Modified influenza virus

Abstract

The present disclosure relates to the field of modified viruses and viral proteins. More specifically, the present disclosure relates to modified hemagglutinin proteins, influenza viruses expressing such proteins, and methods for making them.

Description

[Technical Field]

[0001] The inventions described in this disclosure were made with U.S. Government support under Contract HHSO100200900101C awarded by the Biomedical Advanced Research and Development Authority (BARDA). The U.S. Government may have certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application No. 63 / 398,362, filed August 16, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to the field of modified viruses and viral proteins. More specifically, the present disclosure relates to modified hemagglutinin proteins, influenza viruses expressing such proteins, and methods for making them. [Background technology]

[0004] Influenza is a major respiratory disease in mammalian species, causing significant mortality, morbidity, and economic loss each year. Three broad types of influenza viruses are recognized: A, B, and C, which are defined by the lack of serological cross-reactivity between their internal proteins. Influenza A viruses are further classified into subtypes based on antigenic and genetic differences in the glycoprotein hemagglutinin (HA) and neuraminidase (NA) proteins.

[0005] More recent vaccine production methods involve generating and culturing reassortant influenza viruses in cell culture, such as Madin-Darby canine kidney (MDCK) cells. However, certain influenza viruses appear to be less amenable to growth and replication under such conditions than others, which can lead to challenges in vaccine production. Therefore, there remains a need to develop genetic modifications to the influenza viral genome that confer efficient growth and replication to vaccine virus candidates in cell culture. Summary of the Invention

[0006] This disclosure is based on the surprising discovery that modifications to amino acid residues in the trimer interface region of HA proteins, particularly H2 HA proteins, can be used to improve the growth of influenza viruses cultured in cells such as Madin-Darby canine kidney (MDCK) cells. Without being bound by any theory, such modifications may stabilize or increase the stability of the HA trimer of influenza virions during cell culture.

[0007] Thus, the present disclosure provides a modified hemagglutinin (HA) protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified, and an influenza virus expressing the modified HA protein is capable of growing in a cell.

[0008] Relatedly, the present disclosure also provides modified HA proteins of the H2 subtype comprising an amino acid sequence in which one or more amino acid residues have been modified at a position selected from the group consisting of V39, L219, V233, V320, K383, I388, N390, K391, V392, S394, A405, R416, D430, F450, and any combination thereof, of the full-length H2 amino acid sequence.

[0009] Furthermore, the present disclosure provides a modified HA protein of the H2 subtype, comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified, and an influenza virus expressing the modified HA protein is capable of growing in a cell.

[0010] The present disclosure also provides: An isolated nucleic acid comprising a nucleotide sequence encoding a modified HA protein disclosed herein or a nucleotide sequence complementary thereto; A genetic construct comprising (i) an isolated nucleic acid of the present disclosure or (ii) a nucleotide sequence complementary thereto, operably linked or connected to one or more regulatory sequences; and A host cell transformed with an isolated nucleic acid of the present disclosure or a genetic construct of the present disclosure.

[0011] The present disclosure further provides a method for producing a modified HA protein of the present disclosure, the method comprising the steps of (i) culturing a pre-transformed host cell of the present disclosure, and (ii) isolating the modified HA protein from the host cell cultured in step (i).

[0012] Additionally, the present disclosure provides an isolated influenza virus comprising an HA viral segment encoding a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

[0013] The present disclosure further provides a method for preparing influenza virus in a cell, the method comprising contacting the cell with a genetic construct comprising a nucleic acid encoding a modified HA protein, the modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

[0014] The present disclosure further provides an isolated influenza virus prepared by any of the methods disclosed herein, and a modified HA protein prepared by any of the methods disclosed herein.

[0015] Relatedly, the present disclosure provides a method of making a vaccine composition, comprising the steps of: (a) providing an isolated influenza virus of the present disclosure and / or a modified HA protein of the present disclosure; and (b) combining the isolated influenza virus and / or the modified HA protein with an adjuvant and / or treating the isolated influenza virus with a virus-inactivating agent.

[0016] Accordingly, the present disclosure also provides vaccine compositions produced according to the methods disclosed herein for making vaccine compositions.

[0017] Similarly, the present disclosure provides a vaccine composition, the vaccine composition comprising: (a) an isolated influenza virus of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient; or (b) a modified HA protein of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.

[0018] Furthermore, the present disclosure provides a method for inducing an immune response in a subject, the method comprising administering a therapeutically effective amount of an isolated influenza virus of the present disclosure, a modified HA protein of the present disclosure, or a vaccine composition of the present disclosure to the subject, thereby inducing an immune response in the subject.

[0019] Furthermore, the present disclosure provides a method for preventing and / or treating an influenza-associated disease, disorder or condition in a subject, the method comprising the step of administering a therapeutically effective amount of an isolated influenza virus of the present disclosure, a modified HA protein of the present disclosure, or a vaccine composition of the present disclosure to the subject, thereby preventing and / or treating the influenza-associated disease, disorder or condition.

[0020] The present disclosure also provides a method for identifying or screening for modifications of an HA protein that promote or improve the growth of influenza virus in a cell, the method comprising the steps of: (a) modifying an influenza virus to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified; and (b) testing the ability of the modified influenza virus to grow in cells.

[0021] Relatedly, the present disclosure provides a method for identifying modifications of an HA protein that promote or improve the growth of influenza virus in a cell, the method comprising the steps of: (a) modifying an influenza virus to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified; and (b) Passaging the modified influenza virus expressing the modified HA protein one or more times in the cells.

[0022] Furthermore, the present disclosure provides a method for improving the growth of influenza virus in a cell, the method comprising modifying an influenza virus to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

[0023] The present disclosure also provides a method for improving the stability of an influenza virus strain in a cell, the method comprising modifying the influenza virus strain to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

[0024] Furthermore, the present disclosure provides a method for improving the production of an influenza virus strain, the method comprising modifying an influenza virus strain to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

[0025] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. [Brief explanation of the drawings]

[0026] [Figure 1] Schematic diagram of candidate vaccine virus (CVV) transfection and rescue. [Figure 2] Mapping of observed mutations and variants to the structure of A / Swine / Missouri / 2124514 / 2006_HA. [Figure 3] Diagram of the outer surface and trimer interface of the HA, showing that the HA mutations in the rescued strain of A / Chicken / Ohio / 494832 / 2007 are restricted to the trimer interface region of the molecule. [Figure 4] A close-up structural view of the A405T mutant residue and its proximity to the K423 residue on the adjacent HA monomer.

[0027] Sequence Listing Description [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] DETAILED DESCRIPTION OF THE INVENTION

[0028] General Techniques and Definitions Unless otherwise defined, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., of genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0029] The present disclosure will be carried out without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA techniques, and immunology. Such procedures are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), Volumes I, II, and III in their entirety; DNA Cloning: A Practical Approach, Vols. I and II (D.N. Glover, Second Edition, 1995), IRL Press, Oxford, in its entirety; Oligonucleotide Synthesis: A Practical Approach (M.J. Gait, ed., 1984), IRL Press, Oxford, in its entirety, and in particular in Gait, p. 1-22; Atkinson et al., pp. 35-81; Sproat et al., pp. 83-115; and Wu et al., pp. 135-151, articles therein; 4. Nucleic Acid Hybridization: A Practical Approach (B.D. Hames & S.J. Higgins, eds., 1985), IRL Press, Oxford, in its entirety. Press, Oxford, full text, Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, full text, Perbal, B., A Practical Guide to Molecular Cloning (1984) and the entire series on Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.).

[0030] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations of any two or more of such steps or features.

[0031] The present disclosure is not intended to be limited in scope by the specific embodiments described herein, such embodiments being for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the disclosure as described herein.

[0032] Each feature of any particular aspect or embodiment or embodiment of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment or embodiment of the present disclosure.

[0033] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps or group of compositions of matter should be interpreted as encompassing one and more (i.e., one or more) of those steps, compositions of matter, group of steps or group of compositions of matter.

[0034] As used herein, the singular forms "a," "an," and "the" include the plurals of these words unless the context clearly indicates otherwise. For example, a reference to "a bacterium" includes a plurality of such bacteria, and a reference to an "allergen" is a reference to one or more allergens.

