Broadly reactive immunogens, compositions and methods of use thereof for influenza A (H3N2) virus

Abstract

The present disclosure provides non-naturally occurring, broadly reactive, pan-epitope antigens derived from H3 influenza viruses, which are immunogenic and capable of eliciting a broadly reactive immune response against H3 viruses, such as a broadly reactive neutralizing antibody response, in a subject upon introduction. The present disclosure also provides non-naturally occurring, broadly reactive immunogens, vaccines, virus-like particles (VLPs), and compositions comprising the immunogens and vaccines. The present disclosure provides methods of generating an immune response in a subject by administering an immunogen, vaccine, VLP, or a composition thereof. In particular, the immunogen comprises the hemagglutinin (HA) protein of an H3 influenza virus strain.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the priority and benefit of U.S. Provisional Application No. 62 / 697,846, filed on July 13, 2018, the entire contents of which are hereby incorporated by reference in their entirety. Background of the Invention

[0003] In 2017, the Centers for Disease Control and Prevention in the United States estimated that the effectiveness of seasonal influenza vaccines was only 42%. This limited efficacy was due to mutations in the influenza A (H3N2) vaccine strain, resulting in influenza in infected individuals. In addition, the number of influenza cases caused by influenza B virus increased between 2017 and 2018. Given that a severe influenza season can kill approximately 50,000 people in the United States alone, there is an urgent need for improved immunogens and vaccines to provide broad antiviral protection, especially against currently and future circulating influenza A H3 or H3N2 viruses. Summary of the Invention

[0004] As described below, non - naturally occurring, broadly reactive antigens and antigen sequences derived from H3 influenza viruses (also referred to herein as "H3 influenza", "H3 influenza virus", or "H3 virus"), such as the H3N2 subtype, are provided. Such H3 virus antigens are typically structural proteins or peptides and include, for example, the hemagglutinin (HA) protein, or the HA1 (head) or HA2 (tail or stem) portions of the HA protein, and are effective immunogens that can elicit a broadly reactive immune response against the H3 HA protein and ultimately, against current and future H3 virus strains in a subject. As referred to herein, an H3 virus antigen or antigen sequence that elicits an immune response in a subject is an immunogenic antigen or immunogen. Such H3 immunogens are referred to as broadly reactive and pan - epitope antigens because they can directly elicit broadly reactive antibodies against different subtypes or H3 virus strains with sequence similarity and variability, as well as the diversity of epitopes in their antigens and sequences, particularly the HA antigen.

[0005] On the one hand, the non-naturally occurring H3 virus antigen amino acid sequences and antigens (such as structural antigens) comprising the sequences described herein comprise broadly reactive epitopes that reflect the sequence similarities and variabilities of past, present, and future H3 antigens. Such antigen sequences and antigens comprising the sequences are thus referred to as "non-naturally occurring, broadly reactive, pan-epitope" antigens. The antigens are immunogenic and, when introduced or administered to a subject, elicit broadly reactive antibodies against the H3 virus in the subject, such as neutralizing antibodies, particularly against H3 antigens, such as HA or an antibody-binding portion thereof. In one embodiment, such an H3 antigen sequence is an amino acid sequence. In one embodiment, the H3 antigen sequence is a polynucleotide sequence, such as: a polynucleotide sequence encoding the amino acid sequence of the antigen described herein. For ease of reference, the "non-naturally occurring, broadly reactive, pan-epitope" antigens of the H3 virus described herein are referred to as "broadly reactive antigens".

[0006] The broadly reactive H3 antigens described herein are immunogens because they elicit a broadly reactive immune response in a subject. The immune response is in the form of a neutralizing antibody response, for example: neutralizing antibodies that are specific for the HA antigen of the H3 virus and that neutralize the activity of the HA protein. Accordingly, immunogens and immunogenic compositions comprising the broadly reactive H3 antigens described herein are also provided, immunogenic compositions that induce an immune response against the H3 virus (such as against the HA protein of the H3 virus) in a subject, such as a vaccine (such as a polypeptide or polynucleotide product). For ease of reference, the "non-naturally occurring, broadly reactive, pan-epitope" H3 virus immunogens described herein will be referred to as "broadly reactive immunogens".

[0007] Methods are also provided for inducing an immune response against H3 influenza infection, disease, and / or its symptoms in a subject using the immunogens described herein. In a particular embodiment, the H3 virus antigen is the HA, HA1, or HA2 protein of an H3 influenza virus, or the H3N2 subtype of an influenza virus, or a related virus type, or an antibody-binding portion thereof. Methods for inducing an immune response in a subject using an immunogen are also provided.

[0008] On the one hand, the H3 HA immunogenic antigen has an amino acid sequence that is at least or equal to 85%, at least or equal to 90%, at least or equal to 91%, at least or equal to 92%, at least or equal to 93%, at least or equal to 94%, at least or equal to 95%, at least or equal to 96%, at least or equal to 97%, at least or equal to 98%, or at least or equal to 99% identical to the H3 HA polypeptide (or HA1 or HA2 polypeptide) sequence of one or more of the HA proteins listed in Figure 1 A and 1B (referred to herein as "TJ1 to 9HA").

[0009] In one aspect, a broadly reactive H3 antigen sequence capable of generating an immune response against current and future H3 influenza virus strains can be generated by, for example, the method described in the co-pending provisional patent application No. 62 / 697,803 filed on July 13, 2018, the content of which is incorporated herein by reference, and taking into account the parameters of the H3 antigen sequence, such as the HA antigen sequence, over a time span or range (e.g., a linear time range), such as one or more influenza seasons, and the geographical location(s) where the H3 virus is isolated, such as, for example, the Southern Hemisphere or the Northern Hemisphere.

[0010] In one aspect, there is provided a non-naturally occurring, broadly reactive, pan-epitope antigen of an H3 influenza virus (H3 virus) capable of generating an immune response against current and future H3 virus strains. Wherein the H3 virus antigen comprises an amino acid sequence that is at least 95% identical to the amino acid sequences of the HA antigens (TJ-1 to TJ-9) listed in A to 1C. Figure 1 Amino acid sequences that are at least 95% identical to the amino acid sequences of the HA antigens (TJ-1 to TJ-9) listed in A to 1C.

[0011] In another aspect, there is provided a non-naturally occurring, broadly reactive, pan-epitope antigen of an H3 influenza virus (H3 virus) capable of generating an immune response against current and future H3 virus strains. In one embodiment, the antigen is hemagglutinin (HA), HA1, or HA2, or an antibody-binding protein portion thereof. In one embodiment, the H3 virus antigen comprises an amino acid sequence that is at least 95% identical or at least 98% identical to the amino acid sequences of the HA antigens listed in A to 1C. In a particular embodiment, the H3 virus antigen comprises Figure 1 Amino acid sequences that are at least 95% identical or at least 98% identical to the amino acid sequences of the HA antigens listed in A to 1C. In a particular embodiment, the H3 virus antigen comprises Figure 1 Amino acid sequences of the HA antigens listed in A to 1C.

[0012] It will be understood that there is provided a non-naturally occurring, broadly reactive, pan-epitope immunogen, which for simplicity may be interchangeably referred to herein as a "non-naturally occurring immunogen", a "broadly reactive immunogen", or a "pan-epitope immunogen".

[0013] In another aspect, there is provided a virus-like particle (VLP) comprising an H3 virus immunogenic antigen according to the foregoing aspects. In one embodiment, the VLP comprises a polynucleotide encoding an H3 virus antigen.

[0014] In another aspect, there is provided a non-naturally occurring, pan-epitope immunogen capable of generating an immune response against current and future H3 influenza (H3) virus strains; wherein the immunogen comprises an amino acid sequence that is at least 95% identical to the amino acid sequences of the HA antigens (TJ-1 to TJ-9) shown in A to 1C. Figure 1 Amino acid sequences that are at least 95% identical to the amino acid sequences of the HA antigens (TJ-1 to TJ-9) shown in A to 1C.

[0015] In another aspect, there is provided an immunogen that is non-naturally occurring, broadly reactive, and pan-epitopic, capable of eliciting an immune response against current and future H3 influenza virus strains. In one embodiment, the H3 virus antigen, immunogen, or VLP elicits the production of neutralizing antibodies. In one embodiment, the antibody has hemagglutination inhibition activity. In one embodiment, the H3 virus antigen, immunogen, or VLP elicits the production of T lymphocytes.

[0016] In another aspect, there is provided a pharmaceutically acceptable composition comprising the H3 virus antigen, immunogen, or VLP of any of the foregoing aspects and embodiments, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the composition also comprises an adjuvant.

[0017] In another aspect, there is provided a vaccine or immunogenic composition comprising the H3 virus antigen, immunogen, or VLP of any of the foregoing aspects and embodiments.

[0018] In another aspect, there is provided a method of generating an immune response in a subject, the method comprising administering to the subject an effective amount of the immunogen, pharmaceutical composition, vaccine, or VLP of any of the foregoing aspects and embodiments. In one embodiment, the elicited immune response comprises the production of neutralizing antibodies and / or T lymphocytes.

[0019] In one aspect, the broadly reactive H3 immunogen is isolated and / or purified. In another aspect, the broadly reactive H3 immunogen is formulated for administration to a subject in need thereof. In another aspect, the broadly reactive H3 immunogen or a composition comprising the immunogen is administered to a subject in need thereof in an effective amount to elicit an immune response in the subject. In one embodiment, the immune response elicits neutralizing antibodies. In one embodiment, the immune response is prophylactic or therapeutic.

[0020] In another aspect, there is provided a vaccine or immunogenic composition comprising a broadly reactive H3 immunogen.

[0021] In another aspect, there are provided virus-like particles (VPLs) comprising a broadly reactive H3 immunogen or its sequence. In one embodiment, the sequence is an amino acid sequence. In one embodiment, the sequence is a polynucleotide sequence encoding an amino acid sequence.

[0022] In another aspect, there is provided a method of generating an immune response in a subject, the method comprising administering to the subject an effective amount of the H3 broadly reactive immunogen, vaccine, VLP, or composition of any of the foregoing aspects and embodiments. In one embodiment of the method, an adjuvant is administered to the subject simultaneously.

[0023] Definitions

[0024] 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 this invention belongs or is related. The following references provide one of ordinary skill in the art with general definitions of many of the terms used in this invention: 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, Gene 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 HarperCollins Dictionary of Biology (1991). As used herein, unless otherwise indicated, the following terms have the meanings given to them below.

[0025] "Adjuvant" refers to a substance or vehicle that nonspecifically enhances the immune response against an antigen. Adjuvants can include suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphate) on which the antigen is adsorbed; or water-in-oil emulsions in which the antigen solution is emulsified in mineral oil (e.g., Freund's incomplete adjuvant), sometimes containing killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity.

[0026] 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 biomolecules, such as co-stimulatory molecules. Exemplary biological adjuvants include, but are not limited to, interleukin-1 (IL-2), the protein memory T cell attractant "regulated upon activation, normal T cell expressed and secreted" (RANTES), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), lymphocyte function-associated antigen 3 (LFA-3, also known as CD58), cluster of differentiation 72 (CD72), (a negative regulator of B cell responsiveness), the peripheral membrane protein B7-1 (B7-1, also known as CD80), the peripheral membrane protein B7-2 (B7-2, also known as CD86), the TNF ligand superfamily member 4 ligand (OX40L) or the type II transmembrane glycoprotein receptor belonging to the TNF superfamily (4-1BBL).

[0027] "Administer" means to give, supply, dispense a composition, reagent, therapeutic agent, etc. to a subject, or to apply or bring into contact a composition, etc. with a subject. The administration or application can be accomplished by any of a variety of routes, e.g., but not limited to, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous (IV), (injection), intrathecal, intramuscular, dermal, intradermal, intracranial, inhalation, rectal, intravaginal, or intraocular.

[0028] "Reagent" means any small molecule compound, antibody, nucleic acid molecule, peptide, polypeptide, or fragment thereof.

[0029] "Alter" means a change (increase or decrease) in the expression level or activity of a gene or polypeptide detected by methods known in the art as described herein. As used herein, an alter includes a 5% change in the expression level, a 10% change in the expression level, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in the expression level.

[0030] "Improve" means to reduce, decrease, alleviate, inhibit, attenuate, prevent, or stabilize the development or progression of a disease or pathological condition.

[0031] "Analog" refers to molecules that are different but have similar functional or structural characteristics. For example: a polypeptide analog retains the biological activity of the corresponding naturally occurring polypeptide while having certain biochemical modifications that, relative to the naturally occurring polypeptide, can enhance the function of the analog. Such biochemical modifications can increase the protease resistance, membrane permeability, or half-life of the analog without altering, for example, ligand binding. An analog may contain non-natural amino acids.

