent formulation of broadly reactive recombinant HA immunogen (Cobra): Y2 (H1), (e.g., SEQ ID NO: 15); J4 (H3), (e.g., SEQ ID NO: 6, 8), or NG2 (H3) (e.g., SEQ ID NO: 2, 12); a monovalent formulation of WT rHA: A / Brisbane / 59 / 2007 (Bris / 07) (H1), A / California / 07 / 2009 (Cal / 09) (H1), A / Switzerland / 9715293 / 2013 (Switz / 13) (H3), A / Singapore / IFNIMH-16-0019 / 2016 (Sing / 16) (H3); or 3 μg total (1.5 μg H1 + 1.5 μg H3) of Cobra. Mice were vaccinated with rHA polypeptide immunogen cocktails: Y2, Y2+J4 (H1+H3), or Y2+NG2 (H1+H3); or 3 μg total (1.5 μg H1+1.5 μg H3) WT rHA cocktails: Bris / 07+Switz / 13 (H1+H3), Bris / 07+Sing / 16 (H1+H3), Cal / 09+Switz / 13 (H1+H3), Cal / 09+Sing / 16 (H1+H3); or mock vaccine containing only 25 μL phosphate-buffered saline (PBS) (Corning, Tewksbury, MA, USA).All vaccines (broadly reactive recombinant polypeptide immunogen (Cobra antigen), WT, and mock) were formulated in ADDAVAX™ adjuvant, with a final concentration of 2.5% squalene after mixing 1:1 with rHA. Vaccines were administered intramuscularly in the hind leg of animals on days 30 and 58 in a homologous prime-boost regimen. Blood was collected from the facial vein 14 days after pre-immunization and each vaccination on days 14, 44, and 72. Serum was isolated from the animals' blood by centrifugation at 2,500 rpm for 10 minutes. Clarified serum was removed and frozen at -20±5°C. All DBA / 2J mice were then inoculated with 6.7×10 IgG on day 86. 6 Animals were challenged intranasally with 50 μL of live H3N2 influenza virus A / Kansas / 14 / 2107 (EP4) at a concentration of PFU / 50 μL. After infection, animals were monitored twice daily in the morning and evening for weight loss and clinical signs (labored breathing, lethargy, hunched back, disheveled fur, unresponsiveness to stimuli, and severe respiratory distress) for 14 days post-infection. On day 89, three animals from each group were euthanized and lungs were harvested to assess viral load. Lungs were frozen on dry ice and stored at −80±5°C until viral plaque assays were performed.

[0188] Figures 15A-F show nest reduction assay (FRA) titers of sera from pre-immunized mice at day 72 to assess the presence of antibodies directed against a panel of H3N2 viruses. Sera collected from 136 H1+H3 pre-immunized DBA / 2J mice (n=8 / group) vaccinated with monovalent and bivalent formulations of H1+H3 recombinant influenza immunogenic polypeptides (Cobra antigens) or WT rHA at day 72 post-primary infection were pooled for each group and assessed for FRA neutralization against a panel of historical H3N2 vaccine strains from 2016-2019, as described below. Sera from mice vaccinated with monovalent antigens were tested against the following H1N1: A / Singapore / IFNIMH-16-0019 / 2016 (Figure 15A), A / Kansas / 14 / 2017 (Figure 15B), and A / Hong Kong / 2671 / 2019 (Figure 15C). Sera from mice vaccinated with a cocktail of bivalent H1+H3 antigens were tested against the following H1N1 viruses: A / Singapore / IFNIMH-16-0019 / 2016 (Figure 15D), A / Kansas / 14 / 2017 (Figure 15E), and A / Hong Kong / 2671 / 2019 (Figure 15F). Results showed that broadly reactive influenza immunogenic polypeptides administered to pre-immunized animals enhanced neutralizing antibody responses against recent circulating H3N2 viruses, such as the Sing / 16 virus.

[0189] Pre-immunized mice vaccinated with monovalent formulations of broadly reactive recombinant HA (rHA) immunogenic polypeptides J4 or NG2 induced high (80%) plaque reduction / neutralization titers (PRNTs) against Sing / 16 virus ranging from 10.92 to 11.76. 80 These titers had an average PRNT of 12.46 against the homologously matched Sing / 16 virus (Figure 5A). 80 The neutralization titers were similar to those generated by the monovalent Sing / 16 rHA vaccinated group (Figure 15A). Mice vaccinated with Switz / 13 rHA had neutralization titers approximately six-fold lower than those of the other H3 rHA vaccine antigens, but still had a mean PRNT of 7.47 against the Sing / 16 virus.80 Mice that received monovalent H1 rHA vaccines, Y2, Bris / 07, or Cal / 09, had antibodies that neutralized about 10% of Sing / 16 virus infection, similar to those of mock-vaccinated pre-immunized mice (Figure 15A). Influenza-naive mock-vaccinated mice did not have antibodies capable of neutralizing Sing / 16. (See, e.g., JD Allen et al., 2022, J. Virology, Vol. 96, No. 7: doi.org / 10.1128 / jvi.01652-21.)

[0190] Mice vaccinated with monovalent J4 or NG2 H3 rHA had PRNTs against Kan / 17 virus ranging from 7.56 to 7.82. 50 Mice vaccinated with Switz / 13 rHA produced antibodies with high titers against Kan / 17 (Figure 15B). Mice vaccinated with Switz / 13 rHA had the highest levels of neutralizing antibodies against Kan / 17, and PRNT 50 The titer was 8.71 (Figure 15B). Mean PRNT of Sing / 16 rHA vaccinated mice 50 The titer was 6.34, which is the average PRNT of mice vaccinated with the H3 rHA immunogenic polypeptide antigen. 50 The mean PRNT of the Switz / 13 rHA-vaccinated animals was approximately two-fold lower than that of the control animals. 50 The titers were approximately 4-fold lower than those of the H1 rHA-vaccinated animals (Figure 15B). Animals that received the H1 rHA or mock vaccine had antibodies that neutralized approximately 10% of the Kan / 17 infection, and mock-vaccinated influenza-naive animals were unable to neutralize the Kan / 17 virus (Figure 15B). Pre-immunized mice vaccinated with either J4 or NG2 rHA had PRNTs ranging from 9.64 to 9.89 against HK / 19 v rus. 50 Mice vaccinated with Sing / 16 had similar PRNTs against the HK / 19 virus, ranging from 7.55 to 7.8, which was the highest neutralizing antibody response against this H3N2 strain (Figure 15C). 50Mice vaccinated with Switz / 13 rHA had neutralizing antibody titers, but those titers were approximately 4-fold lower than those generated by J4 and NG2 (FIG. 15C). Mice vaccinated with Switz / 13 rHA had a slightly lower mean PRNT of 7.12. 50 The H3 rHA vaccinated animals had titers lower than any other group of H3 rHA vaccinated animals (Figure 15C). Groups receiving H1 rHA or mock immunogen / vaccine generated antibodies that neutralized approximately 15% of HK / 19 infection (Figure 15C). Mock vaccinated influenza naive animals were unable to neutralize HK / 19 virus at any serum dilution (Figure 15C).