[0035] The term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as providing clear support for both meanings or either meaning.

[0036] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of the stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0037] "Consisting essentially of," in the context of an amino acid sequence, means the referenced amino acid sequence with an additional 1, 2, or 3 amino acids at the N- or C-terminus.

[0038] The term "substantially" does not exclude "completely" (e.g., a composition that is "substantially free" of Y may be completely free of Y).

[0039] The term "about" in reference to a numerical value x is arbitrary and means, for example, any number within 1, 5, or 10% of the specified number. In certain instances, the term "about" encompasses the exact number recited.

[0040] All computer programs, algorithms, patent and scientific literature referred to herein is hereby incorporated by reference.

[0041] For purposes of this disclosure, the database accession numbers or unique identifiers provided herein for genes, proteins, or viral strains, and their associated gene and / or protein sequence or sequences, are hereby incorporated by reference.

[0042] Modified HA protein The present inventors have surprisingly shown that modifying specific amino acid residues in the trimer interface region of the H2 protein can enhance or improve the growth of influenza viruses expressing modified hemagglutinin (HA) proteins in cell culture. These modified proteins may also increase virus production, which may be advantageous for vaccine production.

[0043] Thus, in one aspect, the present disclosure provides a modified hemagglutinin (HA) protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified. Preferably, influenza viruses expressing the modified HA protein are able to grow in cells. In this regard, influenza viruses expressing an unmodified or wild-type form of the modified HA protein are preferably unable to grow in cells or have limited or reduced ability to grow in cells. Thus, the modified HA protein preferably provides influenza viruses expressing the HA protein with the ability to grow in cells or an improved ability to grow in cells.

[0044] In a related aspect, the present disclosure provides a modified HA protein of the H2 subtype, comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified. In some examples, the one or more amino acid residues are modified at positions selected from the group consisting of 39, 219, 233, 320, 383, 388, 390, 391, 392, 394, 405, 416, 430, 450, and any combination thereof, of the full-length H2 amino acid sequence. More specifically, the one or more amino acid residues may be modified at positions selected from the group consisting of V39, L219, V233, V320, K383, I388, N390, K391, V392, S394, A405, R416, D430, F450, and any combination thereof, of the full-length H2 amino acid sequence (e.g., as set forth in SEQ ID NO: 12).

[0045] In another related aspect, the disclosure provides a modified HA protein of the H2 subtype comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified, wherein an influenza virus expressing the modified HA protein is capable of growing in a cell.

[0046] Influenza hemagglutinin (HA) is a glycoprotein encoded by the HA gene segment of influenza viruses. HA is typically expressed as a homotrimer on the surface of the viral capsid and is essential for its infectivity. For this purpose, HA binds to glycans containing the monosaccharide sialic acid, enabling recognition of upper respiratory tract cells or red blood cells. This leads to the internalization of influenza viruses into endosomes by the cells, followed by the reconstitution of the HA trimer. The HA protein then fuses with the endosomal membrane, thereby allowing the release of viral gene segments, present in the form of ribonucleoprotein complexes (RNPs) along with nucleoproteins and polymerase complexes, into the host cell cytoplasm. The RNPs are transported to the host nucleus, where transcription and replication of the viral genome occur. The HA protein, along with other newly produced viral proteins and the replicated genome, then buds from the infected host cell and is incorporated into the influenza virion envelope. The HA protein on the new virus particle remains attached to sialic acid groups on the outer cell surface glycoprotein, and neuraminidase (NA) cleaves these groups, thereby allowing the efficient release of the newly formed virion.

[0047] The terms "hemagglutinin" and "HA" refer to any hemagglutinin protein known to those of skill in the art (e.g., influenza A HA subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16). However, in certain examples, the modified HA proteins described herein are of an H2, H1, H5, H3, H7, or H9 influenza A subtype, or more specifically, an H2, H1, or H5 influenza A subtype. In some examples, the modified HA proteins described herein are of an H2 influenza A subtype. HA can also be derived from an influenza virus isolate from any host species. In various examples, the modified HA protein is derived at least in part from an avian influenza virus isolate or strain. In other examples, the modified HA protein is derived at least in part from a swine influenza virus isolate or strain.

[0048] According to specific examples, the modified HA proteins provided herein are influenza hemagglutinin proteins, such as influenza A hemagglutinin protein or influenza B hemagglutinin protein. Typical hemagglutinin proteins include a signal peptide, a stem or stalk domain, a globular head domain, a luminal domain, a transmembrane domain, and a cytoplasmic domain. In some examples, the modified hemagglutinin proteins provided herein include a single polypeptide chain, such as HA0, HA1, or HA2. In other examples, the modified HA protein includes multiple polypeptide chains in a quaternary association state (e.g., HA1 and HA2). In other examples, the modified hemagglutinin protein lacks a signal peptide (i.e., the modified hemagglutinin protein is a mature hemagglutinin). In alternative examples, the modified hemagglutinin protein includes a signal peptide (i.e., the modified hemagglutinin protein is a full-length hemagglutinin, e.g., as set forth in SEQ ID NO: 12). The modified hemagglutinin proteins provided herein may also be further modified by post-translational processing such as signal peptide cleavage, disulfide bond formation, glycosylation (e.g., N-linked glycosylation), protease cleavage, and lipid modification (e.g., S-palmitoylation).

[0049] As used herein, the term "trimer interface region" in relation to HA proteins refers to the exterior or surface region of an HA monomer that can associate, interact, contact, or bind with adjacent HA monomers during the formation of an HA trimer. In this regard, one or more modified amino acid residues may be adjacent to and / or face the trimer interface region of an additional HA molecule or monomer, such as when in a dimeric or trimeric configuration. Furthermore, one or more modified amino acid residues may modulate structural features, such as the tertiary structure, of the trimer interface region. Preferably, the trimer interface region includes one or more amino acid residues that are close to and participate in the interaction with one or more amino acid residues on an adjacent HA monomer during trimer formation. Such interactions may include, for example, hydrogen bonds, electrostatic interactions, salt bridges, etc. In particular examples, one or more modified amino acid residues in the trimer interface region are within about 15 angstroms or less (e.g., within about 15, 14, 13, 12, 11, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, or 0.5 angstroms, or any range thereof) of one or more residues in an adjacent HA monomer upon trimerization. In other examples, one or more modified amino acid residues in the trimer interface region are within about 11 angstroms or less of one or more residues in an adjacent HA monomer upon trimerization. In various examples, one or more modified amino acid residues in the trimer interface region are within about 8.5 angstroms or less of one or more residues in an adjacent HA monomer upon trimerization.

[0050] It is further contemplated that the amino acid residues in the trimer interface region can be found in any portion, subunit, or domain of an HA protein or molecule, e.g., the globular head domain and / or the stalk domain. In particular examples, one or more of the modified amino acids are present in the lower region of the stalk domain (e.g., one or more of residues V39, K383, I388, N390, K391, V392, S394, and F450 of a full-length H2 HA protein). Preferably, the trimer interface region in the lower region of the stalk domain can comprise, consist of, or consist essentially of amino acid residues 377-397, 439-452, 26-46, and optionally 325-335 of a full-length HA protein of the H2 subtype (e.g., those positions are set forth in SEQ ID NO: 12). Alternatively, the trimer interface region in the lower region of the stalk domain can comprise, consist of, or consist essentially of amino acid residues 383-394, 439-452, 31-40, and optionally 325-335 of a full-length HA protein of the H2 subtype (e.g., those positions are set forth in SEQ ID NO: 12). Still alternatively, the trimer interface region in the lower region of the stalk domain can comprise, consist of, or consist essentially of amino acid residues 26-46, 325-335, 377-397, and 439-452 (e.g., N26-D46, L325-P335, D377-E397, and L439-D452) of a full-length HA protein of the H2 subtype (e.g., those positions are set forth in SEQ ID NO: 12 or SEQ ID NO: 28). In certain examples, the trimer interface region in the lower region of the stalk domain comprises, consists of, or consists essentially of amino acid residues 26-46, 377-397, and 439-452 of a full-length HA protein of the H2 subtype (e.g., N26-D46, D377-E397, and L439-D452).