[0032] "Antibody" refers to an immunoglobulin (Ig) molecule produced by B-lymphoid cells and having a specific amino acid sequence. Antibodies are induced or elicited in a subject (human or other animal or mammal) upon exposure to a specific antigen (immunogen). A subject capable of producing antibodies / immunoglobulins against a specific antigen / immunogen (i.e., an immune response) is said to be immunocompetent. Antibodies are characterized by specifically reacting (e.g., binding) with an antigen or immunogen in a demonstrable manner, with the antibody and antigen / immunogen each defined in terms of the other.

[0033] "Elicit an antibody response" refers to the ability of an antigen, immunogen, or other molecule to induce the production of antibodies. Antibodies have different classes, such as IgM, IgG, IgA, IgE, IgD, and subclasses or isotypes, such as IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4. The antibody / immunoglobulin response elicited in a subject can neutralize a pathogen (e.g., infectious or pathogenic) by binding to an epitope (antigenic determinant) on the pathogen and blocking or inhibiting its activity, and / or by forming a binding complex with a reagent that is cleared from the subject's system (e.g., by the liver).

[0034] As used herein, "broadly reactive" refers to an immune response elicited in a subject against a viral protein (e.g., viral antigen, antigenic sequence, protein, or protein sequence) that is sufficient to block, inhibit, impede, neutralize, or prevent infection by a variety of related influenza viruses (such as most or all influenza viruses within a particular subtype, e.g., viruses related to the H3 influenza virus).

[0035] "Antigen" refers to a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including a composition injected or absorbed into an animal. An antigen reacts with a specific humoral or cellular immune product, including a product induced by a heterologous immunogen. In some embodiments of the disclosed compositions and methods, the antigen is an influenza hemagglutinin (HA) protein. In many cases, an antigen that elicits or stimulates an immune response in a subject is referred to as an "immunogen".

[0036] The term "antigenic drift" refers to the mechanism of biological or microbial variation, such as in a virus, which involves the accumulation of mutations within the gene encoding the antibody binding site (also referred to as the antigenic determinant or epitope). This process gives rise to new strains of the virus / viral particles that cannot be effectively inhibited or blocked by the antibodies initially generated against the viral strain antigen prior to the mutation, thereby making the virus more transmissible in a partially immune population. For example, antigenic drift has occurred in both influenza A and influenza B viruses.

[0037] In the context of a live virus, the term "attenuated" means that the virus is attenuated in a subject if its ability to infect cells or a subject and / or its ability to cause disease is reduced (e.g., decreased, eliminated, or deleted) compared to the wild-type virus's ability to cause disease. Typically, an attenuated virus retains at least some ability to elicit an immune response after administration to an immunocompetent subject. In some cases, the attenuated virus is capable of eliciting a protective immune response without eliciting any 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 or equal to 5%, or at least about or equal to 10%, or at least about or equal to 25%, at least about or equal to 50%, at least about or equal to 75%, or at least about or equal to 80%, or at least about or equal to 85%, or at least about or equal to 90%, or at least about or equal to 95% or higher, compared to the ability of the wild-type virus to cause disease or pathology in the subject.

[0038] The term "clade" refers to different classifications (commonly referred to as subtypes) of known influenza viruses (e.g., influenza A H3N2 virus). Viruses within the H3N2 clade are genetically related but do not share an exact viral genome.

[0039] As will be understood by those skilled in the art, many clades and sub-clades of H3N2 virus subtypes have been designated in the art. For example, one clade is 3C.2a; the sub-clades of this clade include 3C.2a.1, 3C.2a.2, 3C.2a.3, and 3C.2a.4. Additionally, at least 10 different H5N1 virus sub-clades have been designated herein as: 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., New England Journal of Medicine 358:261 - 273, 2008). Clade 2 is further divided into sub-clades (including clade 2.1, clade 2.2, clade 2.3, clade 2.4, and clade 2.5).

[0040] A "codon-optimized" nucleic acid (polynucleotide) refers to a nucleic acid sequence that has been altered such that the codons are optimal for expression in a particular system (e.g., a particular species within a 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.

[0041] In the present disclosure, terms such as "comprises," "comprising," "containing," and "having" can have the meanings given to them under U.S. patent law and can mean "includes," "including," etc.; "consisting essentially of" likewise has the meaning given in U.S. patent law, and the term is open-ended, allowing for more features than those recited as long as the basic or novel features recited are not changed by the presence of the recited features, excluding embodiments of the prior art.

[0042] "Detect" means to identify the presence, absence, or amount of an analyte, compound, reagent, or substance to be detected. A "detectable label" is a composition that, when linked to a molecule of interest, renders it detectable by, for example, 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 density reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxin, or haptens.

[0043] "Disease" means any condition, disorder, or pathology that impairs or interferes with the normal function of cells, tissues, or organs. Examples of diseases include diseases caused by H3 virus infection and symptoms and adverse reactions caused by H3 virus infection of the human body. The influenza virus causes influenza and its symptoms in infected individuals.

[0044] "Effective amount" means the amount of an active therapeutic agent, composition, compound, biological agent (e.g., a vaccine or therapeutic peptide, polypeptide, or polynucleotide) required to improve, alleviate, enhance, abolish, reduce, or eliminate the symptoms and effects of a disease, relative to an untreated patient.

[0045] The effective amount of an immunogen or a composition comprising an immunogen for use in a method of treating a disease, disorder, or pathology caused by an H3 virus depends on the mode of administration, age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosing regimen. The amount is referred to as an "effective" amount.

[0046] "Therapeutically effective amount" means the amount of a specified agent sufficient to achieve the desired effect in a subject being treated with the agent. For example: an amount of an H3 influenza virus immunogen or vaccine can be used to elicit an immune response in a subject and / or to prevent infection by the H3 influenza virus. Ideally, in the context of the present application, the amount of a therapeutically effective amount of an influenza vaccine or an anti-influenza immunogenic composition is sufficient to increase resistance to prevent, ameliorate, reduce, and / or treat infection in a subject caused by an influenza virus without causing substantial cytotoxic effects in the subject. An effective amount of an influenza vaccine of an immunogenic composition can be used to increase resistance to prevent, ameliorate, reduce, and / or treat infection in a subject depending on, for example, the subject being treated, the mode of administration of the therapeutic composition, and other factors, as described above.

[0047] "Fragment" means a portion of a polypeptide or nucleic acid molecule. The portion comprises, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. Fragments can comprise 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 can be a peptide. In the case of an antibody or immunoglobulin fragment, the fragment typically binds to a target antigen.

[0048] "Fusion protein" means a protein produced by expressing a nucleic acid (polynucleotide) sequence engineered from a nucleic acid sequence encoding at least a portion of two different (heterologous) proteins or peptides. To produce 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 comprises an H3 influenza HA protein fused to a heterologous protein.

[0049] "Gene vaccine" means an immunogenic composition comprising a polynucleotide encoding an antigen.

[0050] The term "geographical location or region" means a preselected division of the geographical regions of the Earth, such as by continent or other preselected territory or subdivision (e.g., the Middle East spanning more than one continent). Examples of different geographical regions include countries (e.g., Turkey, Egypt, Iraq, Azerbaijan, China, the United States); continents (e.g., Asia, Europe, North America, South America, Oceania, Africa); recognized geopolitical subdivisions (e.g., the Middle East); or hemispheres of the world (e.g., the Northern Hemisphere, the Southern Hemisphere, the Eastern Hemisphere, or the Western Hemisphere).

[0051] "H3 virus polypeptide" means a polypeptide that binds to Figure 1An amino acid sequence that is at least 85% identical to the amino acid sequence of the HA antigen or a fragment thereof shown in A to 1C is capable of inducing an immune response in an immunized subject. In one embodiment, the H3 virus polypeptide comprises or consists of the TJ1 to 9 HA sequence or a fragment thereof.

[0052] "H3 virus polynucleotide" refers to a nucleic acid molecule encoding an H3 virus polypeptide (antigen or antigenic protein).

[0053] The term "hemagglutinin (HA)" refers to a surface glycoprotein expressed by influenza viruses. HA mediates the binding of virus particles to the cells of a subject and subsequent entry of the virus into the cells of the subject. The nucleotide and amino acid sequences of many influenza HA proteins are known in the art and are publicly available, for example, those deposited in GenBank (see, for example: US Publication No. US2015 / 0030628, Table 1). HA (along with neuraminidase (NA)) is one of the two major influenza virus antigenic proteins having antigenic determinants (epitopes) that are recognized and bound by antibodies / immunoglobulins.

[0054] For example, the hemagglutinin (HA) protein of the H3N2 influenza virus has at least about or equal to 85%, or at least about or equal to 90%, 95%, 98%, 99% or higher amino acid sequence identity to the amino acid sequence of fragment 4 of influenza A virus (A / Hong Kong / 1-4 / 1968 (H3N2)), a polypeptide or a fragment thereof, complete sequence, accession number CY033017, as described below:

[0055] MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPITCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGFIMWACQRGNIRCNICI.

[0056] In addition, the hemagglutinin (HA) protein of the H3N2 influenza virus is encoded by a polynucleotide or a fragment thereof having at least about or equal to 85%, or at least about or equal to 90%, 95%, 98%, 99%, or higher sequence identity with the polynucleotide sequence, as follows:

[0057]

[0058]

[0059] "Hybridization" refers to hydrogen bonding, which can be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding between complementary nucleobases. For example: in DNA, adenine and thymine and cytosine and guanine are complementary nucleobases that pair by forming hydrogen bonds, respectively.

[0060] "Immunogen" refers to a compound, composition, or substance that is capable of initiating or stimulating an immune response under appropriate conditions, such as: generating antibodies and / or T cell responses, in an animal, including a composition that is injected or absorbed into the animal. As used herein, an "immunogenic composition" is a composition that contains an immunogen (e.g., an H3 HA polypeptide or a vaccine containing an H3 HA polypeptide). As will be understood by those skilled in the art, if a medicament is administered to a subject in need thereof before the subject contracts a disease or experiences a full-blown illness, the immunogenic composition can prevent and cause the subject to initiate an immune response, such as: neutralizing antibodies and / or a cellular immune response, to prevent the disease or prevent a more severe disease condition and / or its symptoms. If a medicament is administered to a subject in need thereof after the subject contracts a disease, the immunogenic composition can have a therapeutic effect and cause the subject to initiate an immune response, such as: neutralizing antibodies and / or a cellular immune response, to treat the disease, such as: by reducing, diminishing, abrogating, ameliorating, or eliminating the disease, and / or its symptoms. In one embodiment, the immune response is a B cell response, which results in the production of antibodies, such as: neutralizing antibodies, directly against the immunogen or immunogenic composition containing the antigen or antigen sequence. In a manner similar to the foregoing, in some embodiments, the immunogenic composition or vaccine can be prophylactic. In some embodiments, the immunogenic composition or vaccine can be therapeutic. In one embodiment, the disease is influenza (flu).

[0061] The term "immune response" refers to any response mediated by immune-responsive cells. In one example of an immune response, leukocytes are recruited to perform a variety of different specific functions in response to exposure to an antigen (e.g., a foreign entity). The immune response is a multifactorial process and varies depending on the cell types involved. The immune response includes cell-mediated responses (e.g., T cell responses), humoral responses (B cell / antibody responses), innate responses, and combinations thereof.

[0062] An "immunogenic composition" refers to a composition that contains an antigen, antigen sequence, or immunogen, wherein the composition elicits an immune response in the immunized subject.

[0063] The term "immunize" or "immunization" means to protect a subject from a disease, infectious disease, or pathology, or its symptoms, caused by an H3 virus (e.g., by vaccination).

[0064] The term "influenza virus" refers to a segmented, negative-strand RNA virus belonging to the family of Orthomyxoviridae viruses. Influenza viruses are divided into three types: A, B, and C. Influenza A viruses infect a variety of birds and mammals, including humans, horses, marine mammals, pigs, ferrets, and chickens. In animals, most influenza A viruses cause mild, local infections of the respiratory and intestinal tracts. However, highly pathogenic influenza A strains, such as H3N2, can cause systemic infections in poultry, with a mortality rate that can reach 100%.

[0065] "Inhibitory nucleic acid" refers to double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or an analog thereof, that when administered to mammalian cells results in a decrease in the expression of a target gene (e.g., 5%, 10%, 25%, 50%, 75%, or even 90 to 100%). Generally, 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 described herein.