[0191] Pre-immunized mice vaccinated with rHA formulations containing either bivalent H1+H3 or H3 immunogenic polypeptide antigens (Y2+J4 and Y2+NG2) had antibodies with similar neutralizing ability against Sing / 16 virus, with a mean PRNT of 1.0-1.25. 50 The titers ranged from 12.34 to 12.48 (Figure 15D). These neutralizing antibody titers were similar to, but slightly lower than, those elicited by the homologously matched rHA immunogen / vaccine, Sing / 16, where both bivalent formulations, Bris / 07+Sing / 16 and Cal / 09+Sing16, elicited PRNTs of 12.75 and 12.99, respectively. 50 This is in contrast to mice vaccinated with bivalent combinations containing Switz / 13 rHA, Bris / 07+Switz / 13, and Cal / 09+Switz / 13, which produced approximately 10-fold lower PRNTs of 7.49 and 7.74, respectively, against the Sing / 16 virus. 50 However, mice vaccinated with bivalent vaccines containing Swiss / 13 rHA, Bris / 07+Switz / 13, and Cal / 09+Switz / 13 generated the highest neutralizing antibody titers against Kan / 17 virus, with a mean PRNT of 1.01 and 1.02, respectively. 50Mice vaccinated with bivalent protein formulations containing Sing / 16 H3 rHA, Bris / 07+Sing / 16, and Cal / 09+Sing16 had the lowest mean neutralizing antibody titers of any bivalent vaccination against Kan / 17 virus, with a PRNT of 1.0. 50 Animals vaccinated with rHA formulations containing the H3 immunogenic polypeptide antigen (Y2+J4 and Y2+NG2) had mean PRNTs of 6.92 to 7.51 against the Kan / 17 virus. 50 Mice vaccinated with either Y2+J4 or Y2+NG2 had PRNTs of 8.41 and 8.52, respectively. 50 Pre-immunized animals vaccinated with either Sing / 16 rHA, Bris / 07 1 Sing / 16, and Cal / 09+Sing16 produced similar neutralizing antibody responses to HK / 19 virus, although these titers were approximately 4-fold lower than those produced by either Y2+J4 or Y2+NG2 (Figure 15F). Mice vaccinated with Switz / 13 rHA (Bris / 07+Switz / 13 and Cal / 09+Switz / 13) produced the lowest neutralizing antibody titers to HK / 19 virus, with identical PRNTs. 50 The value was 5.64 (Figure 15F).

[0192] Figures 16A-F show the results of a nest reduction assay (FRA) against a panel of H1N1 influenza viruses performed using sera from pre-immunized DBA / 2J mice on day 72. Mice (n=8 / group) were vaccinated with monovalent and bivalent formulations of H1+H3 recombinant influenza immunogenic polypeptides (Cobra antigens) or WT rHA. Pooled sera from each group were evaluated for FRA neutralization against a panel of historical H1N1 vaccine strains from 2009-2019, as described below. Sera from mice vaccinated with monovalent antigens were tested against the following H1N1 viruses: A / California / 07 / 2009 (Figure 16A), A / Brisbane / 2 / 2018 (Figure 16B), and A / Guangdong Maonan / SWL1536 / 2019 (Figure 16C). Sera from mice vaccinated with a cocktail of bivalent H1+H3 antigens were tested against the following H1N1 viruses: A / California / 07 / 2009 (Fig. 16D), A / Brisbane / 2 / 2018 (Fig. 16E), and A / Guangdong Maonan / SWL1536 / 2019 (Fig. 16F).

[0193] For the study results shown in Figures 16A-16F, an influenza virus foci reduction assay (FRA) was used to determine the ability of broadly reactive immunogenic polypeptide immunogen-elicited antibodies to neutralize live virus infection against a panel of three historical influenza A (H1N1) vaccine strain isolates from 2009-2019. Sera used in the assay were collected from 136 mice (n=8 / group) 72 days after immunization and pooled for each group. Pre-immunized mice vaccinated with a monovalent formulation of the broadly reactive immunogenic polypeptide immunogen Y2 rHA (e.g., SEQ ID NO: 15) demonstrated log2 80% neutralization (80% plaque reduction / neutralization titer [PRNT 80 ]) had antibodies that neutralized Cal / 09 virus infection with mean titers greater than the mark (Figure 16A). Similarly, mice vaccinated with Cal / 09 rHA antigen also had a PRNT 80All other monovalent rHA vaccinated mice did not produce neutralizing antibodies to Cal / 09 viral antigens, with titers similar to mock-vaccinated pre-immunized animals (Figure 16A). Serum collected from mock-immunized / vaccinated influenza naïve mice did not contain antibodies capable of neutralizing Cal / 09 viral antigens at any dilution (Figure 16A). Pre-immunized mice vaccinated with Y2 rHA also produced neutralizing antibodies to Cal / 09 viral antigens at all dilutions, with titers greater than PRNT. 80 Sera collected from mice vaccinated with Cal / 09 rHA antigen also neutralized Bris / 18 virus infection at all dilutions with titers of PRNT 0.01 and 0.02 (Fig. 16B). 80 All other monovalent rHA immunogen / vaccine groups produced antibodies that prevented less than about 20% of cells from infection with Bris / 18, similar to mock-vaccinated pre-immunized animals, at the lowest serum dilutions (Figure 16B). Sera collected from mock-immunized / vaccinated influenza naïve mice did not contain antibodies that were able to neutralize Bris / 18 viral antigens at any serum dilution (Figure 16B). Pre-immunized mice vaccinated with Y2 rHA antigen had the highest neutralization titers of any of the monovalent groups against Guang / 19 virus, with a mean log250% plaque reduction / neutralization titer (PRNT 50 ) titer was 10.14 (FIG. 16C). Mice vaccinated with Cal / 09 rHA had a mean PRNT of 1.08 for Guang / 19. 50All mice vaccinated with monovalent rHA antigen had neutralizing serum antibodies at a titer of 7.75. The titer was approximately 6-fold lower than that induced in Y2 rHA antigen vaccinated animals (Figure 16C). All other groups of mice vaccinated with monovalent rHA antigen produced serum antibodies that prevented approximately 10% of cells from being infected with Guang / 19 virus, similar to mock-vaccinated pre-immunized animals (Figure 16C). Serum from mock-immunized / vaccinated influenza naïve mice was unable to neutralize Guang / 19 virus (Figure 16C).