[0051] Preferably, the modified HA protein comprises a modification to one or more amino acid residues at positions selected from the group consisting of 39, 383, 388, 390, 391, 392, 394, 450, and any combination thereof, of the full-length H2 amino acid sequence (e.g., as set forth in SEQ ID NO:12 or SEQ ID NO:28). In some examples, the modified HA protein comprises an isoleucine modification at amino acid residue 39 of the full-length H2 amino acid sequence. In other examples, the modified HA protein comprises a glutamic acid modification at amino acid residue 383 of the full-length H2 amino acid sequence. In certain examples, the modified HA protein comprises a threonine modification at amino acid residue 388 of the full-length H2 amino acid sequence. In particular examples, the modified HA protein comprises an isoleucine modification at amino acid residue 390 of the full-length H2 amino acid sequence. In other examples, the modified HA protein comprises an arginine or asparagine modification at amino acid residue 391 of the full-length H2 amino acid sequence. In various examples, the modified HA protein comprises an alanine modification at amino acid residue 392 of the full-length H2 amino acid sequence. In some examples, the modified HA protein comprises a tyrosine modification at amino acid residue 394 of the full-length H2 amino acid sequence. In other examples, the modified HA protein comprises a serine modification at amino acid residue 450 of the full-length H2 amino acid sequence.

[0052] More specifically, the modifications to one or more amino acid residues of the modified HA protein are preferably selected from the group consisting of 39I, 383E, 388T, 390I, 391R, 391N, 392A, 394Y, 450S, and any combination thereof, of the full-length H2 amino acid sequence (e.g., as set forth in SEQ ID NO: 12 or SEQ ID NO: 28). Even more specifically, in some examples, the modifications to one or more amino acid residues of the modified HA protein are selected from the group consisting of V39I, K383E, I388T, N390I, K391R, K391N, V392A, S394Y, F450S, and any combination thereof, of the full-length H2 amino acid sequence (e.g., as set forth in SEQ ID NO: 12 or SEQ ID NO: 28).

[0053] In this regard, the trimer interface region can be divided into multiple non-contiguous portions of the amino acid sequence of an HA monomer. For example, one or more amino acid residues of the trimer interface region can be found in the HA1 subunit, and one or more additional amino acid residues of the trimer interface region can be found in the HA2 subunit of an HA protein. Furthermore, one or more amino acid residues of the trimer interface region can be found in the stem domain, and one or more additional amino acid residues of the trimer interface region can be found in the globular head domain of an HA protein.

[0054] The term "modified protein," e.g., "modified HA protein," is understood to refer to a protein containing one or more modifications compared to a parent, consensus, or wild-type protein, e.g., a wild-type HA protein. The sequence of a wild-type HA protein can be determined experimentally or is published in several databases. A suitable example is provided herein as SEQ ID NO: 12. Another example is provided herein as SEQ ID NO: 28. The term "modification" or "modified" in the context of the present disclosure is understood to include chemical modification of a protein as well as genetic manipulation of DNA encoding the protein. Such modifications may be replacement of one or more amino acid side chains in the protein of interest, one or more substitutions, one or more deletions, and / or one or more insertions. Furthermore, these terms are intended to include the screening and / or selection of existing influenza virus isolates that have or express an HA protein that includes one or more amino acid residues described herein as "modified" (e.g., V39, L219, V233, V320, K383, I388, N390, K391, V392, S394, A405, R416, D430 and F450, where these positions are numbered according to the conventional numbering system for a full-length HA protein, such as that exemplified in SEQ ID NO: 12 or SEQ ID NO: 28).

[0055] Preferably, the modified HA proteins described herein exhibit or have altered or modulated stability (e.g., increased stability), e.g., in their trimeric form, compared to a wild-type or unmodified HA protein of a corresponding influenza virus isolate. Thus, phrases such as "increasing HA trimer stability" can mean that, after modification according to the present disclosure, the modified HA protein is capable of forming a more stable homotrimeric configuration when present in cell culture compared to the starting (unmodified) HA protein. Without being bound by any theory, modifications to the trimer interface region described herein may function to improve the ability of modified HA protein monomers to form and maintain a homotrimeric configuration, such as when an influenza virus isolate expressing such modified HA protein is grown in cell culture. Thus, in certain examples, the modified HA protein is capable of forming HA trimers when an influenza virus isolate expressing the modified HA protein is grown in cell culture. In this regard, the interface region of the modified HA protein is preferably capable of interacting with one or more additional HA protein monomers to form the stable trimeric configuration. In some instances, the modified HA protein is capable of forming HA trimers with improved stability compared to a wild-type or unmodified HA protein (e.g., an HA protein that does not contain a respective modification to one or more amino acid residues in the interface region of the modified HA protein).

[0056] Thus, in certain examples, HA trimers containing modified HA proteins described herein exhibit increased stability compared to corresponding HA trimers containing wild-type or unmodified HA proteins. As used herein to describe the stability of a modified HA protein or trimer thereof, "enhanced," "increased," or "upregulated" refers to an increased level of stability compared to a control or reference sample (e.g., an HA trimer containing a wild-type or unmodified HA protein that does not contain one or more of the modifications described herein).

[0057] The stability of a modified HA protein, or more specifically its homotrimer, may be assessed by any means in the art, directly or indirectly, such as by its ability to rescue, grow, and / or replicate when grown in cell culture, for example, in mammalian cells such as MDCK cells. Thus, phrases such as "increasing HA trimer stability" can mean that a virus having a modified HA protein according to the present disclosure has improved ability to rescue, grow, and / or replicate when grown in cell culture (e.g., in mammalian cells such as MDCK cells) compared to a virus having an unmodified or wild-type HA protein. Thus, the term "stability" can also be used with respect to a virus having a modified HA protein according to the present disclosure. For example, a virus having a modified HA protein according to the present disclosure may have improved ability to rescue, grow, and / or replicate when grown in cell culture (e.g., in mammalian cells such as MDCK cells) compared to a virus having an unmodified or wild-type HA protein, and therefore may have increased or improved stability. The degree of improvement in any one or more of these capabilities (e.g., rescue, growth, and / or replication capabilities) can vary, for example, the degree of improvement, such as rescue, growth, and / or replication capabilities, can be greater than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, or at least about 500% greater than the level observed in an influenza virus strain expressing a control HA protein (e.g., an unmodified HA protein or a wild-type HA protein). For example, the degree of improvement can be greater than about 50% compared to a virus having or expressing an unmodified or wild-type HA protein.In another example, improved rescue, growth, and / or replication capacity can be determined by achieving a threshold level of rescue of replicating virus grown on MDCK cells that allows for the production of viral loads for use in vaccine preparation on a commercial scale.

[0058] Further exemplary methods for assessing the stability of a modified HA protein or its homotrimer may include computer or 3D modeling, co-immunoprecipitation, pull-down assays, and far-Western assays. In this regard, the modified HA protein may be comprised in a live or attenuated virus, or virus-like particle (VLP).

[0059] In any method used to detect the stability of a modified HA protein or its homotrimer, the level of stability of the modified HA protein can be relative or absolute. In some examples, the level of stability of the modified HA protein is about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, or at least about 500% higher than that observed in a control HA protein (e.g., an unmodified HA protein or a wild-type HA protein, e.g., SEQ ID NO: 12 or SEQ ID NO: 28).

[0060] The modified HA proteins described herein may be considered isolated. For purposes of this invention, "isolated" refers to material that has been removed from its natural state or that has otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free of components that normally accompany the material in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany the material in its natural state. Isolated material may be in native, chemically synthesized, or recombinant form.

[0061] By "protein" is meant an amino acid polymer. As is well understood in the art, the amino acids can be natural or unnatural amino acids, D-amino acids or L-amino acids.

[0062] The term "protein" includes and encompasses "peptide," which is typically used to refer to proteins having 50 or fewer amino acids (e.g., 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, or 50 or fewer amino acids, and any range therein), and "polypeptide," which is typically used to describe proteins having more than 50 amino acids.