[0066] The terms "isolated," "purified," or "biologically pure" refer to materials that are released from the components that are normally associated with them in their natural state to varying degrees. "Isolate" denotes the degree of separation from the original source or environment. "Purify" denotes a higher degree of separation than isolation. A "purified" or "biologically pure" protein is sufficiently free of other substances so that any impurities do not significantly affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid, protein, or peptide can be purified if it is produced by recombinant DNA techniques and is substantially free of cellular material, debris, unrelated viral material, or culture medium, or if it is produced by chemical synthesis methods and is substantially free of chemical precursors or other chemicals. Purity and homogeneity are typically determined using standard purification methods and analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that a nucleic acid or protein produces a substantially single, sharp band in an electrophoretic gel. For proteins that can be modified, such as phosphorylation or glycosylation, different modifications can result in different isolated proteins, which can be purified separately. The term "isolated" also encompasses recombinant nucleic acids, proteins, or viruses, as well as chemically synthesized nucleic acids or peptides.

[0067] "Isolated polynucleotide" refers to a nucleic acid (e.g., a DNA molecule) that does not contain a gene, where the gene flanks the gene in the naturally occurring genome of the organism from which the nucleic acid molecule of the present invention is derived. Thus, the term includes, for example: recombinant DNA incorporated into a vector; a plasmid or virus that enters autonomous replication; or genomic DNA of a prokaryote or eukaryote; or exists as an independent molecule separate from other sequences (e.g., cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion). Additionally, the term includes RNA molecules transcribed from a DNA molecule, and recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0068] "Isolated polypeptide" refers to a polypeptide of the present invention that has been separated from its naturally associated components. A polypeptide is generally considered isolated when it is at least 40% by weight, at least 50% by weight, at least 60% by weight, free of the naturally associated proteins and natural organic molecules. Preferably, the isolated polypeptide preparation is at least 75% by weight, more preferably at least 90% by weight, most preferably at least 99% by weight, free of the naturally associated proteins and natural organic molecules. Isolated polypeptides can be obtained, for example, by extraction from natural sources; by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemical synthesis of the protein. Purity can be measured by any suitable standard method, such as column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. Isolated polypeptides can refer to a broad range of active viral immunogenic polypeptides produced by the methods described herein.

[0069] "Linker" refers to one or more amino acids that act as a spacer between two polypeptides or peptides of a fusion protein.

[0070] "Marker" refers to any protein or polynucleotide whose expression level or activity is altered in association with a disease, condition, pathology, or disorder.

[0071] "Matrix (M1) protein" refers to an influenza virus structural protein found within the viral envelope. M1 is thought to play a role in viral assembly and budding after cell infection.

[0072] The term "neuraminidase (NA)" refers to an influenza virus membrane glycoprotein. NA is involved in the destruction of the cell receptor for viral HA by cleaving terminal sialic acid residues from carbohydrate moieties on the surface of infected cells. NA can also cleave sialic acid residues from viral proteins, thereby preventing viral aggregation. NA (along with HA) is one of the two major influenza virus antigenic determinants.

[0073] As used herein, "obtain" in "obtain a reagent" includes synthesizing, isolating, purchasing, or otherwise obtaining the reagent.

[0074] The term "operably linked" refers to nucleic acid sequences as used herein. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first and second nucleic acid sequences are in a functional relationship. For example, a promoter is operably linked to a coding sequence if the promoter affects (permits) transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where two protein coding regions are to be ligated, in the same reading frame.

[0075] A "computationally optimized" influenza HA protein typically reflects an HA protein sequence derived by comparing the sequences (amino acid sequences) of two or more viruses, such as sequences of the H3 influenza virus clade, as described, for example, in U.S. Patent Application Publication No. US2015 / 0030628. The nucleotide sequences encoding the H3 HA protein are produced by the described method and can be expressed in mammalian cells by codon optimization and RNA optimization (e.g., increasing RNA stability) using processes and techniques practiced in the art.

[0076] Broadly reactive, pan-epitope immunogens, such as the H3 influenza hemagglutinin (HA) protein, are used to elicit an immune response in a subject and have a set of highly immunogenic epitopes (also called antigenic determinants). The H3 virus HA proteins described herein are "pan-epitope" H3 immunogens suitable for use as vaccines that elicit a broad reactive immune response, e.g., a neutralizing antibody response, that can protect against multiple H3 viruses expressing the HA protein on their surface when introduced into a subject, particularly a human subject infected with an H3 virus. The immunogenic antigen (or vaccine) is useful for providing an anti-H3 virus immunogen (or vaccine) that elicits a broad reactive immune response against the H3 influenza virus HA antigen with antigenic variability and similarity and for treating or preventing infections and diseases caused by more than one H3 influenza virus subtype.

[0077] An "open reading frame (ORF)" refers to a series of nucleotide triplets (codons) that encode amino acids without any stop codons. Such sequences are typically translatable into a peptide or polypeptide.

[0078] As used herein, an influenza virus "outbreak" refers to a collection of virus isolates from a geographic location (e.g., within a single country) over a given time period (e.g., one year).

[0079] The term "pharmaceutically acceptable vehicle" refers to conventional carriers (vehicles) and excipients that are physiologically and pharmaceutically acceptable for use, particularly in mammalian, e.g., human, subjects. Such pharmaceutically acceptable vehicles are known to those skilled in the art and can be readily found in Remington's Pharmaceutical Sciences, E.W. Martin, Mack Publishing Co., Easton, Pennsylvania, 15th Edition (1975) and its updates, which describe vehicles suitable for the delivery of one or more therapeutic compositions (e.g., one or more influenza vaccines) and other pharmaceutical compositions and formulations. Generally, the nature of the pharmaceutically acceptable carrier depends on the particular mode of administration employed. For example, parenteral formulations typically comprise injectable fluids / liquids that contain pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose solutions, glycerol, etc. as vehicles. For solid compositions, e.g., in the form of powders, pills, tablets, or capsules, conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate, which generally stabilize and / or increase the half-life of the composition or drug. In addition to biocompatible carriers, the pharmaceutical compositions to be administered may contain small amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents, e.g., sodium acetate or sorbitan monolaurate.

[0080] A "plasmid" refers to a circular nucleic acid molecule capable of autonomous replication in the cells of a subject.

[0081] By "polypeptide" (or protein) is meant a polymer in which the monomers comprise amino acid residues joined together by amide bonds. When the amino acids are α-amino acids, either the L- or D-optical isomers can be used. The terms "polypeptide" or "protein" as used herein are intended to encompass any amino acid sequence and sequences that include modifications, such as glycoproteins. The term "polypeptide" is particularly used to encompass naturally occurring proteins as well as those produced recombinantly or synthetically. The terms "residue" or "amino acid residue" also refer to an amino acid incorporated into a protein, polypeptide, or peptide.

[0082] Conservative amino acid substitutions refer to those substitutions that have the least effect on the properties of the original protein when made, that is, the structure of the protein, especially the functionally conserved parts, is not significantly altered by such substitutions. Examples of conservative amino acid substitutions are known in the art, for example, as described in U.S. Publication No. 2015 / 0030628. Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the substituted region, such as in a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; and / or (c) most of the side chains.

[0083] Substitutions that are generally expected to produce the greatest changes in protein properties are non-conservative, for example, the following changes: (a) substitution of (or by) a hydrophobic residue such as leucyl, isoleucyl, phenylalanyl, valyl or alanyl with a hydrophilic residue such as seryl or threonyl; (b) substitution of (or by) cysteine or proline with other residues; (c) substitution of (or by) a residue with a positively charged side chain, such as lysyl, arginyl or histidyl, with a negatively charged residue such as glutamyl or aspartyl; (d) substitution of (or by) a residue with a bulky side chain such as phenylalanine with a residue without a side chain such as glycine.

[0084] An "oligonucleotide primer set" refers to a set of oligonucleotides that can be used, for example, in PCR. The primer set should contain 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.

[0085] A "promoter" refers to an array of nucleic acid control sequences that directly transcribe nucleic acids. The promoter contains the essential nucleic acid sequences near the transcription start site. The promoter also optionally contains distal enhancer or repressor sequence elements. A "constitutive promoter" is a promoter that is continuously active and not regulated by external signals or molecules. In contrast, the activity of an "inducible promoter" is regulated by external signals or molecules (e.g., transcription factors). For example, the promoter can be the CMV promoter.

[0086] As will be understood by those skilled in the art, the term "purified" does not require absolute purity. Instead, it is intended as a relative term. Thus, for example: a purified peptide, protein, virus, or other active compound is a peptide that has been separated in whole or in part from the proteins and other contaminants to which it is naturally bound. In certain embodiments, the term "substantially purified" means that it has been separated from cells, cell culture media, or other crude preparations and subjected to conventional methods such as chemical separation, chromatography, or electrophoresis to remove the various components of the initial preparation, such as proteins, cell debris, and other components.

[0087] "Recombinant" nucleic acids, proteins or viruses are sequences that are widely active or have sequences made by the artificial combination of two originally separated sequence fragments. Such artificial combination is usually achieved by chemical synthesis or by artificially manipulating isolated nucleic acid fragments, for example, through genetic engineering techniques. "Non-naturally occurring" nucleic acids, proteins or viruses can be prepared by recombinant techniques, artificial manipulation, or genetic or molecular biology engineering processes and techniques, such as those commonly practiced in the art.

[0088] "Reduce" means a negative change of at least 5%, 10%, 25%, 30%, 40%, 50%, 75%, 80%, 85%, 90%, 95%, 98% or 100%.

[0089] "Reference" means a standard or control condition.

[0090] "Reference sequence" is a defined sequence used as a basis for sequence comparison. The reference sequence can be a subset or all of the designated sequence. For example: a fragment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence is usually 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 the reference nucleic acid sequence is usually 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 or the interval between the two.

[0091] "Specifically bind" means a compound or antibody that recognizes and binds to a polypeptide, such as a viral polypeptide, peptide or vaccine product, but substantially does not recognize and bind to a sample, such as a biological sample, which naturally includes polypeptides, such as viral polypeptides or peptides.

[0092] Nucleic acid molecules useful in the methods described herein include any nucleic acid molecule encoding the polypeptide or a fragment thereof. Such nucleic acid molecules need not be 100% identical to the endogenous nucleic acid sequence, but will generally show substantial identity. Polynucleotides having "substantial identity" to the endogenous sequence are generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" means pairing under various stringent conditions to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes) or portions thereof. (See, e.g., Wahl, G.M. and S.L. Berger, (1987), Methods Enzymol., 152:399; Kimmel, A.R. (1987), Methods Enzymol., 152:507).

[0093] For example, stringent salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions will generally include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Other parameters that can vary, such as hybridization time, concentration of detergents such as sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various stringencies are achieved by combining these various conditions as desired. In a preferred embodiment, hybridization will be carried out at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will be carried out at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In the most preferred embodiment, hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations under these conditions will be apparent to those skilled in the art.

[0094] For most applications, the stringency of the washing step after hybridization also varies. Washing stringency conditions can be defined by salt concentration and temperature. As described above, washing stringency can be increased by decreasing the salt concentration or by increasing the temperature. For example: The stringent salt concentration for the washing step will preferably be 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. The stringent temperature conditions for the washing step generally 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 step will be carried out in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS at 25°C. In a more preferred embodiment, the washing step will be carried out in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 42°C. In an even more preferred embodiment, the washing step will be carried out in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 68°C. Other variations of such conditions will be 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.

[0095] "Substantially identical" means a polypeptide or nucleic acid molecule having at least 50% identity to a reference amino acid sequence (e.g., any amino acid sequence described herein) or nucleic acid sequence (e.g., any nucleic acid sequence described herein). Preferably, such a sequence is at least 60%, or at least 80% or 85%, or at least or equal to 90%, 95% or even 99% identical to the sequence being compared at the amino acid level or nucleic acid level.

[0096] "Sequence identity" refers to the similarity between amino acid or nucleic acid sequences, which is expressed based on the similarity between sequences. Sequence identity is often measured by percent identity (or similarity or homology). The higher the percentage, the more similar the sequences. When aligned using standard methods, homologs or variants of a given gene or protein will have a relatively high sequence identity. Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, Madison, Wis., 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 method for determining the degree of identity, the BLAST program can be used, where the probability score between e -3 and e -100 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. U.S.A. 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. U.S.A. 85:2444; and Altschul et al., 1994, Nature Genet. 6:119-129. The NCBI Basic Local Alignment Search Tool (BLAST TM )(Altschul et al., 1990, J. Mol. Biol. 215:403-410) is available from a variety of sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Maryland), and is used on the Internet in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0097] "Subject" refers to an animal, such as a mammal, including, but not limited to, a human, a non-human primate, or a non-human mammal such as a bovine, a horse, a dog, a sheep or a feline mammal, or a sheep, a goat, a llama, a camel, or a rodent (rat, mouse), a gerbil, or a hamster. In a non-limiting example, the subject refers to a human infected with or at risk of being infected with or susceptible to infection with the H3 virus. In a particular aspect described herein, the subject is a human subject, such as a patient.