[0194] Mice vaccinated with bivalent rHA formulations containing the Y2 HA immunogenic polypeptides, i.e., Y2+J4 and Y2+NG2, all had serum antibodies with similar neutralizing ability against Cal / 09 virus, with a mean PRNT of 1.0-1.25. 80 The titers were 11.08, 11.35, and 11.61, respectively (FIG. 16D). Similarly, pre-immunized mice vaccinated with a mixture containing Cal / 09 rHA (Cal / 09 1 Switz / 13 and Cal / 09 1 Sing / 16) showed high PRNTs of 11.96 to 12.32 against the homologously matched Cal / 09 virus. 80 Mice vaccinated with mixtures containing Bris / 07 H1 rHA (Bris / 07 1 Switz / 13 and Bris / 07 1 Sing / 16) prevented approximately 25% of cells from being infected by Cal / 09 virus at the lowest serum dilution, similar to mock-vaccinated pre-immunized animals (Figure 16D). Mice vaccinated with mixtures containing Cal / 09 rHA (Cal / 09 1 Switz / 13 and Cal / 09 1 Sing / 16) also showed high PRNTs against Bris / 18 virus, ranging from 11.35 to 11.73. 80 All mice vaccinated with mixtures containing the Y2 rHA antigen (such as Y2+J4 and Y2+NG2) produced antibodies with similar neutralizing ability against Bris / 18 virus, and PRNT was 1.0, which was slightly lower than that generated against Cal / 09 virus (Figure 16E). 80Titers ranged from 11.75 to 12.13, slightly higher than those produced by the Cal / 09 vaccine mixture (Figure 16E). Animals vaccinated with mixtures containing Bris / 07 rHA prevented approximately 20% of cells from being infected with Bris / 18 virus at the lowest serum dilution, similar to mock-vaccinated pre-immunized animals (Figure 16E). Mice vaccinated with bivalent formulations containing Cal / 09 rHA antigen (Cal / 09 1 Switz / 13 and Cal / 09 1 Sing / 16) showed PRNTs of 6.59 to 6.78 against Guang / 19 virus, respectively. 50 In contrast, all pre-immunized animals vaccinated with mixtures containing COBRA Y2 H1 rHA antigens (such as Y2+J4 and Y2+NG2) produced antibodies with similar neutralizing ability against Guang / 19 virus, and PRNT 50 The titers were approximately 4-fold higher than those induced in Cal / 09 rHA-vaccinated animals with log2PRNT50 titers between 8.34 and 8.44, respectively (Figure 16F). Animals vaccinated with the mixture containing Bris / 07 rHA prevented approximately 10% of cells from being infected with Guang / 19 virus at the lowest serum dilution, similar to mock-vaccinated pre-immunized animals (Figure 16E). Mean HAI and PRNT 50 Titers were also compared for each group. In general, HAI log2 geometric mean titers (GMTs) of about 5.5 or greater and log2 PRNTs of about 8.34 or greater were associated with 50 We show that the HAI titer (5.32), which correlates with titer and 50% protection in humans, may be a predictive measure of neutralization observed with the COBRA against influenza A (H1N1) viruses. Results demonstrated that the COBRA vaccine enhanced neutralizing antibody responses against contemporary pandemic-like H1N1 viruses.

[0195] Materials, reagents and methods used in Example 2 Virus and HA antigen Influenza A (H3N2) viruses were obtained either by the Influenza Reagents Resource (IRR), BEI Resources, Centers for Disease Control (CDC) or provided by Virapur (San Diego, CA, USA). Following the instructions provided by WHO, viruses were passaged once in the same growth conditions in which they were received, i.e., either in embryonated chicken eggs or in semi-confluent Madin-Darby canine kidney (MDCK) cell cultures. H3N2 virus lots were titrated on 0.75% guinea pig red blood cells in the presence of 20 nM oseltamivir and aliquoted for single-use applications. H1N1 virus lots were titrated on 0.8% turkey red blood cells and aliquoted for single-use applications.

[0196] The A(H3N2) 2012-2019 historical influenza vaccine strain virus panel for HAI analysis included the following eight virus strains: A / Texas / 50 / 2012 (Tx / 12) egg passage 4 (EP4) (clade 3c2), A / Switzerland / 9715293 / 2013 (Switz / 13) EP4 (clade 3c3.a), A / Hong Kong / 4801 / 2014 (HK / 14) EP11 (clade 3c2.a), and A / Singapore / IFNIMH-16-0019 / 2016 (Sing / 16) EP3 (clade 3c2.a1), A / Kansas / 14 / 2017 (Kan / 17) EP1 (clade 3c2.a2). (clade 3c3.a), A / Texas / 71 / 2017 (Tx / 17) MDCK-siat cells passage 1 (MDCK-SP1) (clade 3c3.a), A / Switzerland / 8060 / 2017 (Switz / 17) EP1 (clade 3c3.a2), A / South Australia / 34 / 2019 (SA / 19) EP1 (clade 3c2.a1b / 131K), and A / Hong Kong / 2671 / 2019 (HK / 19) EP1 (clade 3c2.a1b / 137F).

[0197] The A(H1N1) 2007–2019 historical influenza vaccine strain panel for HAI analysis included five influenza A virus (IAV) strains: A / Brisbane / 59 / 2007 (Bris / 07) EP1, A / California / 07 / 2009 (Cal / 09) EP4, A / Michigan / 45 / 2015 (Mich / 15) EP1, A / Brisbane / 02 / 2018 (Bris / 18) EP1, and A / Guangdong-Monan / SWL1536 / 2019 (Guang / 19) EP1. For H1N1 HAI analysis in ferrets, we also used A / Solomon Islands / 03 / 2006 (Genbank accession no. EU100724).

[0198] The IBV 2006–2019 panel for HAI analysis included six virus strains: B / Florida / 04 / 2006 (Genbank accession no. KF009552), B / Massachusetts / 02 / 2012 (Genbank accession no. C892118), and B / Phuket / 3073 / 2013 (NCBI accession no. EPI1799823) for the Yamagata-like lineage, and B / Brisbane / 60 / 2008 (Genbank accession no. FJ766840), B / Colorado / 06 / 2017 (Genbank accession no. CY232066), and B / Washington / 02 / 2019 (Genbank accession no. MK676295) for the Victoria-like lineage.

[0199] The 2004–2014 panel of H5 for HAI analysis included six virus strains: A / Vietnam / 1203 / 2004 (H5N1, Genbank accession number AAW80717.1), A / Wuhan / Mongolia / 244 / 2005 (H5N1, Genbank accession number ACD68156.1), A / Egypt / 321 / 2007 (H5N1, Genbank accession number AEL31632.1), A / Hupei / 01 / 2010 (H5N1, Genbank accession number AEO89181.1), A / Guizhou / 01 / 2013 (H5N1, Genbank accession number EPI420386), and A / Sichuan / 26221 / 2014 (H5N6, Genbank accession number EPI533583).

[0200] Mice were pre-immunized against H1N1 and H3N2 viruses using one historical H1N1 vaccine strain virus, A / Singapore / 6 / 1986 (Sing / 86) EP1, and one historical H3N2 vaccine strain virus, A / Panama / 2007 / 1999 (Pan / 99) EP4, and were also challenged with the H3N2 influenza virus, A / Kansas / 14 / 2017 (Kan / 17) EP1, on day 86 of the pre-immunization study.

[0201] Ferrets were preimmunized against all three seasonal virus subtypes using CA / 09, Pan / 99, and B / Hong Kong / 330 / 2001 virus strains (Genbank accession number AF532549) and challenged with Bris / 18 (H1N1), B / WA / 19 (IBV), or A / Vietnam / 1203 / 2004 (Vn / 04).

[0202] Hemagglutinin inhibition (HAI) assay The hemagglutinin inhibition (HAI) assay was used to evaluate the presence of functional anti-hemagglutinin (HA) antibodies (e.g., in serum obtained from bleeding animals) that could inhibit the agglutination of guinea pig red blood cells for H3N2 viruses and turkey red blood cells for H1N1 viruses. The protocol was adapted from the WHO Laboratory Influenza Surveillance Manual. (See JD Allen et al., 2022, J. Virology, Vol. 96, No. 7: doi.org / 10.1128 / jvi.01652-21.) Guinea pig red blood cells are frequently used to characterize modern A(H3N2) influenza strains that have developed preferential binding to alpha(2,6)-linked sialic acid receptors. To inactivate nonspecific inhibitors, serum samples were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Co., Japan) before testing. Briefly, 3 parts RDE was added to 1 part serum and samples were incubated overnight at 37° C. The RDE was inactivated by incubation at 56° C. for 30 min.