[0063] In particular examples, the modified HA protein comprises about 500 to about 600 amino acid residues, more specifically, about 510 to about 590 amino acid residues, even more specifically, about 520 to about 580 amino acid residues, even more specifically, about 530 to about 570 amino acid residues, or even more specifically, about 540 to about 570 amino acid residues (e.g., about 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 555, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570 amino acid residues, or any range therein).

[0064] It is contemplated that the modified HA proteins described herein preferably do not comprise or consist of the amino acid sequence of a wild-type HA protein, such as a wild-type HA protein of the H2 subtype. In particular examples, the modified HA proteins described herein have at least 70% or 75%, more particularly at least 80% or 85%, or even more particularly at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a reference unmodified or wild-type amino acid sequence of an HA protein, such as that set forth in SEQ ID NO: 12 or alternatively SEQ ID NO: 28.

[0065] Variants of the modified HA proteins described herein are contemplated by the present disclosure. As used herein, a protein, polypeptide, or peptide "variant" shares a definable amino acid sequence relationship with a reference amino acid sequence. In particular examples, the reference amino acid sequence is that of a wild-type HA protein or a modified HA protein, and includes the mature amino acid sequence (i.e., without the signal sequence) and the amino acid sequence including the signal sequence (i.e., the full-length amino acid sequence). The reference amino acid sequence may be, for example, the amino acid sequence of any one of SEQ ID NOs: 12 or 28. A "variant" protein, polypeptide, or peptide may have one or more amino acids of the reference amino acid sequence deleted or substituted with different amino acids. Such modified HA protein variants may, for example, include amino acid residues and / or amino acid sequences of naturally occurring variants and orthologs of HA proteins (e.g., those derived from different influenza strains or different host animals), including consensus sequences. In this regard, it is contemplated that one or more of the amino acid changes described herein (e.g., V39I, L219P, V233A, V320A, V320I, K383E, I388T, N390I, K391R, K391N, V392A, S394Y, A405T, R416G, D430N, and F450S of a full-length HA protein of the H2 subtype) may be introduced into any HA protein known in the art. Additionally, it is contemplated that some amino acids may be substituted or deleted (i.e., conservative substitutions) without altering the activity of the variant protein. Thus, one or more residues of the modified HA proteins described herein, e.g., those defined by SEQ ID NO: 12 or 28, may be conservatively modified (e.g., by amino acid substitution or deletion) to substantially retain the function and / or immunogenicity of the modified HA protein. Furthermore, the modified HA protein may contain additional amino acid changes outside the trimer interface region, for example, in one or more of its hypervariable regions (e.g., the hypervariable head region), as is well known in the art.Such additional amino acid changes can include, for example, changes at V129 (eg, V129I) and / or E184 (eg, E184K) of the full-length H2 HA protein described herein.

[0066] It is envisaged that protein or peptide variants will have at least 70% or 75%, more particularly at least 80% or 85%, or even more particularly at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a reference amino acid sequence such as, for example, that set forth in SEQ ID NO: 12 or alternatively SEQ ID NO: 28.

[0067] Terms generally used herein to describe the sequence relationships between respective proteins and nucleic acids include "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." Because each nucleic acid / protein may contain (1) only one or more portions of the complete nucleic acid / protein sequence shared by the nucleic acids / proteins, and (2) one or more portions that are diverse between the nucleic acids / proteins, sequence comparison is typically performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of, for example, 6, 9, 12, or 20 contiguous residues that are compared to a reference sequence. The comparison window may include no more than about 20% additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the respective sequences. Optimal sequence alignment for aligning a comparison window can be performed by computer implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Geneworks program by Intelligenetics, Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI, USA, which are incorporated herein by reference), or by inspecting and selecting the best alignment (i.e., the one that results in the highest percentage of homology over the comparison window) generated by any of a variety of methods. Reference can also be made to the BLAST family of programs, for example, as disclosed by Altschul et al., 1991, Nucl. Acids Res. 25 3389 (which are incorporated herein by reference). A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

[0068] The term "sequence identity" is used herein in its broadest sense to include the exact number of nucleotide or amino acid matches, taking into account the extent to which sequences are identical across a comparison window, taking into account appropriate alignment using standard algorithms. Thus, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions where identical nucleic acid bases (e.g., A, T, C, G, I) or amino acid residues occur in both sequences to obtain the number of identical positions, dividing the number of identical positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. For example, "sequence identity" can be understood to mean the "percentage of matches" calculated by the DNASIS computer program (version 2.5 for Windows; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA).

[0069] It is contemplated that the trimer interface region may be modified at any amino acid residue therein, as is well known in the art. The modifications described herein may include, but are not limited to, deletions, additions, and substitutions in the amino acid sequence of the HA protein. For example, one class of substitutions is conservative amino acid substitutions. Such substitutions replace a given amino acid in the HA protein with another amino acid of similar characteristics. Conservative substitutions typically include substitutions among the aliphatic amino acids Ala, Val, Leu, and Ile for each other, exchanges of hydroxyl residues Ser and Thr, exchanges of acidic residues Asp and Glu, substitutions between amide residues Asn and Gln, exchanges of basic residues Lys and Arg, and substitutions between aromatic residues Phe and Tyr. Guidance regarding which amino acid changes are considered phenotypically silent can be found, for example, in Bowie et al., Science 247:1306-1310 (1990).

[0070] In certain instances, the modified HA protein comprises a deletion of one or more amino acid residues in the trimer interface region, such as those described herein. The term "deletion" refers to the removal of one or more (or a specified number of) consecutive amino acids from the respective peptide, polypeptide, or protein.

[0071] According to particular examples, the modified HA protein comprises a mutation or substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the trimer interface region, such as those described herein. As used herein, "substituted" and "substitution" refer to the substitution(s) of an amino acid residue in a parent or reference sequence, such as SEQ ID NO: 12, or alternatively, SEQ ID NO: 28. In some examples, the substitution comprises the substitution of a naturally occurring or conserved residue. The modified HA protein encompasses the substitution of one or more amino acid residues in the trimer interface region, as described herein, with any one of the remaining 19 amino acids. In particular examples, the modified HA protein comprises the substitution of an amino acid of a certain size with an amino acid of a different size (e.g., substitution of an amino acid with an amino acid of a relatively larger or smaller size), substitution of an amino acid of a certain hydrophilicity with an amino acid of a different hydrophilicity, substitution of an amino acid of a certain polarity with an amino acid of a different polarity, and / or substitution of an amino acid of a certain acidity with an amino acid of a different acidity. The modification in the modified HA protein may be or may include substituting an amino acid at a particular position identified in any of the examples herein with a respective replacement amino acid identified in any of the examples herein.

[0072] As described above, the modified HA protein may be of the H2 subtype. In this regard, and according to a specific example, the one or more amino acid residue positions in the trimer interface region to be modified, such as by substitution, in the modified HA protein are selected from the group consisting of 39, 219, 233, 320, 383, 388, 390, 391, 392, 394, 405, 416, 430, 450, and any combination thereof (e.g., V39, L219, V233, V320, K383, I388, N390, K391, V392, S394, A405, R416, D430, F450, and any combination thereof), where the amino acid numbering is based on the full-length H2 amino acid sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 28, i.e., the amino acid numbering is based on the first methionine (methionine at position 1, M1) being the first residue). In other examples, the one or more amino acid residues in the trimer interface region that are modified are selected from the group consisting of V39, K383, I388, N390, K391, V392, S394, F450, and any combination thereof, where the amino acid numbering is based on the full-length H2 amino acid sequence. In some examples, the modified HA protein includes one or more of the following substitutions or mutations according to the amino acid numbering of the full-length H2 HA protein (e.g., SEQ ID NO: 12 or SEQ ID NO: 28): 39I, 219P, 233A, 320A, 320I, 383E, 388T, 390I, 391R, 391N, 392A, 394Y, 405T, 416G, 430N, and 450S. In particular examples, the modified HA protein includes one or more of the following substitutions or mutations according to the amino acid numbering of the full-length H2 HA protein (e.g., SEQ ID NO: 12 or SEQ ID NO: 28): V39I, L219P, V233A, V320A, V320I, K383E, I388T, N390I, K391R, K391N, V392A, S394Y, A405T, R416G, D430N, and F450S.In certain examples, the modified HA protein comprises one or more of the following substitutions or mutations according to the amino acid numbering of a full-length H2 HA protein (e.g., SEQ ID NO: 12 or SEQ ID NO: 28): 39I, 383E, 388T, 390I, 391R, 391N, 392A, 394Y, and 450S. In various examples, the modified HA protein comprises one or more of the following substitutions or mutations according to the amino acid numbering of a full-length H2 HA protein (e.g., SEQ ID NO: 12 or SEQ ID NO: 28): V39I, K383E, I388T, N390I, K391R, K391N, V392A, S394Y, and F450S.