[0098] The ranges provided herein should be understood as shorthand for all values within the range, including the recited values at the beginning and end. For example, the range 1 to 50 should be understood to include any number, combination of numbers, or sub-ranges selected from the group consisting of the following numbers: 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 greater, continuously up to 100 or greater.

[0099] As used herein, the terms "treat" ('treat', 'treating', 'treatment', etc.) refer to reducing, alleviating, lessening, abolishing, ameliorating or eliminating a disease, disorder, condition or pathology, and / or the associated symptoms thereof. While not intended to be limiting, "treating" generally involves a therapeutic intervention that occurs after a disease, disorder, condition or pathology, and / or the associated symptoms thereof, have begun to develop, to reduce the severity of the disease, etc. and the associated signs and symptoms. It should be understood that although not excluded, treating a disease or condition does not require the complete elimination of the disease, disorder, condition, pathology, or the associated symptoms thereof.

[0100] As used herein, the terms "prevent" ('prevent', 'preventing', 'prevention'), "prophylactic treatment", etc., refer to inhibiting or blocking the disease state or the overall development of a disease in a subject, or reducing the likelihood of developing a disease, disorder or condition in a subject who has not yet developed the disease but is at risk of or susceptible to developing the disease, disorder or condition.

[0101] As used herein, a "transformed" cell is a cell into which a nucleic acid molecule or polynucleotide sequence has been introduced by molecular biological 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 viral vectors, transformation with plasmid vectors, and introduction of naked nucleic acids by electroporation, lipofection, and particle gun acceleration.

[0102] A "vaccine" refers to a preparation of an immunogenic substance (e.g., a protein or nucleic acid; a vaccine) that is capable of stimulating (eliciting) an immune response and that is administered to a subject to treat a disease, disorder, or pathology, or to prevent a disease, disorder, or pathology, such as an infectious disease (e.g., caused by H3 virus infection). The immunogenic substance can include, for example, an attenuated or killed microorganism (e.g., an attenuated virus), or an antigenic protein, peptide, or DNA derived from such a microorganism. Vaccines can elicit a prophylactic (preventive) immune response in a subject; they can also elicit a therapeutic response immune response in a subject. As described above, the methods of vaccine administration vary according to the vaccine and can include routes or means such as inoculation (intravenous or subcutaneous injection), ingestion, inhalation, or other forms of administration. Inoculation can be delivered by any of a number of routes, including parenteral, such as intravenous, subcutaneous, or intramuscular. Vaccines can also be administered with adjuvants to enhance the immune response.

[0103] As used herein, a "vector" is a nucleic acid (polynucleotide) molecule into which an exogenous nucleic acid can be inserted without disrupting the vector's ability to replicate in and / or integrate into a host cell. A vector can include nucleic acid sequences that permit its replication in a host cell, such as an origin of replication. The inserted vector is capable of inserting itself into the host nucleic acid. A vector can also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to permit transcription and translation of one or more inserted genes in a host cell. In some embodiments of the present application, the vector encodes influenza HA, NA, or M1 protein. In some embodiments, the vector is the 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).

[0104] "Virus-like particle (VLP)" refers to virus particles composed of one or more viral structural proteins, but lacking a viral genome. Since VLPs lack a viral genome, they are non-infectious and can produce safer and potentially more economical vaccines and vaccine products. Additionally, VLPs can typically be produced by heterologous expression and can be easily purified. Most VLPs contain at least the viral core protein, which drives budding of the host cell and release of the particles. An example of such a core protein is M1 influenza. In some embodiments herein, H3 influenza VLPs contain HA, NA, and M1 proteins. As described herein, H3 influenza VLPs can be produced by transfecting host cells with plasmids encoding the H3 HA, NA, and M1 proteins. After incubating the transfected cells for an appropriate time to allow protein expression (e.g., about 72 hours), the VLPs can be isolated from the cell culture supernatant. For example, clinical experiments for purifying or isolating influenza VLPs from cell supernatants include 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 are generally smaller than viruses and constitute particles without a viral capsid or genome.

[0105] Unless specifically stated or obvious from the context, as used herein, the term "or" shall be understood to be inclusive. Unless specifically stated or obvious from the context, as used herein, the terms "a", "an", and "the" shall be understood to be singular or plural. Similarly, unless the context clearly indicates otherwise, the word "or" is intended to include "and". Thus, "comprising A or B" means including A, or B, or A and B. It should also be understood that all base sizes or amino acid sizes and all molecular weight or molecular weight values given for nucleic acids or polypeptides are approximate values and are provided for description.

[0106] Unless specifically stated or obvious from the context, as used herein, the term "about" shall be understood to be within the normal tolerances in the art, e.g., within 2 standard deviations of the mean. About can 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 recited value. Unless the context clearly indicates otherwise, all numerical values provided herein are modified by the term "about".

[0107] In any definition of a variable herein, a list of chemical groups includes any single group of the listed groups or a combination of the listed groups that defines the variable. A 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 part thereof.

[0108] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein. Brief Description of the Drawings

[0109] Figure 1 A through 1C show the amino acid sequences of nine representative HA polypeptides (proteins) of influenza A H3 virus strains, referred to herein as TJ1-9 HA, which are broad-spectrum immunoreactive immunogens that elicit an immune response against H3 viruses and the HA proteins of H3 viruses. The nucleic acid sequences encoding these polypeptides can be used to produce virus-like particles (VLPs) containing H3 protein antigens, which are used as immunogens / vaccines to generate neutralizing antibodies in an immunized subject. As Figure 1 shown in A through 1C, the total length of the TJ1-9 HA polypeptides is 566 amino acids.

[0110] Figures 2A to 2C Shows the hemagglutination inhibition titers of serum antibodies (e.g., mouse serum) against VLPs from different H3 virus HA protein sources after incubation with different H3 virus strains (historical strains) in a hemagglutination inhibition assay (HAI), where these VLPs are used as immunogens (vaccines). Figure 2A Shows the hemagglutination inhibition assay (HAI) titration graphs of serum antibodies against VLPs produced from and used as immunogens (vaccines) of the TJ-2, TJ-3, TJ-5, TJ-6, TJ-7, TJ-8, and TJ-9 HA sequences (the "TJ" VLPs) described (shown in Figure 1 A through 1C). Antibodies generated against several TJ VLPs have broad reactivity against different historical strains (x-axis) of H3 virus strains. HAI analysis was performed using serum from immunized mice bled on day 77, 0.75% guinea pig red blood cells, and 20 nM oseltamivir (Tamiflu). Figure 2B Shows the results of HAI analysis using VLPs containing other H3 HA sequences (e.g., Bris / 07, Perth / 09, Vic / 11, Tx / 12, Switz / 13, HK / 14). Figure 2C Shows the results of HAI analysis using a PBS control. Detailed Description of the Invention

[0111] H3 influenza viruses typically circulate among humans and cause seasonal influenza epidemics. H3 viruses typically cause severe influenza illness and adapt to escaped disease by constantly changing their surface proteins (such as HA proteins). During the 2017-2018 influenza season, H3 influenza A viruses were found to be the dominant strains in the United States and globally (e.g., Australia and the United Kingdom). Treatment of H3 virus strains is particularly difficult due to their unusually high mutation rate and the inability to generate vaccines that effectively counter the relatively rapid changes occurring in their HA surface proteins (e.g., during the production of vaccines against this strain).

[0112] The present disclosure features synthetic (non-naturally occurring) immunogenic antigens, such as protein and glycoprotein antigens, derived from the influenza (“flu”) hemagglutinin (HA) protein of influenza A virus H3 strains, which can elicit a strong, broad, and persistent immune response in a subject (particularly a human subject). These immunogenic antigens are also referred to herein as “immunogens”.

[0113] Immunogens are provided that can prevent disease caused by H3 influenza virus strains or seasonal H3 influenza virus strains over several years, including drift strains that do not yet exist. In one embodiment, fully synthetic protein antigens, such as H3 influenza virus HA protein antigens, are provided. Such H3 HA antigens are synthetic proteins not found in nature, but they retain all the functions of the native H3 HA viral protein and are immunogenic, i.e., they can elicit an immune response, particularly, upon administration or delivery to or introduction into a subject, a broad and active immune response in the form of neutralizing antibodies and / or reactive T lymphocytes. Immunogenic compositions, such as vaccines, are also provided that comprise the synthetic H3 viral protein antigen or a nucleic acid encoding the antigen.

[0114] The H3 HA amino acid sequence and the protein antigen having said sequence are particularly useful as immunogens or in immunogenic compositions, such as vaccines, that elicit a broad reactive immune response in a subject to which the composition or vaccine is administered, particularly a human subject. The H3 virus immunogen contains antigenic determinants representing different “antigenic spaces” derived from the sequences of many H3 virus strains analyzed based on seasonal time periods (overlapping or non-overlapping seasonal time periods). Such overlapping or non-overlapping seasonal time periods may include different time intervals, such as 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 10 years or more, including the intervals therebetween.

[0115] The H3 virus antigens described herein comprise seasonal, pan-epitopic, broadly reactive antigens of H3 influenza viruses and their subtypes, in particular antigens containing sequences based on H3 drift variants, wherein said antigens are designed to generate an immunological response with broad activity, particularly in the form of neutralizing antibodies in a subject, particularly a human subject. Such antigens are useful as immunogens, which elicit an immune response against H3 viruses (e.g., the production of neutralizing antibodies), where multiple H3 strains are circulating simultaneously at one time. The broadly active H3 immunogenic antigens can be derived from H3 viruses, which often mutate a portion of their genome to evade immune pressure and thus evade immune surveillance in subjects who are not immunized or whose immune system is not primed to produce antibodies against the epitopes on the H3 antigen after infection. Thus, synthetic H3 antigens, such as H3 HA antigens, contain amino acid (or polynucleotide) sequences which, compared to wild-type antigen sequences, will elicit a greater number of neutralizing antibodies against potential H3 drift variants over multiple seasons and across multiple seasons.

[0116] The H3HA immunogenic protein, or immunogen, as described herein can be used in an immunogenic composition or as a vaccine, which can provide protection against many H3 virus strains for several years. The advantage of the broadly active H3 influenza immunogens and vaccines described herein is that they are designed to provide broader and more persistent protection against several seasonal H3 influenza strains (or clades) prevalent in different geographical locations. As described herein, what is provided by the immunogen and its sequence is a universal and broad H3 influenza vaccine, which can reduce the need for seasonal influenza vaccines (immunogenic compositions) against H3 strains and subtypes of influenza virus administered annually.

[0117] The immunogenic H3 virus HA antigen described herein can be used in an immunogenic composition (such as an influenza vaccine) capable of providing protective immunity against H3 influenza infection and disease in a subject.

[0118] Influenza virus

[0119] Influenza viruses are segmented, negative-strand RNA viruses belonging to the family Orthomyxoviridae. There are three types of influenza viruses: A, B, and C. Influenza A viruses infect a variety of birds and mammals, including humans, horses, marine mammals, pigs, ferrets, and chickens. In animals, most influenza A viruses cause mild local infections of the respiratory and intestinal tracts. However, highly pathogenic influenza A strains, such as H3, cause systemic infections in poultry, with a mortality rate that can reach 100%. Animals infected with influenza A are often used as subjects for influenza viruses, and it has been shown that certain subtypes cross the species barrier in humans, resulting in severe disease and devastating influenza outbreaks, leading to the death of infected human subjects.

[0120] Influenza A viruses can be classified into subtypes based on allelic variations in the antigenic regions of two genes encoding surface glycoproteins, namely hemagglutinin (HA) and neuraminidase (NA), which are required for viral attachment and cell release. Currently, 16 subtypes of HA (H1 to H16) and 9 antigenic variants of NA (N1 to N9) of influenza A viruses are known. Previously, only three subtypes have circulated in humans (H1N1, H1N2, and H3N2). However, in recent years, for example, as reported in the records in Hong Kong, China in 1997 and 2003, the pathogenic H5N1 subtype of avian influenza A can cross the species barrier and infect humans, resulting in the death of many patients.

[0121] In humans, avian influenza viruses infect cells of the respiratory tract as well as the intestine, liver, spleen, kidney, and other organs. Symptoms of avian influenza infection include fever, difficulty in breathing, including shortness of breath and coughing, lymphopenia, diarrhea, and difficulty in regulating blood sugar levels. In contrast to seasonal influenza, the most at-risk population is healthy adults, who make up the majority of the total population. Due to the high pathogenicity of certain avian influenza A subtypes (especially H3), and their ability to cross-infect humans, the economic and public health risks associated with these virus strains are very high, including the threat of a true epidemic and pandemic.