[0203] RDE-treated sera were diluted in a series of two-fold serial dilutions in v-bottom microtiter plates. An equal volume of each A(H3N2) virus, adjusted to approximately 8 hemagglutinin units (HAU) / 50 μl in the presence of 20 nM oseltamivir carboxylate, was added to each well. Plates were covered and incubated at room temperature for 30 min, then 0.75% guinea pig red blood cells (Lampire Biologicals, Pipersville, PA, USA) in PBS were added. Before use, red blood cells (RBCs) were washed twice with PBS, stored at 4°C, and used within 24 hours (h) of preparation. Plates were mixed by gentle agitation, covered, and RBCs were allowed to settle for 1 h at room temperature. HAI titers were determined by reciprocal dilutions of the last well containing unagglutinated RBCs. Positive and negative serum controls were included on each plate.

[0204] In a separate assay, RDE-treated sera were diluted in a series of two-fold serial dilutions in v-bottom microtiter plates. An equal volume of each A(H1N1) virus, adjusted to approximately 8 hemagglutinin units (HAU) / 50 μl, was added to each well. Plates were covered and incubated at room temperature for 20 min, then 0.8% turkey red blood cells (Lampire Biologicals, Pipersville, PA, USA) in PBS were added. Before use, RBCs were washed twice with PBS, stored at 4°C, and used within 24 h of preparation. Plates were mixed by gentle agitation, covered, and RBCs were allowed to settle at room temperature for 30 min. HAI titers were determined by reciprocal dilutions of the last well containing unagglutinated RBCs. Positive and negative serum controls were included on each plate.

[0205] All mice were negative for pre-existing antibodies to human influenza virus prior to infection or vaccination (HAI≦1:10) and for the animal studies described above, a positive HAI response (HAI+) or "seroprotection" was defined as an HAI titer of ≧1:40, while "seroconversion" refers to a four-fold increase in titer compared to baseline according to the WHO and European Committee for Medicinal Products for evaluating influenza vaccines.

[0206] Foci reduction assay (FRA) The foci reduction assay (FRA) used for the animal studies was originally developed by the WHO collaborating Centre in London, UK, and subsequently modified by the US Centers for Disease Control and Prevention (CDC). MDCK-SIAT1 cells (Sigma, St. Louis, MO, USA) were cultured at 2.5–3 × 10 5 Cells were plated at 100 cells / ml (100 μL / well in 96-well plates). Cells were cultured overnight in Dulbecco's modified Eagle's medium (DMEM) containing 5% heat-inactivated fetal bovine serum and antibiotics in 96-well flat-bottom plates to form a 95-100% confluent monolayer. The next day, cell monolayers were washed with 0.01 M phosphate-buffered saline (PBS), pH 7.2 (Gibco, Waltham, MA, USA), followed by the addition of two-fold serial dilutions of RDE-treated serum (50 μL per well) starting from a 1:20-fold dilution in virus growth medium containing TPCK-treated trypsin (1 μg / ml) (Thermo Fisher, Waltham, MA, USA), VGM-T (i.e., DMEM containing 0.1% BSA, 1% penicillin / streptomycin (100 U / mL penicillin, 100 μg / mL streptomycin solution), and 1 μg / ml TPCK-treated trypsin) (Sigma, St. Louis, MO, US). Wells of each plate were incubated with 50 μL of A(H3N2) influenza virus (1.2 × 10 410 ...

[0207] Then, after a 2-h incubation period at 37°C in 5% CO2, the cells in each well were overlaid with an equal volume of 100 μL of 1.2% Avicel RC / CL (type: RC581 NF; FMC Health and Nutrition, Philadelphia, PA, USA) in 2x modified Eagle's medium containing 1 μg / ml TPCK-treated trypsin, 0.1% BSA and antibiotics. The plates were incubated at 37°C, 5% CO2 for 18-22 h. The overlay was then removed from each well and the monolayer was washed once with PBS to remove any residual Avicel. The plates were then fixed with ice-cold 4% formalin in PBS for 30 min at 4°C, followed by a PBS wash and permeabilized using 0.5% Triton-X-100 in PBS / glycine for 20 min at room temperature (RT). Plates were washed three times with washing buffer (PBS, 0.1% TWEEN-20; PBST) and then incubated for 1 h with a monoclonal antibody (FR-1217) (1 mg / mL) directed against influenza A nucleoprotein obtained from Influenza Reagent Resource (IRR) (Manassas, VA, USA) and diluted 1:2000 in ELISA buffer (PBS, 10% horse serum, 0.1% TWEEN-80). After washing (3x PBST), cells were incubated with goat anti-mouse peroxidase-labeled IgG (Sera Care, Inc., Milford, MA, USA) (KPL 474-1802) (1 mg / mL) and diluted 1:2000 in ELISA buffer for 1 h at room temperature. Plates were then washed again (3x PBST) and infectious viral foci were visualized using TrueBlue substrate (SeraCare, Inc., Milford, MA USA) containing 0.03% H2O2 and incubated for 10 min at room temperature. The reaction was stopped by washing five times with dH20. Plates were air-dried and foci were enumerated using a CTL BioSpot Analyser with ImmunoCapture 6.4.87 software (CTL, Shaker Heights, OH, USA).FRA titers were reported as the reciprocal of the highest dilution of serum corresponding to a 50% foci reduction compared with the virus control minus the cell control.

[0208] For a plate to pass quality control (QC), the mean of the octuplet virus control wells (VC) and the mean of the octuplet cell control wells (CC) must both pass QC. The virus controls must fall within 200-1600 foci and the cell controls must contain no foci. In addition, positive control A(H1N1) and A(H3N2) historical influenza vaccine strain viruses must be run in triplicate plates in each individual assay and at least two of the three plates must pass the VC and CC criteria. 1 × 10 6 Homologous mouse antisera, previously generated by infection with homologously matched A(H1N1) and A(H3N2) influenza viruses at FFU / mL and collected 14 days after infection, should have the same titer across plates. Each assay plate (one virus per plate) contained a panel of mouse reference antisera, as well as a human influenza vaccine serum control to assess overall assay consistency. The percentage of infected cells reported in the assay was calculated by averaging the foci counts from the positive control (virus and cells only) wells and dividing the foci counts in each experimental well by the average of the positive controls.

[0209] Influenza virus plaque assay MDCK cells (Sigma, St. Louis, MO, USA) were cultured at 1 × 10 6Cells were seeded into each well of a 6-well plate at a concentration of 100 cells / well. On the day of the assay, frozen lung tissue was thawed on ice, weighed, and homogenized in 1 ml of DMEM (Thermo Fisher, Waltham, MA, USA). The homogenate was centrifuged at 2,000 rpm for 5 min to remove tissue debris. The supernatant was collected and serially diluted 10-fold in DMEM supplemented with 1% penicillin-streptomycin (DMEM+P / S) (Thermo Fisher, Waltham, MA, USA). When MDCK cells reached 90% confluency in each well, the plates were washed twice with DMEM+P / S and infected with 100 μL of each dilution of homogenate supernatant. The plates were then shaken every 15 min for 1 h. After 1 h of incubation, the supernatant was removed and the cells were washed twice with fresh DMEM+P / S. After the second wash, a solution of 2x MEM and 1.6% agarose (Thermo Fisher, Waltham, MA, USA) mixed 50:50 (v / v) and supplemented with 1 μg / mL TPCK trypsin (Thermo Fisher, Waltham, MA, USA) was added to each well. The plates were then incubated at 37°C + 5% CO2 for 72 h. After 72 h, the gel overlay was removed from each well and the cells were fixed with 10% buffered formalin for 10 min and stained with 1% crystal violet (Thermo Fisher, Waltham, MA, USA) for 10 min at room temperature. The plates were then rinsed thoroughly five times with fresh water to remove excess crystal violet. The plates were air-dried for 24 h and viral plaques were enumerated as the reciprocal of each dilution. Lung viral titers were calculated and presented as plaque-forming units (PFU) / g of lung tissue.