[0073] According to certain examples, the modified HA protein comprises, consists of, or consists essentially of an amino acid sequence selected from SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68, or a fragment, variant, or derivative thereof.

[0074] Preferably, the modified HA protein comprises a modification, such as a substitution, at a position corresponding to position 405 of the full-length H2 HA protein. More specifically, the modified HA protein preferably comprises a modification, such as a substitution, of an alanine at a position corresponding to position 405 of the full-length H2 HA protein. In various examples, the modified HA protein comprises a substitution of threonine for alanine at a position corresponding to position 405 of the full-length H2 HA protein (i.e., A405T). More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 38, or a fragment, variant, or derivative thereof. Influenza virus isolates (e.g., reassortant influenza viruses) expressing such modified HA proteins may be particularly suitable or suited for large-scale production thereof.

[0075] Preferably, the modified HA protein comprises a modification, such as a substitution, at a position corresponding to position 39 of the full-length H2 HA protein. More specifically, the modified HA protein preferably comprises a modification, such as a substitution, of valine at a position corresponding to position 39 of the full-length H2 HA protein. In one particular example, the modified HA protein comprises a substitution of isoleucine for valine at a position corresponding to position 39 of the full-length H2 HA protein (i.e., V39I). More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 42, or a fragment, variant, or derivative thereof.

[0076] Preferably, the modified HA protein comprises a modification, such as a substitution, at a position corresponding to position 394 of the full-length H2 HA protein and / or at a position corresponding to position 416. More specifically, the modified HA protein preferably comprises a modification, such as a substitution, of one or more of a serine at a position corresponding to position 394 of the full-length H2 HA protein and an arginine at a position corresponding to position 416. In one particular example, the modified HA protein comprises the following substitutions: (a) a tyrosine instead of a serine at the position corresponding to position 394 (i.e., S394Y), and / or (b) Alanine instead of arginine at the position corresponding to position 416 (i.e., R416G).

[0077] In some examples, the modified HA protein comprises a modification of one or more amino acid residues at the following positions in a full-length H2 HA protein: (a) S394, (b) R416, or (c) S394 and R416. In other examples, the modified HA protein comprises a modification of a full-length H2 HA protein at the following positions: (a) S394Y, (b) R416G, or (c) S394Y and R416G. More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 36, or a fragment, variant, or derivative thereof. Furthermore, influenza virus isolates (e.g., reassortant influenza viruses) expressing such modified HA proteins may be particularly suitable or amenable to large-scale production thereof.

[0078] Preferably, the modified HA protein comprises a modification, such as a substitution, at a position corresponding to position 233 of the full-length H2 HA protein and / or at a position corresponding to position 320. More specifically, the modified HA protein preferably comprises a modification, such as a substitution, of one or more of a valine at a position corresponding to position 233 of the full-length H2 HA protein and a valine at a position corresponding to position 320. In one particular example, the modified HA protein comprises the following substitution: (a) an alanine instead of a valine at the position corresponding to position 233 (i.e., V233A), and / or (b) Alanine instead of valine at the position corresponding to position 320 (i.e., V320A).

[0079] In some examples, the modified HA protein comprises a modification of one or more amino acid residues at the following positions in a full-length H2 HA protein: (a) V233, (b) V320, or (c) V233 and V320. In other examples, the modified HA protein comprises a modification of the following positions in a full-length H2 HA protein: (a) V233A, (b) V320A, or (c) V233A and V320A. More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 40, or a fragment, variant, or derivative thereof.

[0080] Preferably, the modified HA protein comprises a modification, such as a substitution, at a position corresponding to position 383 of the full-length H2 HA protein. More specifically, the modified HA protein preferably comprises a lysine modification, such as a substitution, at a position corresponding to position 383 of the full-length H2 HA protein. In various examples, the modified HA protein comprises a substitution of glutamic acid for lysine at a position corresponding to position 383 of the full-length H2 HA protein (i.e., K383E). More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 48, or a fragment, variant, or derivative thereof.

[0081] Preferably, the modified HA protein comprises a modification, such as a substitution, at a position corresponding to position 320, a position corresponding to position 390, and / or a position corresponding to position 391 of the full-length H2 HA protein. More specifically, the modified HA protein preferably comprises a modification, such as a substitution, of one or more of a valine at a position corresponding to position 320, an asparagine at a position corresponding to position 390, and a lysine at a position corresponding to position 391 of the full-length H2 HA protein. In certain examples, the modified HA protein comprises the following substitutions: (a) isoleucine instead of valine at the position corresponding to position 320 (i.e., V320I); (b) isoleucine instead of asparagine at the position corresponding to position 390 (i.e., N390I), and / or (c) Arginine instead of lysine at the position corresponding to position 391 (i.e., K391R).

[0082] In some examples, the modified HA protein comprises a modification of one or more amino acid residues at the following positions of a full-length H2 HA protein: (a) V320, (b) N390, (c) K391, (d) V320 and N390, (e) V320 and K391, (f) N390 and K391, or (g) V320, N390, and K391. In other examples, the modified HA protein comprises a modification of a full-length H2 HA protein at: (a) V320I, (b) N390I, (c) K391R, (d) V320I and N390I, (e) V320I and K391R, (f) N390I and K391R, or (g) V320I, N390I, and K391R. More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 46, or a fragment, variant, or derivative thereof.

[0083] Preferably, the modified HA protein comprises a modification, such as a substitution, at a position corresponding to position 219 of the full-length H2 HA protein. More specifically, the modified HA protein preferably comprises a modification, such as a substitution, of leucine at a position corresponding to position 219 of the full-length H2 HA protein. In various examples, the modified HA protein comprises a substitution of proline for leucine at a position corresponding to position 219 of the full-length H2 HA protein (i.e., L219P). More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 50, or a fragment, variant, or derivative thereof.

[0084] Preferably, the modified HA protein comprises a modification, such as a substitution, at a position corresponding to position 450 of the full-length H2 HA protein and / or at a position corresponding to position 391. More specifically, the modified HA protein preferably comprises a modification, such as a substitution, of one or more of a phenylalanine at a position corresponding to position 450 of the full-length H2 HA protein and a lysine at a position corresponding to position 391. In one particular example, the modified HA protein comprises the following substitutions: (a) alanine instead of valine at the position corresponding to position 233 (i.e., V233A); (b) serine instead of phenylalanine at the position corresponding to position 450 (i.e., F450S), and / or (c) asparagine instead of lysine at the position corresponding to position 391 (i.e., K391N);

[0085] In some examples, the modified HA protein comprises modifications of one or more amino acid residues at the following positions of a full-length H2 HA protein: (a) V233, (b) F450, (c) K391, (d) V233 and F450, (e) V233 and K391, (f) F450 and K391, or (g) V233, F450, and K391. In other examples, the modified HA protein comprises modifications of a full-length H2 HA protein at the following positions: (a) V233A, (b) F450S, (c) K391N, (d) V233A and F450S, (e) V233A and K391N, (f) F450S and K391N, or (g) V233A, F450S, and K391N. More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 54, or a fragment, variant, or derivative thereof.

[0086] Preferably, the modified HA protein comprises a modification, such as a substitution, at a position corresponding to position 450 of the full-length H2 HA protein and / or at a position corresponding to position 391. More specifically, the modified HA protein preferably comprises a modification, such as a substitution, of one or more of a phenylalanine at a position corresponding to position 450 of the full-length H2 HA protein and a lysine at a position corresponding to position 391. In one particular example, the modified HA protein comprises the following substitutions: (a) serine instead of phenylalanine at the position corresponding to position 450 (i.e., F450S), and / or (b) Asparagine instead of lysine at the position corresponding to position 391 (i.e., K391N).