[0122] The influenza A virus genome encodes nine structural proteins and one non-structural protein (NS1) with regulatory functions. The segmented genome of influenza virus contains eight negative-sense RNA (nsRNA) gene segments (PB2, PB1, PA, NP, M, NS, HA, and NA), which encode at least ten polypeptides, including RNA-directed RNA polymerase proteins (PB2, PB1, and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin, such as the subunit HA1, usually referred to as the "head" subunit; and HA2, usually referred to as the "tail" or "stem" subunit; matrix proteins (M1 and M2); and non-structural proteins (NS1 and NS2) (see, for example: Krug et al., 1989, In: The Influenza Viruses, edited by R.M. Krug, Plenum Press, New York, pp. 89 - 152).

[0123] The ability of influenza viruses (such as H3) to cause widespread disease is due to their ability to evade the immune system by undergoing antigenic changes, which is believed to occur when a subject is simultaneously infected with an animal influenza virus and a human influenza virus. During the process of mutation and reassortment in the subject, the virus may integrate the HA and / or NA surface protein genes of another virus into its genome, thereby generating a new influenza subtype and evading the immune system.

[0124] Due to antigenic variations (drifts) in circulating strains of H3 influenza virus, particularly in the HA and NA proteins of the virus, the efficacy of vaccines against H3 influenza virus often fails to reach optimal and sub-optimal levels. The methods described herein provide broadly reactive, pan-epitope HA or NA antigens of H3 influenza virus, which generate a broad immune response, particularly in the form of neutralizing antibodies that bind to H3 virus antigens and neutralize viral activity (e.g., its ability to infect cells), to more effectively treat H3 influenza and its symptoms.

[0125] Influenza virus hemagglutinin (HA) and neuraminidase (NA) proteins

[0126] HA is a viral surface glycoprotein that typically contains approximately 560 amino acids (e.g., 566 amino acids) and accounts for 25% of the total viral protein. As described herein, HA is a protein antigen that is highly effective as an immunogen against H3 virus because it contains multiple epitopes for antibodies that are generated in a subject or subjects that encounter the H3 HA antigen during infection.

[0127] HA is responsible for attaching virus particles to subject cells at an early stage of infection and penetrating into subject cells, particularly in the respiratory epithelium. In order for the virus to infect cells, cleavage of the viral HA0 precursor into HA1 and HA2 subfragments is a necessary step. Thus, cleavage is required to convert newly formed virus particles in subject cells into virions capable of infecting new cells. Cleavage is known to occur during the transport of the intact HA0 membrane protein from the endoplasmic reticulum of infected cells to the plasma membrane. During transport, HA undergoes a series of co-translational and post-translational modifications, including proteolytic cleavage of the precursor HA into an amino-terminal fragment HA1 ("head") and a carboxyl-terminal HA2 ("tail" or "stalk"). One of the main difficulties in growing H3 influenza strains in primary tissue culture or established cell lines is triggered by activation of proteolytic cleavage of influenza hemagglutinin in subject cells.

[0128] Although it is known that uncleaved HA can mediate the binding of the virus to its sialic acid-containing receptor on the cell surface, it cannot mediate fusion, which is the next step in the infection cycle. It has been reported that exposure of the hydrophobic amino terminus of HA2 through cleavage is required so that it can be 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.

[0129] Proteolytic activation of HA involves cleavage at arginine residues by trypsin-like endoproteases, which are typically intracellular enzymes, calcium-dependent and having a neutral optimal pH. Since the activating protease is a cellular enzyme, the type of infected cell determines whether HA is cleaved. The HA of mammalian influenza viruses and non-pathogenic avian influenza viruses is susceptible to proteolytic cleavage only in a limited number of cell types. Due to the differences in hemagglutinin cleavability, there are also differences in the range of subjects, which is related to the pathogenic characteristics of the virus.

[0130] Neuraminidase (NA) is the second membrane glycoprotein of influenza viruses. The presence of viral NA has been shown to be important for the generation of a multifaceted protective immune response against the infected virus. For most influenza A viruses, NA is 413 amino acids in length 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, and all subtypes are found in wild birds. NA is involved in the destruction of the cellular receptor of viral HA by cleaving terminal neuraminic acid (also known as sialic acid) residues from the carbohydrate moieties on the surface of infected cells. NA can also cleave sialic acid residues from viral proteins, thus preventing virus aggregation. Presumably using this mechanism, NA promotes the release of virus progeny by preventing newly formed virus particles from accumulating along the cell membrane and by facilitating the transport of the virus through the mucus present on mucosal surfaces. NA is an important antigenic determinant and undergoes antigenic variation.

[0131] In addition to the surface proteins HA and NA, H3 influenza viruses also contain 6 other internal genes, which produce 8 different proteins, including the polymerase genes PB1, PB2, and PA, the matrix proteins M1 and M2, the nucleoprotein (NP), and the non-structural proteins NS1 and NS2 (see, e.g., Horimoto et al., 2001, Clin Microbiol Rev. 14(1): 129-149).

[0132] For packaging into progeny virus particles, H3 viral RNA is transported from the nucleus as a ribonucleoprotein (RNP) complex, which consists of three influenza virus polymerase proteins, the nucleoprotein (NP), and viral RNA, as well as the influenza virus matrix 1 (M1) protein and the nuclear export protein (Marsh et al., 2008, J Virol, 82: 2295-2304). The M1 protein located within the envelope is thought to play a role in assembly and budding. A limited number of M2 proteins are incorporated into virions (Zebedee, 1988, JVirol 62: 2762-2772). These M2 proteins form a structure with H +a tetramer of ion channel activity that, when activated by the low pH in endosomes, acidifies 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 interfering with M2 ion channel activity, thus inhibiting viral uncoating.

[0133] NS1 is a non-structural protein with multiple functions, including regulating the splicing and nuclear export of cellular mRNAs and stimulating translation. The main function of NS1 seems to be to counteract the interferon activity of the subject, since NS1 knockout viruses are viable, although their growth efficiency in interferon-free cells is lower than that of the parental virus (Garcia-Sastre, 1998, Virology 252: 324-330).

[0134] The NS2 non-structural protein has been detected in virus particles (Richardson et al., 1991, Arch. Virol. 116: 69-80; Yasuda et al., 1993, Arch. Virology 196: 249-255). The average number of NS2 proteins in virus particles is estimated to be 130-200 molecules. In vitro binding assays have demonstrated a direct protein-protein contact between M1 and NS2. The NS2-M1 complex has also been detected in virus-infected cell lysates by immunoprecipitation. The NS2 protein is thought to play a role in the export of RNP from the nucleus through interaction with the M1 protein (Ward et al., 1995, Archives of Virology 140: 2067-2073).

[0135] Broadly reactive influenza proteins and virus-like particles (VLPs)

[0136] provide non-naturally occurring, broadly active, pan-epitope H3 influenza HA immunogenic polypeptides (immunogens) and influenza virus-like particles (VLPs) that contain an H3 HA immunogen containing multiple epitopes (antigenic determinants) and can, upon administration of the medicament and delivery to a susceptible subject, confer the ability to elicit an immune response with broad activity against HA antigens for influenza and its symptoms for prophylaxis or treatment. For example, by practicing the methods described herein, representative H3 HA immunogenic antigen sequences are produced, given in Figure 1 A to 1C herein. In certain instances, the broadly reactive, pan-epitope H3 HA polypeptide is administered as part of a VLP.

[0137] It should be understood that whether or not these characteristics and features are explicitly stated, the H3 influenza virus immunogens and sequences described and provided herein are non-naturally occurring, broadly reactive, and pan-epitopic. It should also be understood that the H3 antigen proteins, such as HA, HA1, or HA2, described herein and used as immunogens are non-naturally occurring or synthetic antigens that elicit an immune response, such as neutralizing antibodies, in a subject.

[0138] Broadly reactive and immunogenic H3 antigen sequences that are capable of generating an immune response against H3 influenza virus strains, including current and future H3 viruses, can be generated by methods such as those described in the co-pending provisional patent application No. 62 / 697,803 filed on July 13, 2018, the content of which is incorporated herein by reference and which relates to the consideration of H3 antigen sequence parameters, such as the HA antigen sequence, over a time span or range (e.g., a linear time range), such as one or more influenza seasons, and the geographical location(s) from which the H3 virus was isolated, such as the Southern or Northern Hemisphere.

[0139] In one embodiment, the H3 influenza VLP contains the viral HA protein. In an embodiment, the VLP can contain the HA1 and / or HA2 proteins. It should be understood that in some cases, the H3 influenza virus VLP can contain the viral 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 transfecting a subject's cells with one or more plasmids containing polynucleotide sequences encoding the HA, NA, and M1 proteins. After incubating the transfected cells for an appropriate period of time to allow protein expression (e.g., about 72 hours), the H3 VLPs can be isolated from the cell culture supernatant. The H3 influenza VLPs can be purified from the cell supernatant using steps practiced in the art, e.g., the VLPs can be separated by low-speed centrifugation (to remove cell debris), vacuum filtration, and ultracentrifugation through 20% glycerol.

[0140] Influenza VLPs can be used as an immunogenic composition or influenza vaccine to elicit an immune response against H3 influenza virus. In particular, the components of the immunogenic composition or vaccine (or VLP), the broadly reactive, pan-epitopic H3 influenza HA polypeptide contains antigenic (pan-epitopic) determinants that are broadly reactive and elicit an immune response in a subject (e.g., generate neutralizing antibodies and / or activated T cells), can treat a subject infected with an H3 virus (e.g., neutralize the infecting virus) and / or protect the subject from a full-blown infection with the virus or its signs and symptoms.

[0141] In one embodiment, the antigenic sequences of the H3 influenza antigens with broad reactivity and immunogenicity described herein, such as the H3 HA antigen, contain various epitope determinants, which can reflect antigenic drift and sequence variability in the H3 viral antigen proteins, for example: at different seasons (times) and different geographical locations. In particular, the H3 viral HA antigens described herein can contain amino acid sequences that contain antigenic determinants (epitopes) derived from influenza virus strains with diverse sequences, including drift variants, against which broadly reactive neutralizing antibodies can be generated, especially when the antigen is used as an immunogenic product, (immunogen), for example: an antiviral vaccine, and is introduced into a subject.

[0142] In one aspect, the H3 viral antigen amino acid sequence provides a complex, immunogenic antigen sequence, which contains the ultimate determinants derived from viral infections or diseases in past and recent seasons, and / or viruses from different geographical locations, and / or different subtypes or clades of the H3 virus, that is, a "pan-epitope" antigen, which, when used as an immunogen, vaccine, or VLP, elicits a broadly reactive immune response. In one embodiment, the immunogenic H3 viral HA antigen sequence contains epitopes that are due to antigenic changes in the H3 HA surface antigen sequence caused by point mutations during the viral replication process. As a result, administering an H3 immunogen as described herein to a subject can elicit a broadly reactive immune response in the subject against epitopes that reflect such antigenic changes.

[0143] The broadly reactive H3 HA antigens and their sequences as described herein, when used as an immunogen or immunogenic composition, such as a vaccine, elicit a broadly reactive immune response in immunocompetent subjects, and thus, they provide an excellent vaccine that can capture the antigenic epitopes of many different H3 influenza virus isolates (subtypes or strains) and generate a broadly active immune response against them (for example: broadly active neutralizing antibodies). It should be noted that the terms "broadly active" and "broadly reactive" are used synonymously herein.

[0144] In one embodiment, the H3 viral antigen as described herein is a polypeptide or a peptide antigen of the H3 virus that currently causes diseases or infections and their symptoms, such as seasonal H3 influenza, and is native to a specific geographical region. In another embodiment, the H3 viral antigen is a polypeptide or peptide antigen that will cause diseases and symptoms of H3 infection in the future. In one embodiment, the H3 viral antigen is a polynucleotide sequence. In one embodiment, the H3 viral antigen is a polynucleotide sequence encoding the polypeptide or peptide antigen as described herein. For example, representative broadly reactive H3 viral HA immunogens are shown in Figure 1 A to 1C.

[0145] In another embodiment, the H3 immunogen sequences described herein are expressed as polypeptides, proteins, or peptides in cells. In one embodiment, the H3 immunogen is isolated and / or purified. In one embodiment, the immunogen is formulated for administration to a subject in need thereof. In one embodiment, the immunogen is administered to a subject in need thereof in an effective amount to elicit an immune response in the subject. In one embodiment, the immune response elicits neutralizing antibodies. In one embodiment, the immune response is prophylactic or therapeutic.