[0210] Similarly, for the ferret nasal wash plaque assay, nasal wash samples were thawed on the day of the assay and serially diluted 10-fold in DMEM+P / S. 100 μL of each dilution was placed onto 90% confluent MDCK cells that had been washed twice with DMEM+P / S. Plates were shaken every 15 min for 1 h as described above. Plates were washed and incubated in an agarose overlay at 37°C + 5% CO2 for 72 h. The overlay was then removed and cells were fixed, stained and enumerated as above. Viral titers were calculated and presented as PFU / mL of nasal wash sample.

[0211] Enzyme-linked immunosorbent assay (ELISA) Antibody reactivity against different H1N1 HA strains was assessed using ELISA and was performed as previously described. Briefly, Immulon 4HBX plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated overnight at 4°C in a humidified chamber with a solution of 1 μg / mL of different rHA (A / California / 07 / 2009, A / Brisbane / 02 / 2018), or cH6 / 1 purified rHA and 5 μg / ml of bovine serum albumin (BSA) in carbonate buffer (pH 9.4) with 50 μl per well. 5 μg / ml of BSA (50 μl per well) was coated alone as a negative control. Plates were blocked with ELISA blocking buffer in a volume of 200 μl / well for 1 h at 37°C. Serum samples were serially diluted 3-fold in blocking buffer starting from a dilution of 1:100 and then added to the HA protein-coated plates. After overnight incubation at 4°C, goat anti-mouse IgG (Southern Biotech, Birmingham, AL, USA) secondary antibody diluted 1:2000 was added to each well in a volume of 100 μl and incubated at 37°C for 1 h. Finally, 50 μl of ABTS substrate (VWR Corporation) was added to each well and the plate was further incubated at 37°C for 15–20 min. The colorimetric conversion was stopped by adding 50 μl of 1% SDS to each well. OD values ​​(OD414) were measured by a spectrophotometer (PowerWave XS, BioTek) at 414 nm.

[0212] Similarly, ELISA was used to evaluate antibody reactivity in ferret sera against influenza HA or NA polypeptide antigens (Cobra antigens). Plates were coated overnight at 4° C. in carbonate buffer containing 1 μg / mL of different rHA and rNA at 50 μl per well in a humidified chamber. Plates were blocked for 1 h at 37° C. with a volume of 200 μl / well of ELISA blocking buffer. Serum samples were serially diluted 3-fold in blocking buffer starting from a dilution of 1:100 and then added to the protein-coated plates. After overnight incubation at 4° C., plates were washed five times in wash buffer (0.05% TWEEN-20 in PBS). 100 μl of goat anti-ferret IgG H&L HRP (Abcam, Boston, MA) diluted 1:4000 in blocking buffer was added and plates were incubated for 1 h at 37° C. Plates were washed five times with wash buffer. 50 μl of ABTS substrate (VWR, Radnor, PA) was added to each well and the plate was further incubated at 37°C for 15-20 min. The colorimetric conversion was stopped by adding 50 μl of 1% SDS to each well. OD values ​​(OD414) were measured by a spectrophotometer (PowerWave XS, BioTek) at 414 nm.

[0213] statistical analysis Data are presented as absolute mean ± standard error of the mean (SEM). One-way ANOVA and two-way ANOVA with multiple comparisons were used to analyze statistical differences between groups using GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p value of less than 0.05 was defined as statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001).

[0214] Example 3: Mouse studies using recombinant influenza virus HA immunogenic peptides (influenza H1N1 virus) as immunogens (vaccine) Similar studies to those described in Example 2 were performed in vivo in pre-immunized and naive mice using recombinant influenza virus HA immunogenic polypeptides (influenza H1N1 virus) as immunogens (vaccines) to determine the immunogenicity and efficacy of recombinant influenza HA immunogenic polypeptides in eliciting broadly protective immune responses against seasonal and pandemic influenza viruses using in vivo mouse models. As described in Examples 1 and 2, the broadly reactive non-natural HA polypeptide immunogen(s) (and recombinant HA polypeptide immunogen(s)) comprise influenza virus hemagglutinin (HA) antigenic amino acid sequences derived from influenza H1 types, such as H1N1, containing HA sequences representative of those of seasonal or pandemic influenza viruses.

[0215] H1N1 influenza viruses used in this study included A / Chile / 1 / 1983 (GenBank accession number CY121261.1), A / Singapore / 6 / 1986 (GenBank accession number CY020477.1), A / Beijing / 262 / 1995 (GenBank accession number CY033614.1), A / New Caledonia / 20 / 1999 (GenBank accession number EF566076.1), A / California / 07 / 2009 (GISAID accession number EPI516535), A / Brisbane / 02 / 2018 (GISAID accession number EPI1799929), and A / Guangdong-Maonan / SWL 1536 / 2019 (GISAID accession number EPI1921671). All viruses were obtained from Virapur (NY) or BEI Resources. Each virus was amplified in fertilized chicken eggs.

[0216] Animal Vaccination / Vaccination and Infection BALB / c and DBA / 2J mice (female, 6-8 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME, USA), housed in microisolator units and allowed free access to food and water. Animals were cared for under USDA guidelines for laboratory animals. All procedures were reviewed and approved by the University of Georgia Institutional Animal Care and Use Committee (IACUC) (no. A2018 06-018-Y3-A16). Eighty-eight BALB / c mice were randomly divided into eight groups, each containing 11 mice. Mice were vaccinated intramuscularly with 1 μg of recombinantly produced, non-natural, broadly reactive H1 HA polypeptide immunogen (Cobra) Brisbane / 59 / 2007, California / 07 / 2009, Brisbane / 02 / 2018 virus-like particles (VLPs); or PBS formulated with ADDAVAX™ (oil-in-water emulsion) (InvovoGen, San Diego, CA, USA) at a 1:1 ratio in a final volume of 50 μL. Mice were boosted intramuscularly with the same amount of VLPs or PBS at weeks 4 and 8 after the first vaccination. (Y. Huang et al., 2021, Vaccines, 9(7):793, the contents of which are incorporated herein by reference).

[0217] Another group of 40 DBA / 2J mice were divided into 5 groups (n=8 / group) and vaccinated with 1 μg of the corresponding soluble recombinant HA protein described above using the same vaccination regimen. Blood was collected 6 and 10 weeks after the first vaccination, and serum was separated and stored at -20°C for future use. At week 12, all mice were vaccinated with 5×10 4 PFU of wild-type H1N1 A / California / 07 / 2009 (CA / 09) or 8.75 × 10 6Mice were infected intranasally with PFU of H1N1 A / Brisbane / 02 / 2018 (Bris / 18). Animals were monitored and their body weights were recorded daily for 14 days post-infection. On days 3 and 6 post-infection, three mice from each group were euthanized and lungs were harvested. The left lung was inflated with 10% neutral formalin for histopathology, and the right lung lobe was snap frozen on dry ice and then stored at -80°C for viral titer assessment. Mice were humanely euthanized when they reached a humane endpoint by losing 20% ​​of their original body weight or accumulated a clinical disease score of 3. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, and Biosafety in Microbiological and Biomedical Laboratories.