[0087] In some examples, the modified HA protein comprises a modification of one or more amino acid residues at the following positions in a full-length H2 HA protein: (a) F450, (b) K391, or (c) F450 and K391. In other examples, the modified HA protein comprises a modification of a full-length H2 HA protein at the following positions: (a) F450S, (b) K391N, or (c) F450S and K391N. More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 52, or a fragment, variant, or derivative thereof.

[0088] Preferably, the modified HA protein comprises a modification, such as a substitution, at a position corresponding to position 39 of the full-length H2 HA protein and / or at a position corresponding to position 430. More specifically, the modified HA protein preferably comprises a modification, such as a substitution, of one or more of a valine at a position corresponding to position 39 of the full-length H2 HA protein and an aspartic acid at a position corresponding to position 430. In one particular example, the modified HA protein comprises the following substitutions: a valine at a position corresponding to position 39 of the full-length H2 HA protein and / or an aspartic acid at a position corresponding to position 430 of the full-length H2 HA protein. (a) isoleucine instead of valine at the position corresponding to position 39 (i.e., V39I), and / or (b) asparagine instead of aspartic acid at the position corresponding to position 430 (i.e., D430N);

[0089] In some examples, the modified HA protein comprises a modification of one or more amino acid residues at the following positions in a full-length H2 HA protein: (a) V39, (b) D430, or (c) V39 and D430. In other examples, the modified HA protein comprises a modification of a full-length H2 HA protein at the following positions: (a) V39I, (b) D430N, or (c) V39I and D430N. More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 56, or a fragment, variant, or derivative thereof.

[0090] Preferably, the modified HA protein comprises a modification, such as a substitution, at a position corresponding to position 39, a position corresponding to position 388, and / or a position corresponding to position 392 of the full-length H2 HA protein. More specifically, the modified HA protein preferably comprises a modification, such as a substitution, of one or more of a valine at a position corresponding to position 39 of the full-length H2 HA protein, an isoleucine at a position corresponding to position 388, and a valine at a position corresponding to position 392 of the full-length H2 HA protein. In certain examples, the modified HA protein comprises the following substitutions: (a) isoleucine instead of valine at the position corresponding to position 39 (i.e., V39I); (b) a threonine instead of an isoleucine at the position corresponding to position 388 (i.e., I388T), and / or (c) Alanine instead of valine at the position corresponding to position 392 (i.e., V392A).

[0091] In some examples, the modified HA protein comprises modifications of one or more amino acid residues at the following positions of a full-length H2 HA protein: (a) V39, (b) I388, (c) V392, (d) V39 and I388, (e) V39 and V392, (f) I388 and V392, or (g) V39, I388, and V392. In other examples, the modified HA protein comprises modifications of a full-length H2 HA protein at the following positions: (a) V39I, (b) I388T, (c) V392A, (d) V39I and I388T, (e) V39I and V392A, (f) I388T and V392A, or (g) V39I, I388T, and V392A. More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 58, or a fragment, variant, or derivative thereof.

[0092] Preferably, the modified HA protein comprises a modification, such as a substitution, at a position corresponding to position 39, 391, and / or 430 of the full-length H2 HA protein. More specifically, the modified HA protein preferably comprises a modification, such as a substitution, of one or more of a valine at a position corresponding to position 39, a lysine at a position corresponding to position 391, and an aspartic acid at a position corresponding to position 430 of the full-length H2 HA protein. In certain examples, the modified HA protein comprises the following substitutions: (a) isoleucine instead of valine at the position corresponding to position 39 (i.e., V39I); (b) an asparagine instead of a lysine at the position corresponding to position 391 (i.e., K391N), and / or (c) asparagine instead of aspartic acid at the position corresponding to position 430 (i.e., D430N).

[0093] In some examples, the modified HA protein comprises a modification of one or more amino acid residues at the following positions of a full-length H2 HA protein: (a) V39, (b) K391, (c) D430, (d) V39 and K391, (e) V39 and D430, (f) K391 and D430, or (g) V39, K391, and D430. In other examples, the modified HA protein comprises a modification of a full-length H2 HA protein at the following positions: (a) V39I, (b) K391N, (c) D430N, (d) V39I and K391N, (e) V39I and D430N, (f) K391N and D430N, or (g) V39I, K391N, and D430N. More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 60, or a fragment, variant, or derivative thereof.

[0094] Additionally, modified HA proteins having one or more additional amino acid modifications or substitutions at other positions compared to the respective wild-type HA proteins are contemplated. Thus, modified HA proteins may have at least about 2, 3, 4, 5, 6, 7, or more different residues at other positions compared to the respective wild-type HA proteins. As will be understood by those skilled in the art, the number of additional positions that may have amino acid substitutions depends on the wild-type HA protein or encoding nucleic acid used to generate the variant. To this end, the modified HA proteins provided herein may be derived from any known HA sequence derived from influenza isolates known in the art. For example, the National Center for Biotechnology Information (NCBI) maintains a database of known HA sequences (https: / / www.ncbi.nlm.nih.gov / genomes / FLU / Database / ). Additionally, a database of influenza HA wild-type or MDCK cell and egg passage sequences is available from the Global Initiative on Sharing All Influenza Database (GISAID) EpiFlu database.

[0095] Modified HA proteins can be produced by any means known in the art, including, but not limited to, chemical synthesis, recombinant DNA techniques including site-directed mutagenesis, or replacing a portion of the HA coding sequence with a portion containing a hallmark residue(s) and proteolytic cleavage to generate peptide fragments.

[0096] Chemical synthesis includes solid-phase synthesis and liquid-phase synthesis. Such methods are well known in the art, and reference is made to examples of chemical synthesis techniques provided in Chapter 9 of Synthetic Vaccines Ed. Nicholson (Blackwell Scientific Publications) and Chapter 15 of Current Protocols in Protein Science Eds. Coligan et al. (John Wiley & Sons, Inc. NY USA 1995-2008). In this regard, reference is also made to International Publication Nos. WO99 / 02550 and WO97 / 45444.

[0097] Recombinant proteins can be conveniently prepared by those skilled in the art using standard protocols as described, for example, in Sambrook et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), especially Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. NY USA 1995-2008), especially Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. NY USA 1995-2008), especially Chapters 1, 5, and 6. Typically, recombinant protein preparation involves expression of a nucleic acid encoding the protein in a suitable host cell. Modified HA proteins can be obtained, for example, by mutating a gene or genes (i.e., viral gene segments) encoding the HA protein of interest by site-directed or random mutagenesis. Such mutations can include point mutations, deletion mutations, and insertion mutations. For example, one or more point mutations (e.g., substitution of one or more amino acids with one or more different amino acids) can be used to construct the modified HA proteins described herein.

[0098] According to particular examples, the modified HA protein has been modified or mutated by one or more passages (e.g., 1, 2, 3, 4, 5, 6, 7, etc.), e.g., serial passaging, in cells and / or eggs such as those provided herein, of an influenza virus isolate expressing an unmodified or wild-type HA protein. In some examples, the modified HA protein has been modified or mutated by one or more passages (e.g., 1, 2, 3, 4, 5, 6, 7, etc.) of an influenza virus isolate expressing an unmodified or wild-type HA protein in mammalian cells, such as MDCK cells.

[0099] Suitably, the modified HA proteins described herein are immunogenic. Thus, the modified HA proteins may be suitable for use as immunogens in vaccines for treating or preventing influenza virus infection in humans or animals (e.g., avian animals or pigs). As used herein, the term "immunogenic" will be understood to mean that the composition induces or generates an immune response.

[0100] In certain examples, the modifications provided herein do not, or do not substantially, alter or modulate (i.e., increase or decrease) the immunogenicity / antigenicity of the modified HA protein (e.g., relative to or compared to a wild-type or unmodified version thereof). To this end, it should be noted that the trimer interface region is internalized upon HA trimer formation and is typically not accessible by the host's immune system during influenza virus infection. The immunogenicity or antigenicity of a modified HA protein can be assessed by any means known in the art, for example, by determining the presence or amount of neutralizing antibodies or antibodies that recognize the modified HA protein (e.g., in a trimeric configuration) using standard immunoassays and / or by determining predicted or actual T-cell reactivity.