[0146] In one embodiment, a non-naturally occurring H3 viral immunogen (immunogen sequence), such as a vaccine, is provided that elicits a broad immune response in a subject after the immunogen is introduced, administered, or delivered to the subject. The route of introduction, administration, or delivery is not limited and can include, for example, intravenous, subcutaneous, intramuscular, oral, and other routes. The vaccine can be therapeutic (e.g., administered to a subject after the symptoms of a disease (influenza) caused by the H3 virus), or prophylactic (protective) (e.g., administered to a subject before the subject has or exhibits symptoms of a disease (influenza), or a full-blown disease caused by the H3 virus).

[0147] In one embodiment, the final amino acid sequence of the antigen (e.g., HA) is reverse-translated and optimized for expression in mammalian cells. As will be understood by those skilled in the art, optimization of the nucleic acid sequence includes optimization of the codons for expressing the sequence in mammalian cells and optimization of the RNA (e.g., RNA stability).

[0148] In one embodiment, an isolated nucleic acid molecule (polynucleotide) comprising a nucleotide sequence encoding a polypeptide or peptide antigen, such as an H3 influenza HA polypeptide (or HA1 or HA2 polypeptide), is provided. In certain embodiments, the nucleotide sequence encoding the H3 HA polypeptide is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the polynucleotide encoding the HA polypeptide (or HA1 or HA2 polypeptide) sequence shown in A to 1C. Figure 1 A to 1C.

[0149] In other embodiments, the nucleotide sequence encoding the H3 influenza HA polypeptide (or HA1 or HA2 polypeptide) is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the polynucleotide encoding the H3 HA polypeptide (or HA1 or HA2 polypeptide) sequence shown in A to 1C, but lacks the start codon encoding the N-terminal methionine. Figure 1 A to 1C.

[0150] A vector is provided that contains a nucleotide sequence encoding a non-naturally occurring, broadly reactive polypeptide or peptide antigen, such as an H3 influenza HA polypeptide (or HA1 or HA2 polypeptide). In some embodiments, the vector contains a nucleotide sequence encoding a polypeptide or peptide antigen, such as an influenza H3 HA polypeptide antigen, that is at least 94%, at least %, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polynucleotide encoding the H3 HA polypeptide (or HA1 or HA2 polypeptide) sequence shown in A through 1C. In some embodiments, the vector further contains a promoter operably linked to the nucleotide sequence encoding the H3 HA polypeptide (or HA1 or HA2 polypeptide). In a particular embodiment, the promoter is the 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 the H3 HA polypeptide sequence shown in A through 1C. In specific embodiments, the nucleotide sequence of the vector contains a polynucleotide encoding the H3 HA polypeptide (or HA1 or HA2 polypeptide) sequence shown in A through 1C. In 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. Figure 1 A to a polynucleotide encoding the H3 HA polypeptide (or HA1 or HA2 polypeptide) sequence shown in 1C is at least 94%, at least %, at least 96%, at least 97%, at least 98%, or at least 99% identical. In some embodiments, the vector further contains a promoter operably linked to the nucleotide sequence encoding the H3 HA polypeptide (or HA1 or HA2 polypeptide). In a particular embodiment, the promoter is the 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 the H3 HA polypeptide sequence shown in A through 1C. In specific embodiments, the nucleotide sequence of the vector contains a polynucleotide encoding the H3 HA polypeptide (or HA1 or HA2 polypeptide) sequence shown in A through 1C. In 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. Figure 1 A to a polynucleotide encoding the H3 HA polypeptide sequence shown in 1C is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical. In specific embodiments, the nucleotide sequence of the vector contains a polynucleotide encoding the H3 HA polypeptide sequence shown in A through 1C. In 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. Figure 1 A to the H3 HA polypeptide (or HA1 or HA2 polypeptide) sequence shown in 1C. In 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.

[0151] A vector for expressing an H3 viral antigen, such as an H3 viral protein, such as an HA protein, as described herein, can be any suitable expression vector known and used in the art. The vector can be, for example, a mammalian expression vector or a viral vector. In some embodiments, the vector is the pTR600 expression vector (U.S. Patent Application Publication No. 2002 / 0106798, incorporated herein by reference; Ross et al., 2000, Nat Immunol. 1(2):102 - 103; and Green et al., 2001, (e.g., Vaccine 20:242 - 248).

[0152] An H3 influenza virus-derived non-naturally occurring polypeptide antigen is provided, such as an H3 influenza HA polypeptide antigen, or an HA1 or HA2 polypeptide antigen, which is produced by transfecting a subject's cells with an expression vector known and used in the art under conditions sufficient to permit expression of the polypeptide in the cells, such as an H3 HA, HA1, or HA2 polypeptide. An isolated cell containing the vector is also provided.

[0153] Also provided are non-naturally occurring, broadly reactive, pan-epitope H3 antigen polypeptides as described herein, such as pan-epitope, broadly reactive H3 influenza HA polypeptides. In certain embodiments, the amino acid sequence of the polypeptide is at least 95% to 99% (inclusive) identical to the amino acid sequence of an HA, HA1, or HA2 polypeptide, such as Figure 1 shown in A to 1C. In a particular embodiment, the amino acid sequence of an H3 influenza HA, HA1, or HA2 polypeptide is at least 95% to 99% (inclusive) identical to the amino acid sequence of the HA, HA1, or HA2 polypeptide shown in Figure 1 A to 1C, but lacks the N-terminal methionine residue. In a specific embodiment, the amino acid sequence of an H3 influenza HA polypeptide is at least 95% to 99% (inclusive) identical to the Figure 1 1-566 amino acids of the H3 HA polypeptide shown in A to 1C.

[0154] In some embodiments, the fusion protein comprises a broadly reactive, pan-epitope H3 viral antigen polypeptide as described herein, such as, but not limited to, the H3 influenza HA polypeptide disclosed herein. In some embodiments, the H3 influenza HA polypeptide can be fused with any heterologous amino acid sequence to form a fusion protein. For example, the HA1 and HA2 polypeptides can be produced independently and then fused together to produce an H3 HA polypeptide antigen, such as, comprising 566 amino acids.

[0155] Also provided are virus-like particles (VLPs), particularly H3 influenza VLPs, which contain pan-epitope, broadly reactive protein antigens, such as the H3 influenza HA, HA1, or HA2 proteins described herein. In certain embodiments, the HA protein of the VLP is at least or equal to 94%, at least or equal to 95%, at least or equal to 96%, at least or equal to 97%, at least or equal to 98%, at least or equal to 99% or 100% identical to the Figure 1 H3 HA protein shown in A to 1C. The virus or influenza virus VLP can also comprise any other viral or influenza proteins required to form the viral particle. In certain embodiments, the virus or influenza VLP further comprises an influenza neuraminidase (NA) protein, an influenza matrix (M1) protein, or both.

[0156] Also provided are H3 influenza VLPs comprising an H3 influenza HA, HA1, or HA2 polypeptide as described herein, which are produced by transfecting a subject's cells with a vector containing a polynucleotide encoding the H3 HA, HA1, or HA2 polypeptide. In one embodiment, also provided is an H3 influenza VLP containing an H3 influenza HA polypeptide, or an HA1 or HA2 polypeptide, as described herein, produced by transfecting a subject's cells with a vector encoding the H3 HA, HA1, or HA2 polypeptide, a vector encoding an influenza NA protein, and a vector encoding an influenza M1 protein under conditions sufficient to permit the expression of the H3 HA, NA, and M1 proteins. AsFigures 2A to 2C as observed in Figure 1 such VLPs that contain the sequences shown in A to 1C and are used as immunogens generate antibodies with high hemagglutination inhibition (HAI) titers against different H3 influenza virus strains.

[0157] Collection of plasmids (vectors) is also contemplated. In certain embodiments, the collection of plasmids includes a plasmid encoding H3 influenza NA, a plasmid encoding H3 influenza MA, and a plasmid encoding a broadly reactive H3 HA protein as described herein. In some embodiments, the nucleotide sequence encoding the H3 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 Figure 1 the polynucleotide encoding the H3 HA amino acid sequence shown in A to 1C. In some embodiments, the nucleotide sequence encoding the codon-optimized H3 influenza HA protein in the HA-encoding plasmid is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to Figure 1 the polynucleotide encoding the H3 HA amino acid sequence shown in A to 1C.

[0158] In the context of the present application, "broadly reactive" or "broadly active" means that the H3 protein (e.g., H3 HA protein sequence) is immunogenic and contains a diversity of epitopes (antigenic determinants; pan-epitopes) that elicit an immune response in a subject sufficient to treat a disease or infection (e.g., neutralizing antibodies against the H3 virus HA epitope diversity, often accompanied by a T cell response), and / or inhibits, neutralizes, or prevents infection caused by most or all of the H3 influenza viruses or related virus strains within a particular subtype. In embodiments, a non-naturally occurring H3 viral antigen protein, e.g., an HA protein, is capable of eliciting a protective immune response against most or all of the known H3 influenza virus isolates, e.g., about 80%, about 85%, about 90%, about 95%, or about 96% to 99% of the known H3 influenza virus isolates.

[0159] Compositions for administration and pharmaceutical compositions

[0160] Compositions are provided that comprise a broadly reactive, pan-epitope H3 influenza HA protein, or a fusion protein, or VLPs that comprise such a broadly reactive H3 influenza HA protein as described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient, or vehicle. In some embodiments, an adjuvant (a drug or immunological reagent that modifies or enhances an immune response, such as by generating more persistent antibodies) is also used. For example, but not limited to, the adjuvant can be an inorganic compound such as alum, aluminum hydroxide, or aluminum phosphate; a mineral or paraffin oil; squalene; a detergent such as Quil A; a plant saponin; Freund's complete or incomplete adjuvant, a biological adjuvant (e.g., a cytokine such as IL-1, IL-2, or IL-12); a bacterial product such as killed Bordetella pertussis, or a toxoid; or an immunostimulatory oligonucleotide (e.g., a CpG oligonucleotide).

[0161] Compositions and formulations (e.g., physiologically or pharmaceutically acceptable compositions) for parenteral administration that contain the non-naturally occurring, broadly active, pan-epitope H3 influenza HA polypeptide and H3 influenza virus-like particles (VLPs) include, but are not limited to, sterile water 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 rapeseed oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, an alcohol / water solution, an emulsion, or a suspension, including saline and buffered media. Parenteral vehicles include, for example: sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include, for example: fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives such as anti-microbial agents, antioxidants, chelating agents, coloring agents, stabilizers, inert gases, etc. may also be present in such compositions and formulations.

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

[0163] The pharmaceutical compositions provided herein comprise a therapeutically effective amount of a non-naturally occurring, broadly reactive, pan-epitope, H3 viral protein antigen, or H3 influenza VLP, which can be used alone or in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile, and the formulation is suitable for the mode of drug administration. The composition may 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. The liquid or aqueous composition can be lyophilized and reconstituted with a solution or buffer before use. The composition can be formulated into suppositories with conventional binders and carriers such as triglycerides. Oral formulations can contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Any common and known pharmaceutical carrier, such as sterile saline solution or sesame oil, can be used. The medium can also contain conventional pharmaceutical adjuvants such as, for example, pharmaceutically acceptable salts that regulate osmotic pressure, buffers, preservatives, and the like. Other media that can be used in the composition and method of administration are physiological saline and sesame oil.

[0164] Methods of Treatment, Drug Administration, and Delivery

[0165] Methods are provided herein for treating a disease or infection caused by an H3 influenza virus, or its symptoms. The methods comprise administering to a subject (e.g., a mammal), particularly a human subject, a therapeutically effective amount of a broadly reactive, pan-epitope immunogen as described herein or a pharmaceutical composition or vaccine (e.g., a VLP vaccine) comprising the immunogen. One embodiment relates to a method of treating a subject having or at risk of or susceptible to a disease or infection caused by an H3 influenza virus, or its symptoms. The method comprises administering to the subject (e.g., a mammalian subject) an amount or therapeutic dose of an immunogenic composition or vaccine comprising a non-naturally occurring, broadly reactive, pan-epitope H3 viral antigen polypeptide, such as an HA polypeptide, or an HA polypeptide VLP, under conditions effective to treat a disease, infection, and / or its symptoms caused by an H3 influenza virus.