[0218] ELISA was used to evaluate antibody reactivity against the different H1N1 HA strains and was performed as described in GA Kirchenbaum GA et al., (2017, J. Immunol.;199:3798-3807). Briefly, Immulon 4HBX plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated overnight at 4 °C in a humidified chamber with a solution of carbonate buffer (pH 9.4) containing 1 μg / mL of different rHA (A / California / 07 / 2009, A / Brisbane / 02 / 2018) and 5 μg / mL of bovine serum albumin (BSA) with 50 μL per well. An amount of 5 μg / mL of BSA (50 μL per well) was coated alone as a negative control. Plates were blocked with a volume of 200 μL / well of ELISA blocking buffer for 1 h at 37 °C. Serum samples were serially diluted 3-fold in blocking buffer starting from a dilution of 1:100 and then added to the HA protein-coated plates. After overnight incubation at 4°C, goat anti-mouse IgG (Southern Biotech, Birmingham, AL, USA) secondary antibody (diluted at 1:2000) was added to each well in a volume of 100 μL, and the plates were incubated at 37°C for 1 h. Finally, 50 μL of ABTS substrate (VWR Corporation) was added to each well, and the plates were further incubated at 37°C for 15–20 min. The colorimetric conversion was stopped by adding 50 μL of 1% SDS to each well. OD values ​​(OD414) were measured using a spectrophotometer (PowerWave XS, BioTek) at 414 nm.

[0219] The samples were analyzed by performing a plaque assay. For the plaque assay, MDCK cells within 20 passages were cultured at 1 × 10 6Cells were seeded into each well of a 6-well plate at a concentration of 10 cells / well. Frozen lung tissue was thawed on ice and homogenized in 1 mL of DMEM. The homogenate was centrifuged at 2,000 rpm for 5 min to remove tissue debris, and the supernatant was collected and serially diluted 10-fold in DMEM supplemented with 1% penicillin-streptomycin. MDCK cells with 90% confluency in each well were infected with 100 μL of each diluted homogenate supernatant. The plates were then shaken every 15 min for 1 h. After 1 h of incubation, the supernatant was removed and the cells were washed twice with fresh DMEM. Finally, 2 mL of 2×MEM and 0.8% agarose overlay (Cambrex, East Rutherford, NJ, USA) was added to each well, and the plates were incubated at 37° C., 5% CO2 for an additional 72 h. The overlay was then removed from each well, and cells were fixed with 10% buffered formalin for 20 min and stained with 1% crystal violet (Fisher Science Education, Waltham, MA, USA) for 15 min at room temperature (RT). Plates were then rinsed thoroughly using tap water to remove excess crystal violet. Plaques were enumerated and lung viral titers were calculated and presented as PFU / mL.

[0220] A hemagglutinin inhibition (HAI) assay was used to assess the presence of functional antibodies capable of binding to the HA protein and inhibiting hemagglutination. The protocol was adapted from the WHO Laboratory Influenza Surveillance Manual (WHO Global Influenza Surveillance Network. Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza. World Health Organization; Geneva, Switzerland: 2011). In this study, the HAI assay was performed against a panel of seven H1N1 influenza viruses, including A / Chile / 1 / 1983, A / Singapore / 6 / 1986, A / Beijing / 262 / 1995, A / New Caledonia / 20 / 1999, A / California / 07 / 2009, A / Brisbane / 02 / 2018, and A / Guangdong-Monan / SWL 1536 / 2019. HAI assays were performed as described by DM Carter et al. (2016, J. Virol. 2016;90:4720-4734). Briefly, sera were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Co., Tokyo, Japan) and then tested to remove nonspecific inhibitors by incubating overnight at 37°C. RDE was then further inactivated at 56°C for 45 min. A volume of 25 μL of PBS was added to a 96-well V-bottom plate in rows 2-12, 50 μL of RDE-treated serum was added to row 1, and then two-fold serial dilutions were performed across the plate. An equal volume of H1N1 virus with approximately 8 hemagglutinin units (HAU) / 50 μL was added to each well. After the plate was incubated at room temperature for 30 min, a 50 μL volume of 0.8% turkey red blood cells in PBS was added to each well. Plates were mixed by vortexing and further incubated for 30 min at room temperature. HAI titers were determined as reciprocal dilutions of the last well containing unagglutinated RBCs. Positive and negative serum controls were included on each plate.An HAI titer of >1:40 was defined as seroprotection, and a four-fold increase in HAI titer compared to baseline was considered as seroconversion according to the WHO and European Committee for Medicinal Products guidelines for evaluating influenza vaccines (European Medicines Agency, 2014, Guideline on Influenza Vaccines: Non-Clinical and Clinical Module (Draft) European Medicines Agency; London, UK).

[0221] This study used the foci reduction assay (FRA) originally developed by the World Health Organization collaborating center in London, UK, and modified by the US Centers for Disease Control and Prevention (CDC). Briefly, serum samples were processed with RDE as described above. First, 3 × 10 5 100 μL of MDCK cells at a concentration of 10 cells / mL were seeded into each well in a 96-well flat-bottom plate. After 24 h, the cells were allowed to reach 95%-100% confluence and then washed with PBS. Next, 50 μL of two-fold serially diluted serum samples were added to each well, starting from a 1:20 dilution in virus clarified medium (VGM) supplemented with 1 μg / mL tosylsulfonylphenylalanyl chloromethyl ketone (TPCK)-treated trypsin (VGM-T) (Sigma, St. Louis, MO, USA). Influenza virus was then diluted in VGM-T to a concentration of 1.2 × 10 450 μL of virus solution at a concentration of FFU / mL was added to each well, and VGM-T alone was also added to cell control wells. Plates were incubated at 37°C for 2 h, and then 100 μL of overlay was added to each well. The overlay contained equal volumes of 1.2% Avicel RC / CL (FMC Health and Nutrition, Philadelphia, PA, USA) and 2x MEM supplemented with 1 μg / mL TPCK-treated trypsin, 0.1% BSA, and 1% penicillin-streptomycin. After 18–22 h of incubation at 37°C, the overlay was removed and cells were washed twice using PBS. Cells were then fixed with ice-cold 4% formalin in PBS for 30 min at 4°C, followed by one wash with PBS and permeabilization with 0.5% Triton-X-100 for 20 min at RT. The monolayers were washed three times with PBS containing 0.1% Tween 20 (wash buffer) and incubated with mouse anti-IAV nucleoprotein monoclonal antibody at 37°C for 1 h. ((Y. Huang et al., 2021, Vaccines, 9(7):793). After washing three times with wash buffer, cells were incubated with the secondary antibody goat anti-mouse peroxidase-labeled IgG (SeraCare, Inc., Milford, MA, USA) for 1 h at RT. Cells were then washed three times with wash buffer and TrueBlue substrate (SeraCare, Inc., Milford, MA, USA) containing 0.03% HO was added and incubated for 10–15 min at RT. The reaction was stopped by washing the plates five times with distilled water. Plates were air-dried and foci were counted using a CTL BioSpot Analyzer with ImmunoCapture 6.4.87 software (CTL, Cleveland, OH, USA). FRA titers were presented as the reciprocal of the highest dilution of serum corresponding to a 50% foci reduction compared to virus control wells minus cell control wells.