[0101] It is further contemplated that the modified HA protein may contain one or more additional modifications known in the art. For example, the modified HA protein may be further modified to remove a determinant that makes the virus highly pathogenic (e.g., a highly basic region around the HA1 / HA2 cleavage site). In some examples, the modified HA protein is further modified to remove a polybasic cleavage site therein. This site allows for trypsin-independent HA maturation, typically defining "highly pathogenic" versus "lowly pathogenic" avian influenza.

[0102] Modified HA proteins can also be engineered to constitute chimeric HA proteins (i.e., contain amino acid sequences from multiple influenza strains). For example, preferably, in addition to one or more modified amino acid residues in the trimer interface region, the chimeric HA can contain the cytoplasmic portion, or the cytoplasmic and transmembrane portions, of HA from one influenza strain and at least the extracellular antigenic portion of HA from a different influenza strain. This approach has been previously described as a technique for producing influenza viruses containing antigenic portions of HA proteins in situations where unmodified HA segments may be produced in low yield.

[0103] In other examples, the modified HA protein is a non-chimeric HA protein. In other words, the HA sequence comprises cytoplasmic, transmembrane, and extracellular domains from the same influenza strain. In such examples, the modified HA protein sequence is a non-chimeric sequence, but may include other modifications described herein.

[0104] Coding nucleic acids The present disclosure also provides isolated nucleic acids encoding the modified HA proteins described herein.

[0105] The term "nucleic acid" as used herein refers to single-stranded or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA, and autocatalytic RNA. Nucleic acids can also be DNA-RNA hybrids. Nucleic acids typically include nucleotide sequences containing nucleotides containing A, G, C, T, or U bases. However, nucleotide sequences can include other bases, such as modified purines (e.g., inosine, methylinosine, and methyladenosine) and modified pyrimidines (e.g., thiouridine and methylcytosine).

[0106] It is contemplated that the encoding nucleic acids described herein encode the modified HA proteins of the present disclosure directly, e.g., via a viral segment or viral mRNA, or indirectly, e.g., via a viral segment or a complementary DNA sequence encoding the viral segment. In particular examples, the isolated nucleic acid is or comprises an HA viral segment (i.e., an influenza RNA segment) encoding a modified HA protein provided herein. Suitably, the HA viral segment comprises, consists of, or consists essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, or 67, or a fragment, derivative, or variant thereof. In some examples, the isolated nucleic acid is or comprises a nucleotide sequence complementary to an HA viral segment encoding a modified HA protein provided herein. In alternative examples, the isolated nucleic acid is or comprises a DNA or cDNA sequence encoding an HA viral segment (i.e., viral RNA) encoding a modified HA protein provided herein. In various examples, the isolated nucleic acid is or includes a viral mRNA sequence that encodes a modified HA protein provided herein.

[0107] As used herein, a "polynucleotide" generally refers to a nucleic acid having 80 or more contiguous nucleotides, while an "oligonucleotide" generally refers to an oligonucleotide having fewer than 80 contiguous nucleotides. A "primer" is usually a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, that is capable of annealing to a complementary nucleic acid "template" and being extended in a template-dependent manner by the action of a DNA polymerase (such as Taq polymerase, reverse transcriptase, or Sequenase™). A "probe" can be a single- or double-stranded oligonucleotide or polynucleotide, suitably labeled, for the purpose of detecting complementary sequences, e.g., in Northern or Southern blotting.

[0108] Also contemplated herein are isolated nucleic acid fragments, variants, and derivatives. Variants can include nucleotide sequences that have at least 70%, at least 75%, preferably at least 80%, at least 85%, and more preferably at least 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity to any nucleotide sequence encoding a variant or modified HA protein of the present disclosure (e.g., SEQ ID NOs: 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, or 67). Nucleic acid derivatives can include chemically modified nucleic acids, modified internucleotide linkages, nucleic acid analogs, artificial nucleic acids, and combinations thereof, as known in the art.

[0109] Isolated nucleic acid fragments can comprise or consist of up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95-99% of the contiguous nucleotides present in any nucleotide sequence encoding a modified HA protein of the present disclosure, such that they encode at least a portion of the modified HA protein (e.g., encode at least a portion of the trimer interface region of the HA protein). Generally, fragments encode portions of the modified HA proteins described herein (e.g., SEQ ID NOs: 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, or 67) up to 150, 165, 180, 195, 210, 225, 240, 255, 270, 285, 300, 315, 330, 345, 360, 375, 390, 405, 420, 435, 450, 465, 480, 495, 500, 515, 520, 535, 540, 550, 565, 570, 580, 595, 600, 615, 620, 635, 640, 655, 660, 675, 680, 695, 700, 715, 720, 735, 740, 755, 760, 775, 780, 795, 800, 815, 820, 835, 840, 855, 860, 875, 880, 895, 900, 915, 920, 935, 940, 955, 960, 975, 980, 995, 1000, 1015, 1020, 1035, 1040, 1055, 1060, 1 It may comprise, consist essentially of, or consist of 10, 525, 540, 555, 570, 585, 600, 615, 630, 645, 660, 675, 690, 705, 720, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800 contiguous nucleic acids.

[0110] In particular examples, the isolated nucleic acids described herein can be modified to include, or alternatively not include, the 5' non-coding region (e.g., AGC[A / G]AAAGCAGG (SEQ ID NO: 73), where [A / G] indicates an A or G nucleotide mutation at that position) and / or the 3' non-coding region (e.g., CCTTGTTTCTACT (SEQ ID NO: 74)) of an influenza virus, as known in the art.

[0111] The present disclosure also provides nucleic acids modified by exploiting codon sequence redundancy. In more specific examples, codon usage can be modified to optimize expression of the nucleic acid in a particular organism or cell type.

[0112] The isolated nucleic acids disclosed herein can be conveniently prepared using standard protocols, such as those described in Chapters 2 and 3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al. John Wiley & Sons NY, 1995-2008).

[0113] Nucleic acids of the present disclosure can be produced, isolated, detected, and / or subjected to recombinant DNA techniques using nucleic acid sequence amplification methods.

[0114] Suitable nucleic acid amplification techniques, covering both thermal and isothermal methods, are well known to those skilled in the art and include, but are not limited to, polymerase chain reaction (PCR); strand displacement amplification (SDA); rolling circle replication (RCR); nucleic acid sequence-based amplification (NASBA), Q-beta replicase amplification, recombinase polymerase amplification, and helicase-dependent amplification (RPA).

[0115] Gene constructs The present disclosure also provides a genetic construct comprising the aforementioned isolated nucleic acid herein. The genetic construct may be a vector.

[0116] In certain examples, a genetic construct comprises an isolated nucleic acid operably linked or connected to one or more other genetic components. The genetic construct may be suitable for therapeutic delivery of the isolated nucleic acid (e.g., a DNA or RNA vaccine) or for recombinant production of the modified HA protein of the present disclosure in a host cell. Furthermore, the genetic construct may be used to produce or generate influenza viruses (e.g., variant or modified strains of influenza virus isolates or reassortant influenza virus isolates) that express the modified HA protein.

[0117] Generally, the genetic construct will be in the form of, or contain genetic components of, a plasmid, bacteriophage, cosmid, yeast, or bacterial artificial chromosome, as is well understood in the art. The genetic construct may be suitable for maintenance and propagation of isolated nucleic acids in bacteria or other host cells for manipulation by recombinant DNA technology and / or expression of the nucleic acids or encoded proteins of the present disclosure. The vector may also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA in the same strand, poly-lysine-linked DNA or RNA, peptide-linked DNA or RNA, liposome-linked DNA, etc. Such vectors may or may not be autonomously replicating.

[0118] For purposes of host cell expression, the genetic construct is an expression construct. Preferably, the expression construct comprises a nucleic acid of the present disclosure operably linked to one or more additional sequences in an expression vector. An "expression vector" can be either a self-replicating extrachromosomal vector, such as a plasmid, or a vector that integrates into a host genome. Alternatively, the expression construct can be a linear expression construct. Such linear expression constructs typically do not contain any amplification and / or selection sequences. However, linear constructs containing such amplification and / or selection sequences are also within the scope of the present disclosure. The linear expression construct can, for example, comprise a separate linear expression construct for each viral segment. It is also possible to include multiple viral segments, for example, two, three, four, five, or six, on the same linear expression construct.