[0166] In one embodiment, the methods described herein include administering to a subject, including a human subject determined to be in need of such treatment, an effective amount of a non-naturally occurring, broadly reactive, pan-epitope H3 viral antigen polypeptide, such as an H3 viral HA polypeptide, or a vaccine, or a composition described herein, to produce such an effect. The treatment methods are appropriately administered to a subject having, being susceptible to, or at risk of developing a disease, disorder, infection, or symptoms thereof, namely influenza or flu, particularly a human. Identifying a subject in need of such treatment can be based on the judgment of the subject or a healthcare professional and can be subjective or objective. Briefly, those subjects in need of treatment or "at risk" or "susceptible" can be determined by any objective or subjective judgment, including the opinion of the subject or healthcare provider, through diagnostic tests (e.g., genetic testing, enzyme or protein marker detection), signature analysis, family history, etc. Non-naturally occurring, broadly reactive pan-epitope H3 immunogens, such as the H3HA polypeptide immunogens and vaccines described herein, can also be used to treat any other disorder that may involve an infection or disease caused by an H3 influenza virus. The subject being treated can be a non-human mammal, such as a veterinary subject or a human subject (also referred to as a "patient").

[0167] Additionally, a prophylactic method is provided for preventing or protecting against a disease, infection, or symptoms thereof caused by an H3 influenza virus. Such a method includes administering a therapeutically effective amount of a pharmaceutical composition comprising an H3 immunogenic composition or vaccine (e.g., an H3 VLP vaccine) as described herein to a subject (e.g., a mammal such as a human), particularly, prior to infection or onset of disease in the subject, such as an H3 virus-related disease.

[0168] In another embodiment, a method is provided for monitoring the progression of an H3 virus infection or disease caused by an H3 virus, or for monitoring the treatment of an H3 infection or disease. The method includes determining the level or diagnostic measurement of a diagnostic marker or biomarker in a subject having or susceptible to an infection, disease, or symptoms thereof associated with an H3 influenza virus, to which a sufficient amount of a non-naturally occurring, broadly reactive, pan-epitope H3 viral HA protein as described herein, or a vaccine as described herein, has been administered to treat the infection, disease, or symptoms thereof.

[0169] The level or amount of a marker or biomarker (e.g., a protein) determined in the method can be compared to the known level of the marker or biomarker in a healthy, normal control sample; in a pre-infection or pre-disease sample of the subject; or in other diseased / infected / ill patients to determine the disease status of the treatment subject. For monitoring, a second level or amount of the marker or biomarker in a sample obtained from the subject is determined at a time point later than when the first level or amount was determined, and the levels or amounts of the two markers or biomarkers can be compared to monitor the course of the disease or infection, or the efficacy of a therapy / treatment. In certain embodiments, as described, a pre-treatment level of the marker or biomarker in the subject (e.g., in a sample obtained from the subject) is determined before treatment is initiated; then the pre-treatment level of the marker or biomarker can be compared to the level of the marker or biomarker in the subject after and / or during treatment to determine the efficacy of the disease treatment (monitor efficacy).

[0170] The non-naturally occurring, broadly reactive, pan-epitope H3 virus antigen polypeptide, such as the H3 virus HA polypeptide as described, and the VLP or composition thereof comprising the H3 HA polypeptide, can be administered to a subject by any route under normal circumstances for introducing a recombinant protein, a composition comprising the recombinant protein, or a recombinant virus into the subject. Routes of administration and methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intrathecal, parenteral, such as intravenous (IV) or subcutaneous (SC), vaginal, rectal, intranasal, inhalation, intraocular, intracranial, or oral. Parenteral administration of a drug, such as subcutaneous, intravenous, or intramuscular administration of a drug, is typically achieved by injection (immunization). Injectables can be prepared in conventional forms and formulations, can be liquid solutions or suspensions, can also be solid forms (e.g., lyophilized forms) suitable for existing as a solution or suspension before injection, or can be emulsions. Injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets. Administration of the drug can be systemic or local.

[0171] The non-naturally occurring, broadly reactive, pan-epitope H3 virus polypeptide, such as the H3 virus HA polypeptide as described, and the VLP or composition thereof comprising the H3 HA polypeptide, can be administered in any suitable manner, such as with a pharmaceutically acceptable carrier as described supra. The pharmaceutically acceptable carrier depends in part on the particular immunogen or composition being administered and the particular method for administering the composition. Accordingly, a variety of suitable and physiologically and pharmaceutically acceptable formulations can be used to prepare a pharmaceutical composition comprising a non-naturally occurring, broadly active, pan-epitope H3 virus polypeptide, such as the H3 virus HA polypeptide, and the VLP or composition thereof comprising the H3 HA polypeptide.

[0172] A broadly reactive, pan-epitope H3 viral antigen polypeptide, such as an H3 viral HA polypeptide, and a VLP comprising the HA polypeptide, or a composition thereof, can be administered by a single dose or multiple doses. The dose administered to a subject should be sufficient over time to induce a beneficial therapeutic response in the subject, such as inhibiting, blocking, reducing, ameliorating, guarding against, or preventing a disease or infection by an H3 influenza virus. It depends on the type, age, weight, and general condition of the subject, on the severity of the infection being treated, on the particular composition being used, and on the mode of administration of the medicament, and the required dose will vary between subjects. A suitable dose can be determined by a person skilled in the art, such as a clinician or a practicing physician, using only routine experimentation.

[0173] Further provided is a method of eliciting an immune response in a subject against an H3 influenza virus by administering to the subject a non-naturally occurring, broadly active, pan-epitope H3 influenza HA protein, a fusion protein comprising an H3 influenza HA protein, a VLP comprising an influenza HA protein, or a composition thereof as described herein. In some embodiments, the H3 HA protein, HA fusion protein, or VLP can be administered using any suitable route of administration, such as: by intramuscular injection. In some embodiments, the H3 HA protein, fusion protein, or VLP is administered in the form of a composition comprising a pharmaceutically 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 (such as a CpG oligonucleotide). In other embodiments, the composition can be administered in combination with another therapeutic agent or molecule.

[0174] Also provided is a method of immunizing a subject against an infection or disease or its symptoms caused by an H3 influenza virus, wherein the method comprises administering to the subject a VLP comprising a non-naturally occurring, pan-epitope, broadly reactive H3 influenza HA protein, or an immunogenic composition as described herein. In some embodiments of the method, the composition further comprises a pharmaceutically acceptable carrier and / or an adjuvant. In some embodiments of the method, the composition further comprises a pharmaceutically 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 (such as a CpG oligonucleotide). In one embodiment, the H3 VLP (or its composition) is administered intramuscularly.

[0175] In some embodiments of methods of eliciting an immune response or immunizing a subject against an infection or disease caused by or associated with an H3 influenza virus, a subject is administered at least 1 μg of a VLP comprising a non-naturally occurring, broadly reactive pan-epitope H3 viral HA protein, such as 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 VLP comprising a non-naturally occurring, broadly reactive pan-epitope H3 viral HA protein, such as from about 1 to about 50 μg or from about 1 to about 25 μg of a VLP comprising an H3 HA protein. In certain instances, a subject is administered from about 5 to about 20 μg of a VLP, or from about 10 to about 15 μg of a VLP. In certain but non-limiting instances, a subject is administered about 15 μg of a VLP. However, those skilled in the art can determine a therapeutically effective amount of a VLP suitable for administering a medicament to a subject in need of treatment or prevention of a viral infection (e.g., an amount that provides a therapeutic effect or protection against an H3 influenza virus infection).

[0176] It is expected that administration of a VLP comprising a non-naturally occurring, broadly reactive pan-epitope H3 HA protein as described herein will elicit high titers of neutralizing antibodies against the diversity of antigenic determinants on the H3 HA protein immunogen, as well as protection levels of H3 HA-inhibiting (HAI) antibodies against many representative H3 isolates, and will provide overall protection against a lethal challenge with an H3 virus and / or related H3 virus types. The VLP comprising a non-naturally occurring, broadly reactive pan-epitope H3 influenza HA protein described herein elicits a broader immune response (e.g., compared to the immune response elicited by a multivalent H3 influenza virus vaccine, can elicit neutralizing antibodies against a broader range of H3 virus isolates).

[0177] Adjuvants and combination therapies

[0178] The H3 virus immunogen or immunogenic composition comprising an H3 protein antigen (e.g., an H3 HA antigen), or a VLP comprising an H3 virus as described herein, can be used alone or in combination with other therapeutic agents to enhance antigenicity or immunogenicity, i.e., increase the immune response of a subject, such as elicit specific antibodies. For example, an H3 influenza VLP can be administered with an adjuvant, such as alum, Freund's incomplete adjuvant, Freund's complete adjuvant, a biological adjuvant, or an immunostimulatory oligonucleotide (such as a CpG oligonucleotide).

[0179] One or more cytokines, such as interleukin-1 (IL-2), interleukin-6 (IL-6), interleukin-12 (IL-12), the protein memory T cell attractant "regulated on activation normal T cell expressed and secreted factor" (RANTES), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ); one or more growth factors, such as GM-CSF or granulocyte colony-stimulating factor (G-CSF); if desired or necessary, one or more molecules can be used as biological adjuvants, such as TNF ligand superfamily member 4 ligand (OX40L) or type 2 transmembrane glycoprotein receptor belonging to the TNF superfamily (4-1BBL), or a combination of such molecules. (See, e.g., Salgaller et al., 1998, Journal of Surgical Oncology 68(2):122-38; Lotze 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, Progress in Experimental Medicine and Biology 465:381-90). Such molecules can be administered systemically (or locally) to a subject.

[0180] Several methods for inducing cellular responses in vitro and in vivo are known and practiced in the art. Lipids have been identified as reagents capable of assisting in the in vivo priming of cytotoxic lymphocytes (CTLs) against various antigens. For example: palmitic acid residues can be linked (e.g., via one or more linking residues, such as glycine, glycine-glycine, serine, serine-serine, etc.) to the α and ε amino groups of lysine residues and then conjugated to an immunogenic peptide (U.S. Patent No. 5,662,907). The lipidated peptide can then be injected directly in micellar form, incorporated into liposomes or emulsified in an adjuvant. As another example, Escherichia coli lipoproteins, such as tripalmitoyl-S-glyceryl-cysteine-serine-serine, can be used to prime tumor-specific CTLs when covalently attached to a suitable peptide (see, e.g., Deres et al., 1989, Nature 342:561).). In addition, the induction of neutralizing antibodies can also be primed with the same molecule conjugated to a peptide displaying the appropriate epitope, and two compositions can be combined to elicit a humoral response and a cell-mediated response, where such a combination is considered desirable.

[0181] Although the methods of treatment may involve administering VLPs comprising the non-naturally occurring, broadly reactive, pan-epitope H3 HA immunogenic protein described herein, those skilled in the art will understand that the non-naturally occurring, broadly reactive, pan-epitope H3 HA immunogenic protein, the H3 influenza HA protein itself (in the absence of viral particles), as a component of a pharmaceutically acceptable composition or as a fusion protein, can be administered to a subject in need thereof to elicit an immune response in the subject.

[0182] Kit

[0183] Also provided herein are kits that contain a non-naturally occurring, broadly reactive, pan-epitope H3 viral immunogen, or a vaccine, or a pharmaceutically acceptable composition comprising an immunogen, and a pharmaceutical composition of a pharmaceutically acceptable carrier, diluent, or excipient, e.g., for administering a medicament to a subject. As described herein, the immunogen can be in the form of an H3 viral protein (polypeptide) or a polynucleotide (a polynucleotide encoding an H3 viral polypeptide, e.g., an H3 HA protein). Also provided herein are kits that contain one or more plasmids or a collection of plasmids as described herein. As will be understood by those skilled in the art, such kits can contain one or more containers for the immunogen, vaccine, or composition, a diluent or excipient if necessary, and instructions for use.

[0184] Recombinant polypeptide expression

[0185] Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques have been well described in the literature, such as "Molecular Cloning: A Laboratory Manual", 2nd 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); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the present invention and, for example, can be taken into account in carrying out and practicing the present invention. Particularly useful techniques for specific embodiments will be discussed in the following sections.

[0186] Examples

[0187] The following examples are provided to illustrate specific features and / or embodiments. The examples should not be construed as limiting the application to the specific features or embodiments described.

[0188] Example 1

[0189] Hemagglutination inhibition (HAI) assay

[0190] The hemagglutination inhibition (HAI) assay is used to evaluate functional antibodies against the HA protein, which are capable of inhibiting the agglutination of guinea pig, horse, or turkey red blood cells (RBCs).

[0191] The sera used for the assay contain antibodies generated after immunizing animals (mice) with VLPs containing the H3 virus HA antigen sequence (as described herein), such as TJ2, TJ-3, TJ5-9 ( Figure 1 A to 1C), other H3 virus HA sequences (e.g., Bris / 07, Perth / 09, Vic / 11, Tx / 12, Switz / 13, HK / 14), wild-type H3 HA sequences, and / or PBS controls.