[0222] All data are presented as absolute mean ± standard error of the mean (SEM). One-way ANOVA was used to analyze statistical differences between groups using GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p value of less than 0.05 was defined as statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

[0223] FIG. 17 shows a timeline of the study described in Example 3. FIG. 18A-D presents graphs showing weight and survival curves following influenza virus infection of mice vaccinated with broadly reactive recombinant influenza HA immunogens according to the study described in Example 3 and the timeline and protocol shown in FIG. 17. Weight and survival results provided in FIG. 18A-D show that animals vaccinated with broadly reactive recombinant influenza HA immunogens described herein did not lose significant weight and showed 100% survival following virus challenge. FIG. 19A-E show serum HAI antibody titers in mice following vaccination against a panel of H1N1 viruses. Sera from animals vaccinated with VLPs encoding broadly reactive recombinant influenza H1N1 HA immunogen Y2 (e.g., SEQ ID NO: 15) or H1N1 wild type CA / 09 or Bris / 18 had high HAI titers against CA / 09, Bris / 18, and Guangdong / 19 virus antigens following infection. FIG. 20A and FIG. 20B show serum neutralizing antibody titers and neutralizing activity of antibodies elicited in immunologically naive Balb / c mouse serum after immunization / vaccination with a broadly reactive HA immunogenic polypeptide antigen as described herein (e.g., Y2 (SEQ ID NO: 15) or Y4 (SEQ ID NO: 17), ("H1 COBRA HA"), or wild-type Bris / 07, CA / 09, or Bris / 18 VLP immunogen / vaccine. Mice vaccinated with PBS and wild-type Bris / 07 VLP immunogen had no detectable neutralizing antibody titers against either CA / 09 virus (FIG. 20A) or Bris / 18 virus (FIG. 20B). Mice immunized / vaccinated with H1 COBRA HA VLP immunogen had high neutralizing antibody titers against both CA / 09 and Bris / 18 viruses. COBRA HA Antisera from VLP-vaccinated mice had log2 titers of 11.32 (50% inhibition) against CA / 09 virus and 9.32 (50% inhibition) against Bris / 18 virus.Figures 21A-C show total IgG antibody responses in mice immunized / vaccinated with broadly reactive recombinant influenza HA VLP immunogen (H1 Cobra HA, e.g., Y2 or Y4 rHA VLP immunogen), wild type influenza rHA VLP immunogen, or PBS formulated with ADDAVAX™ adjuvant. IgG antibody titers were determined against (Figure 21A): A / California / 07 / 2009 rHA protein, (Figure 21B): A / Brisbane / 02 / 2018 rHA protein immunogen, or (Figure 21C): cH6 / 1 HA protein immunogen with a globular head from A / California / 07 / 2009 HA and a stalk from subtype H6 influenza virus rHA protein immunogen: chimeric rHA. Mice vaccinated with the different VLPs had significantly higher IgG antibody titers against CA / 09 HA than control mice receiving PBS. (FIG. 21A.) Antisera from CA / 09-rHA vaccinated mice had the highest total IgG titers against the homologously matched CA / 09 HA protein, while antisera from mice vaccinated with H1 Cobra HA protein immunogens (e.g., Y2 or Y4 immunogens) had statistically similar IgG antibody titers compared to CA / 09 vaccinated mice. (FIG. 21A.) Bris / 18 rHA vaccinated mice had lower IgG antibody titers compared to mice vaccinated with CA / 09 rHA immunogen and mice vaccinated with H1 Cobra HA protein immunogen (FIG. 21A), suggesting that the H1 Cobra HA protein antigen on VLPs efficiently elicited IgG titers similar to those generated by the homologously matched CA / 09 HA antigen. Antisera from mice vaccinated with H1 Cobra HA protein immunogen VLPs (Y2 or Y4) had statistically similar total IgG titers against Bris / 18 rHA compared to Bris / 18- and CA / 09-vaccinated mice (Figure 21B).Figures 22A and 22B show viral titers in lung tissues of BALB / c and DBA / 2J mice 3 days after infection with H1N1 A / California / 07 / 2009 or A / Brisbane / 02 / 2018 viruses. BALB / c mice were vaccinated intramuscularly with broadly reactive HA immunogenic polypeptides (H1 Cobra HA VLP vaccines), such as Y2 or Y4 immunogens, or wild-type HA VLP vaccines (e.g., Bris / 18 HA, CA / 09 HA, Bris / 07 HA) (x-axis), and then challenged with H1N1 A / California / 07 / 2009 virus (Figure 22A) 12 weeks after vaccination. Lung samples (n=3 mice / group) were collected 3 days after infection and lung viral titers were determined. Another set of DBA / 2J mice immunized / vaccinated with the same vaccines as above, delivered in rHA format, were challenged with A / Brisbane / 02 / 2018 virus 12 weeks after vaccination (Figure 22B). Lung samples (n=3) were taken 3 days after infection and lung virus titers were measured (Figure 22B). From this study, it was determined that for A / California / 07 / 2009 ("CA / 09") virus challenge, PBS-vaccinated mice had the highest lung virus titers 3 days after infection, and mice vaccinated with Bris / 07 HA VLPs had statistically similar lung virus titers compared to PBS-vaccinated animals. Mice vaccinated with broadly reactive HA immunogenic polypeptide VLP vaccines (e.g., Y2) had the lowest lung virus titers, which were statistically similar to those of CA / 09-vaccinated mice (Figure 22A). With A / Brisbane / 02 / 2018 ("Bris / 18") virus challenge, PBS-vaccinated mice had the highest lung virus titers on day 3 post-infection, mice vaccinated with a broadly reactive HA immunogenic polypeptide VLP vaccine (e.g., Y2) or Bris / 18 had essentially no virus titers in the lungs, while mice vaccinated with CA / 09 had significantly lower lung virus titers than PBS-vaccinated mice.Mice vaccinated with Bris / 07 had similar virus titers as mice vaccinated with PBS (Figure 22B). These results demonstrated that animals immunized / vaccinated with a broadly reactive HA H1N1 immunogenic polypeptide vaccine have reduced viral loads in the lungs after challenge and infection with influenza virus.

[0224] As described in the Examples above, the non-natural broadly reactive H1 and H3 immunogenic polypeptides used as vaccines in the vaccines described herein (e.g., Y2, J4, and NG2) were evaluated in mice previously exposed to influenza A(H1N1) and A(H3N2) viruses, i.e., pre-immunized animals. The impact of pre-immunization on influenza vaccination has not been extensively studied, but such evaluation is important because most humans are infected with influenza viruses before the age of 5 years. Thus, a large proportion of the target vaccine population will already have an immune history against one or both of these virus subtypes prior to vaccination. In the pre-immunization model, the J4 immunogen improved the breadth of HAI-reactive antibodies compared to other vaccine candidates in the influenza naive model. Pre-immunized mice vaccinated with the J4 immunogen also generated serum antibodies with HAI activity against Switz / 13, Kan / 17, Tx / 17, and HK / 19 virus types that were not present in influenza naive J4 HA-vaccinated mice. A similar breadth of expansion occurred in pre-immunized mice vaccinated with Sing / 16, generating serum antibodies in the pre-immunized model with HAI activity against HK / 19 that were not present in influenza-naive mice.

[0225] Without wishing to be bound by theory, seasonal influenza vaccination in the context of pre-immunization is strongly biased by contributions from immune memory compartments, primarily stimulating memory B cells leading to a transient surge in the number of antibody-secreting plasmablasts. Thus, the observed broadening of reactive antibodies (antibody breadth) is likely due to memory B cell responses that recognize shared epitopes between the rHA vaccine antigen and the HA protein of the H3N2 priming strain, since broad HA group-level immune memory arises when lymphocytes target conserved HA epitopes. Furthermore, pre-immune imprinting induces immune memory at both conserved and variable sites on different influenza virus antigens. Thus, shared HA epitopes between vaccine antigens and priming strains may also be present in H3N2 viruses of the historical HAI panel. Upon vaccine-induced immune recall, specific memory B cells are stimulated in pre-immunized animals to produce HAI-reactive antibodies that are not elicited by the vaccine in the absence of influenza pre-immunization.

[0226] Other embodiments From the above description, it will be apparent that variations and modifications may be made to the various aspects and embodiments described herein to adapt them to various uses and conditions, such embodiments still falling within the scope of the following claims.

[0227] The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0228] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

1. A non-naturally occurring and immunogenic influenza virus antigen or immunogenic portion thereof, or a polynucleotide encoding said antigen, wherein said antigen comprises or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of a hemagglutinin (HA) protein antigen of any one of SEQ ID NOs: 1, 15, 2-14, 16 and 17.

2. 2. The influenza virus antigen or polynucleotide of claim 1, wherein the antigen comprises or consists of an amino acid sequence that is at least 98% identical to the amino acid sequence of an HA protein antigen of any one of SEQ ID NOs: 1, 15, 2-14, 16 and 17.

3. 3. The influenza virus antigen or polynucleotide of claim 1 or 2, wherein the antigen comprises or consists of the amino acid sequence of a full-length HA protein antigen set forth in any one of SEQ ID NOs: 1 to 8.

4. 3. The influenza virus antigen or polynucleotide of claim 1 or 2, wherein the antigen comprises or consists of the amino acid sequence of a soluble HA (sHA) protein antigen set forth in any one of SEQ ID NOs: 15, 9-14, 16 and 17.

5. 3. The influenza virus antigen of claim 1, wherein the influenza virus is an H1 or H3 influenza virus.

6. A virus-like particle (VLP) comprising the influenza virus antigen of claim 1 or 2 or a polynucleotide encoding said influenza virus antigen.

7. A non-naturally occurring immunogen capable of generating an immune response against current and future influenza virus strains, said immunogen comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of a hemagglutinin (HA) antigen set forth in any one of SEQ ID NOs: 1, 15, 2-14, 16 and 17.

8. 8. The immunogen of claim 7, comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of a hemagglutinin (HA) antigen set forth in any one of SEQ ID NOs: 1, 15, 2-14, 16 and 17.

9. 3. The viral antigen of claim 1 or 2, which (i) generates an immune response that includes the production of neutralizing antibodies; (ii) generates an immune response that includes the production of antibodies having hemagglutinin-inhibiting activity; and / or (iii) generates an immune response that includes the production of T lymphocytes.

10. An immunogenic composition or vaccine comprising a viral antigen or polynucleotide according to claim 1 or 2, or a virus-like particle comprising a viral antigen or polynucleotide according to claim 1 or 2.

11. 11. The immunogenic composition or vaccine of claim 10, comprising: (i) a pharmaceutically acceptable carrier, diluent, or excipient; or (ii) a pharmaceutically acceptable carrier, diluent, or excipient and an adjuvant.

12. A pharmaceutical composition comprising an effective amount of the immunogenic composition or vaccine of claim 10 for generating an immune response in a subject.

13. A pharmaceutical composition comprising an effective amount of the immunogenic composition or vaccine of claim 11 for generating an immune response in a subject.

14. A pharmaceutical composition comprising an effective amount of the immunogenic composition or vaccine of claim 10 for treating or protecting a subject from disease and / or symptoms thereof caused by influenza virus infection.

15. A pharmaceutical composition comprising an effective amount of the immunogenic composition or vaccine of claim 11 for treating or protecting a subject from disease and / or symptoms thereof caused by influenza virus infection.

16. 15. The pharmaceutical composition of claim 14, wherein the subject is infected with, at risk of infection by, or susceptible to influenza virus.

17. 16. The pharmaceutical composition of claim 15, wherein the subject is infected with, at risk of infection by, or susceptible to influenza virus.

18. 15. The pharmaceutical composition of claim 14, wherein the subject generates an immune response comprising the production of neutralizing antibodies, a cellular immune response, and / or the production of T lymphocytes.

19. 16. The pharmaceutical composition of claim 15, wherein the subject generates an immune response comprising the production of neutralizing antibodies, a cellular immune response, and / or the production of T lymphocytes.

20. The polynucleotide of claim 1 or 2, wherein the polynucleotide is RNA or DNA.

21. A pharmaceutical composition comprising the polynucleotide of claim 1 or 2, or a virus-like particle (VLP) containing said polynucleotide, and a pharmaceutically acceptable carrier, diluent, or excipient.

22. A monovalent immunogen comprising the non-natural and immunogenic influenza virus antigen of claim 1 or 2.

23. the immunogenic influenza virus antigen Y2 antigen comprising the sequence set forth in SEQ ID NO: 15, J1 antigen comprising the sequence set forth in SEQ ID NO: 3, 7, or 9, J2 antigen comprising the sequence set forth in SEQ ID NO: 4, J3 antigen comprising the sequence set forth in SEQ ID NO: 5, J4 antigen comprising the sequence set forth in SEQ ID NO: 6 or 8, NG1 antigen comprising the sequence set forth in SEQ ID NO: 11, NG2 antigen comprising the sequence set forth in SEQ ID NO: 2 or 12, or NG3 antigen comprising the sequence set forth in SEQ ID NO: 13 23. The monovalent immunogen of claim 22, comprising:

24. A multivalent immunogen comprising at least two of the non-natural and immunogenic influenza virus antigens of claim 1 or 2.

25. 25. The multivalent immunogen of claim 24, wherein the immunogen is bivalent and comprises a combination of a Y2 antigen comprising the sequence set forth in SEQ ID NO: 15 and a J4 antigen comprising the sequence set forth in SEQ ID NO: 6 or 8, or a combination of a Y2 antigen comprising the sequence set forth in SEQ ID NO: 15 and a NG2 antigen comprising the sequence set forth in SEQ ID NO: 2 or 12.

26. 25. The multivalent immunogen of claim 24, wherein the immunogen comprises eight of the non-natural and immunogenic influenza virus antigens.

27. 25. A virus or virus-like particle (VLP) comprising one or more polynucleotides encoding the immunogenic influenza virus antigen of claim 24.

28. 25. A composition comprising the multivalent immunogen of claim 24.

29. 28. A composition comprising the viral particle or VLP of claim 27.

30. 30. The composition of claim 28, further comprising a pharmaceutically acceptable carrier, excipient, or vehicle.

31. 30. The composition of claim 29, further comprising a pharmaceutically acceptable carrier, excipient, or vehicle.

32. A pharmaceutical composition comprising an effective amount of the pharmaceutically acceptable composition of claim 30 for generating an immune response in a subject and / or treating or protecting a subject from disease and / or symptoms thereof caused by influenza virus infection.

33. 33. The pharmaceutical composition of claim 32, wherein the influenza virus is an H1 and / or H3 influenza virus.

34. 33. The pharmaceutical composition of claim 32, wherein the subject generates an immune response comprising the production of neutralizing antibodies, a cellular immune response, and / or the production of T lymphocytes.

35. A pharmaceutical composition comprising an effective amount of the pharmaceutically acceptable composition of claim 31 for generating an immune response in a subject and / or treating or protecting a subject from disease and / or symptoms thereof caused by influenza virus infection.

36. 36. The pharmaceutical composition of claim 35, wherein the influenza virus is an H1 and / or H3 influenza virus.

37. 36. The pharmaceutical composition of claim 35, wherein the subject generates an immune response comprising the production of neutralizing antibodies, a cellular immune response, and / or the production of T lymphocytes.

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