[0192] For immunization, 5 mice were used per group. Each animal received 3 μg of HA antigen / VLP per dose, along with the squalene adjuvant (AF03) (MF Klucker, 2012, J. Pharm. Sci., 101(12):4490-4500). A homologous antigen, adjuvant, and booster immunization / administration regimen was employed. The animals were boost-immunized with the immunogen on day 56 and then bled on day 77, as shown in Table 1 below.

[0193] Table 1

[0194]

[0195]

[0196] The protocol was adapted from the WHO Influenza Laboratory Surveillance Manual (Gillim-Ross and Subbarao, 2006, Clin. Microbiol. Rev. 19(4):614-636) and used the subject species that are commonly used to characterize contemporary H3N2 strains, which have a preferential binding ability to α(2,6)-linked sialic acid receptors. Turkey or guinea pig red blood cells were used to compare whether there were differences in HAI depending on the type of red blood cells used.

[0197] To inactivate nonspecific inhibitors, sera were treated with receptor-destroying enzyme (RDE) (Toa Gosei Kogyo Co., Ltd., Japan) prior to testing. (Bright et al., 2005, Lancet 366(9492):1175-1181; Bright et al., 2003, Journal of Virology 308(2):270-278; Bright et al., 2006, Journal of the American Medical Association 295(8):891-894; Mitchell et al., 2004, Vaccine 21(9-10):902-914; Ross et al., 2000, Nature Immunology 1(2):127-131). Briefly, three parts of RDE were added to one part of serum and incubated overnight at 37°C. The RDE was inactivated by incubating at 56°C for approximately 30 minutes (~30 minutes). The RDE-treated sera were serially two-fold diluted in V-bottom microtiter plates. Equal volumes of each virus, such as H3N2 virus, adjusted to approximately 8 hemagglutination units (HAU / 50 μl), were added to each well. The plates were covered and incubated for 20 minutes at room temperature, followed by the addition of 0.75% or 0.8% guinea pig red blood cells (Lampire Biologicals, Pipersville, PA, USA) in phosphate-buffered saline (PBS). The erythrocytes were stored at 4°C and used within 72 hours of preparation.

[0198] The plates were mixed and covered by agitation and the RBCs were allowed to settle at room temperature for 1 hour. The HAI titer was determined by the reciprocal dilution of the last well containing unagglutinated RBCs. Each plate contained positive and negative serum controls. Prior to vaccination, all mice were seronegative for currently circulating human influenza viruses (HAI ≤ 1:10). According to the WHO and European Medicines Agency definitions for the evaluation of influenza vaccines, seroprotection was defined as an HAI titer >1:40 and seroconversion was defined as a four-fold increase in titer compared to baseline.

[0199] A more stringent threshold of >1:80 was often examined. Since the mice were antigen-naive and seronegative at the time of vaccination, seroconversion and seroprotection rates were interchangeable in the experiment.

[0200] Figures 2A to 2C showed that compared to VLPs produced using different H3 virus HA sequences ( Figure 2B ) or PBS ( Figure 2C ), the hemagglutination inhibition antibody titers of antibodies generated against VLPs containing the H3 virus HA immunogen (immunogen sequence) using the immunization / administration regimens described above and herein ( Figure 2A ).

[0201] Example 2

[0202] Preparation of virus-like particles (vaccines)

[0203] Using methods described above (see, e.g., U.S. Patent Application Publication No. US2015 / 0030628), a plasmid expressing influenza virus neuraminidase (A / mallard / Alberta / 24 / 01, H7N3), HIV p55 Gag sequence, or a broadly reactive HA expression plasmid (e.g., containing the encoding Figure 1 Each of the three mammalian expression plasmids (sequences of the HA immunogen shown in A to 1C) was transfected into mammalian 293T cells. After incubation at 37°C for 72 hours, the supernatant from the transiently transfected cells was collected, centrifuged to remove cell debris, and filtered through a 0.22 μm pore membrane. Mammalian virus-like particles (VLPs) were purified and precipitated by ultracentrifugation at 135,000xg at 4°C for 4 hours on a 20°C glycerol cushion. The VLPs were resuspended in phosphate-buffered saline (PBS) and the total protein concentration was assessed using a conventional bicinchoninic acid assay (BCA). The hemagglutination activity of each VLP preparation was determined by adding an equal volume of turkey or guinea pig red blood cells (RBCs) to a V-bottom 96-well plate and incubating with serially diluted VLPs for 30 minutes at room temperature (RT). The highest dilution of the VLP was considered the endpoint HA titer for fully agglutinated RBCs.

[0204] Example 3

[0205] ELISA to determine HA content

[0206] Coat high-affinity, 96-well, flat-bottom ELISA plates with 5 to 10 μg of total VLP protein, and add recombinant H3 antigen (3006_H3_Vc, Protein Sciences, Meriden, CT) serially diluted in ELISA carbonate buffer (50 mM carbonate buffer, pH 9.5) to the wells. Incubate the plates overnight at 4°C on a rocker. The next morning, wash the plates with PBS with 0.05% Tween-20 (PBST), and block non-specific epitopes with 1% bovine serum albumin (BSA) in PBST solution for 1 hour at room temperature. Remove the buffer, and add the stem-specific group 2 antibody CR8020 (Tharakaraman, K. et al., 2014, Journal of Cellular Subjects and Microbes, Vol. 15, pp. 644-651; Ekiert, D.C. et al., 2012, Science Journal, 333(6044): 843-850; Creative Biolabs, Shirley, NY) to the plates, followed by incubation at 37°C for 1 hour. Wash the plates, and then detect with goat anti-human IgG horseradish peroxidase-conjugated secondary antibody (2040-05, Southern Biotech, Birmingham, AL) for 1 hour at 37°C.

[0207] Wash the plates. Add the substrate of freshly prepared o-phenylenediamine dihydrochloride (OPD) (P8287, Sigma, City, State, USA) in citrate buffer (P4922, Sigma) to the wells, and then add 1N H2SO4 stop reagent. Read the plates at 492 nm absorbance using a microplate reader (Powerwave XS, Biotek, Winooski, VT). Subtract the background signal from the negative wells. Perform linear regression standard curve analysis using recombinant standard antigen of known concentration to estimate the HA content in many VLPs.

[0208] Example 4

[0209] Mouse and ferret studies

[0210] Mouse studies

[0211] BALB / c mice (Mus musculus, female, 6 to 8 weeks old) were purchased from Jackson Laboratories (Bar Harbor, Maine, USA), housed in microisolator units and allowed free access to food and water. Animals were cared for according to the University of Georgia Research Animal Resources University Laboratory Animal Care Guidelines. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). Mice (5 mice per group) were vaccinated with purified viroplasms (3.0 μg / mouse) according to the HA content quantified by ELISA, and the VLP immunogen (vaccine) was delivered to the animals by intramuscular injection at week 0. At weeks 4 and 8, the animals were boosted with the same immunogen (vaccine) at the same dose. Each dose of vaccine was formulated with emulsified squalene-in-water adjuvant (Sanofi Pasteur, Lyon, France). The final concentration after mixing with VLP 1:1 was 2.5% squalene. Blood samples were collected by submandibular cheek 28 days after each vaccination and transferred to microcentrifuge tubes. The tubes were centrifuged at 10,000 rmp for 10 minutes. Serum samples were removed and frozen at -20°C ± 5°C.

[0212] Ferret studies

[0213] Ferrets (Mustela putorius furo, female, 6 to 12 months old) were purchased from Marshall Farms (Sale, Pennsylvania, USA). Ferrets were housed in pairs in stainless steel cages (Shor-line, Kansas City, Kansas, USA) containing Sani-chips laboratory animal bedding (PJ Murphy Forest Products, Montville, New Jersey, USA). Ferrets were provided with Teklad Global ferret diet (Harlan Teklad, Madison, Wisconsin, USA) and fresh water ad libitum.

[0214] Purified VLP was diluted in PBS at pH 7.2 to achieve the final concentration. At week 0, ferrets (n = 3) were inoculated with 15 μg of purified VLP in a volume of 0.25 ml by intramuscular injection into the quadriceps according to the HA content determined by densitometry, and then boosted at week 3 with the same dose. The vaccine was stored at -80°C before use and alum adjuvant ( Alum; Pierce Biotechnology, Inc., Rockford, Illinois, USA) or co-formulated with the above emulsified squalene adjuvant. During the vaccination protocol, animals were monitored weekly for adverse events, including weight loss, decreased body temperature, decreased mobility, runny nose, sneezing, and diarrhea. Before vaccination, animals were confirmed to be seronegative for influenza A (e.g., H1N1) and influenza B viruses by HAI assay. Fourteen to twenty-one days after each vaccination, blood was collected from anesthetized ferrets via the anterior vena cava and transferred to microcentrifuge tubes. The tubes were centrifuged; the serum was removed and frozen at -20 ± 5 °C.

[0215] Other embodiments

[0216] From the foregoing description, it will be apparent that variations and modifications can be made to the invention described herein to adapt it to various uses and conditions. Such embodiments are also within the scope of the appended claims.

[0217] The recitation of a list of elements in any definition of a variable herein includes defining the variable as any single element or combination (or sub-combination) of the listed elements. The recitation of an embodiment herein includes the embodiment as any single embodiment or in combination with any other embodiment or portions thereof.

[0218] All patents and publications mentioned herein are hereby incorporated by reference to the same extent as if each individual patent and publication were specifically and individually indicated to be incorporated by reference.

Claims

1. An isolated, recombinant, non-naturally occurring, broadly reactive, pan-epitope H3 influenza virus antigen that is capable of eliciting an immune response against current and future H3 virus strains; Wherein, The isolated and recombinant H3 influenza virus antigen consists of the amino acid sequence shown in SEQ ID NO.10 or 11.

2. The H3 influenza virus antigen according to claim 1, Wherein, The antigen is a hemagglutinin antigen.

3. A virus-like particle comprising the H3 influenza virus antigen according to claim 1 or 2, which is capable of eliciting an immune response against current and future H3 virus strains.

4. The virus-like particle according to claim 3, which comprises a polynucleotide encoding the H3 influenza virus antigen.

5. An isolated, recombinant, non-naturally occurring, pan-epitope immunogen that is capable of eliciting an immune response against current and future H3 influenza virus strains; Wherein, The isolated and recombinant immunogen consists of the amino acid sequence shown in SEQ ID NO,10 or 11.

6. The H3 influenza virus antigen according to claim 1 or 2, the immunogen according to claim 5, or the virus-like particle according to claim 3 or 4, Wherein, The immune response comprises the production of neutralizing antibodies.

7. The H3 influenza virus antigen according to claim 1 or 2, the immunogen according to claim 5, or the virus-like particle according to claim 3 or 4, Wherein, The immune response comprises the production of antibodies having hemagglutinin inhibitory activity.

8. The H3 influenza virus antigen according to claim 1 or 2, the immunogen according to claim 5, or the virus-like particle according to claim 3 or 4, Wherein, The immune response further comprises the production of T lymphocytes.

9. A pharmaceutically acceptable composition comprising the H3 influenza virus antigen according to claim 1 or 2, the immunogen according to claim 5, or the virus-like particle according to claim 3 or 4, and a pharmaceutically acceptable carrier, diluent or excipient.

10. The pharmaceutically acceptable composition according to claim 9, further comprising an adjuvant.

11. An immunogenic composition or vaccine comprising the H3 influenza virus antigen according to claim 1 or 2, the immunogen according to claim 5, or the virus-like particle according to claim 3 or 4.

12. A pharmaceutically acceptable composition comprising the immunogenic composition or vaccine according to claim 11, and a pharmaceutically acceptable carrier, diluent or excipient.

13. The pharmaceutically acceptable composition according to claim 12, further comprising an adjuvant.

14. Use of an effective amount of the H3 influenza virus antigen according to claim 1 or 2, the immunogen according to claim 5, or the virus-like particle according to claim 3 or 4 in the preparation of a medicament for eliciting an immune response in a subject.

15. Use of an effective amount of the pharmaceutically acceptable composition according to any one of claims 9, 10, 12 and 13 in the preparation of a medicament for eliciting an immune response in a subject.

16. Use of an effective amount of the immunogenic composition or vaccine according to claim 11 in the preparation of a medicament for eliciting an immune response in a subject.

17. The use according to any one of claims 14 to 16, wherein, the immune response comprises the production of neutralizing antibodies.

18. The use according to claim 17, wherein, the immune response further comprises the production of T lymphocytes.

19. The use according to any one of claims 14 to 16, wherein, the adjuvant is administered to the subject simultaneously.

20. A polynucleotide encoding an H3 influenza virus antigen according to claim 1 or 2.

21. A composition comprising the polynucleotide according to claim 20 and a pharmaceutically acceptable carrier, diluent or excipient.

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