[0119] Expression constructs suitable for use in the disclosed methods can be unidirectional or bidirectional. Because influenza viruses require proteins for infectivity, it is generally preferable to use bidirectional expression constructs, as this reduces the total number of expression constructs required by the host cell. A bidirectional expression construct contains at least two promoters that drive expression in different directions (i.e., both 5' to 3' and 3' to 5') from the same construct. The two promoters can be operably linked to different strands of the same double-stranded DNA. Preferably, one of the promoters is a Pol I promoter and at least one of the other promoters is a Pol II promoter. Thus, the disclosed methods can utilize at least one bidirectional expression construct in which at least one gene or cDNA is located between an upstream Pol II promoter and a downstream non-endogenous Pol I promoter. Transcription of the gene or cDNA from the Pol II promoter generates capped, positive-strand viral mRNA that can be translated into protein, while transcription from the non-endogenous Pol I promoter generates negative-strand viral RNA (vRNA).

[0120] By "operably linked" is meant that the additional nucleotide sequence(s) in question are positioned relative to the nucleic acid of the disclosure so as to preferably initiate, regulate, or otherwise control transcription.

[0121] Regulatory nucleotide sequences are generally appropriate for the host cell used for expression. As described herein, numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Expression vectors can be designed for expression of the modified HA proteins described herein using prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., insect cells (using baculovirus expression vectors, see, e.g., Treanor et al., 2007, JAMA, 297(14):1577-1582, incorporated herein by reference in its entirety), yeast cells, plant cells, algae, or mammalian cells).

[0122] Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosomal binding site, a polyadenylation sequence, a transcription start and stop sequence, a translation start and stop sequence, and an enhancer or activator sequence. Constitutive, repressible, or inducible promoters, as known in the art, are contemplated by the present disclosure.

[0123] In some instances, the genetic construct comprises one or more untranslated 5' and / or 3' regions operably linked or connected to the HA viral segment encoding the modified HA protein. To this end, the UTRs can be derived from the same influenza virus isolate as the modified HA protein or from a different influenza virus isolate.

[0124] The expression construct may also include an additional nucleotide sequence (typically provided by the expression vector) encoding a fusion partner so that the recombinant protein is expressed as a fusion protein.

[0125] The expression construct may also include a selectable marker, such as, but not limited to, an amp R , neo R , or kan R The polypeptide may comprise an additional nucleotide sequence encoding the polypeptide.

[0126] Suitably, the genetic constructs provided herein are suitable or compatible for use in producing or generating reassortant influenza viruses that express modified HA proteins by reverse genetics or hybrid reverse genetics-classical reassortment methods. Accordingly, one or more genetic constructs provided herein can be introduced into a host cell using any method for introducing expression construct(s) by known reverse genetics techniques.

[0127] The genetic constructs provided herein can be introduced into host cells using any technique known to those skilled in the art. For example, the genetic constructs can be introduced into host cells using electroporation, DEAE-dextran, calcium phosphate precipitation, liposomes, microinjection, or particle bombardment. In some examples, the genetic constructs can be in the form of naked nucleic acid. The naked nucleic acid can be purified from influenza virus. In another example, the genetic constructs can be in the form of transcribed RNA (e.g., viral mRNA). In other examples, the genetic constructs can be in the form of one or more shuttle vectors. Examples of shuttle vectors include non-influenza viruses and replicons, such as alphavirus-based replicons.

[0128] The gene constructs provided herein may include an RNA transcription termination sequence. The termination sequence may be an endogenous termination sequence or a termination sequence that is not endogenous to the host cell. Suitable termination sequences will be apparent to those skilled in the art and include, but are not limited to, RNA polymerase I transcription termination sequences, RNA polymerase II transcription termination sequences, and ribozymes. Furthermore, the expression construct may include one or more polyadenylation signals for mRNA, particularly at the end of a gene whose expression is controlled by a Pol II promoter.

[0129] In another aspect, the present disclosure also provides a plurality of genetic constructs, including a genetic construct comprising a nucleic acid encoding a modified HA protein described herein and one or more additional genetic constructs that can be utilized in preparing reassortant viruses, including 6:1:1 reassortants, 6:2 reassortants, and 7:1 reassortants. The additional genetic constructs may comprise or encode one or more of the NA, PA, PB1, PB2, NP, NS, and M viral segments (i.e., encoding the NA, PA, PB1, PB1-F2, PB2, NP, NS1, NEP, M1, and M2 viral proteins).

[0130] host cell The present disclosure also provides host cells transformed with the isolated nucleic acids and / or genetic constructs described herein.

[0131] In a related aspect, the present disclosure relates to a method of producing a modified HA protein provided herein, the method comprising the steps of (i) culturing a previously transformed host cell as described herein, and (ii) isolating the modified HA protein from the host cell cultured in step (i).

[0132] The host cell can be any known in the art. One well-known method for propagating influenza virus uses specific pathogen-free (SPF) embryonated chicken eggs, with the virus inoculated into the egg contents (i.e., the allantoic fluid), propagated, and purified therefrom. Influenza virus can also be propagated in animal cell culture, and this culture method is preferred for reasons of replication fidelity, speed, and patient allergies.

[0133] When referring to cells described herein, the methods typically use cell lines, although primary cells can alternatively be used. Such cells or cell lines can be bacteria, insect cells, yeast cells, plant cells, algae, or mammalian cells. Examples of yeast host cells include, but are not limited to, S. pombe and S. cerevisiae. Examples of mammalian host cells include, but are not limited to, Crucell Per.C6 cells, Vero cells, CHO cells, VERY cells, BHK cells, HeLa cells, COS cells, MDCK cells, 293 cells, 3T3 cells, or WI-38 cells. In certain examples, the host cell is a myeloma cell, such as an NSO cell, a 45.6TG1.7 cell, an AF-2 clone 9B5 cell, an AF-2 clone 9B5 cell, a J558L cell, a MOPC315 cell, a MPC-11 cell, an NCI-H929 cell, an NP cell, an NSO / 1 cell, a P3 NS1 Ag4 cell, a P3 / NS1 / 1-Ag4-1 cell, a P3U1 cell, a P3X63Ag8 cell, a P3X63Ag8.653 cell, a P3X63Ag8U.1 cell, an RPMI8226 cell, a Sp20-Ag14 cell, a U266B1 cell, a X63AG8.653 cell, a Y3.Ag.1.2.3 cell, or a YO cell. Non-limiting examples of insect cells include SJ9, SJ21, Trichoplusia ni, Spodoptera fugiperda, and Bombyx mori. Exemplary plant cell systems for expression of modified HA proteins are provided in U.S. Patent Nos. 7,504,560, 6,770,799, 6,551,820, 6,136,320, 6,034,298, 5,914,935, 5,612,487, and 5,484,719, and U.S. Patent Application Publication Nos. 2009 / 0208477, 2009 / 0082548, 2009 / 0053762, 2008 / 0038232, 2007 / 0275014, and 2006 / 0204487.

[0134] In certain examples, the host cell is mammalian. Suitable mammalian cells include, but are not limited to, hamster, bovine, primate (including human and monkey), and canine cells. As known in the art, various cell types, such as kidney cells, fibroblasts, retinal cells, and lung cells, can be used. An example of a suitable hamster cell is a cell line designated BHK21 or HKCC. Suitable monkey cells include African green monkey cells, such as kidney cells of the Vero cell line (Kistner et al. (1998) Vaccine 16:960-8; Kistner et al. (1999) Dev Biol Stand 98:101-110; Bruhl et al. (2000) Vaccine 19:1149-58). Suitable canine cells include canine kidney cells, such as in the CLDK and MDCK cell lines (W097 / 37000, Brands et al. (1999) Dev Biol Stand 98:93-100, Halperin et al. (2002) Vaccine 20:1240-7, Tree et al. (2001) Vaccine 19:3444-50). Thus, suitable cell lines include, but are not limited to, MDCK, CHO, 293T, BHK, Vero, MRC-5, PER.C6, and WI-38 cell lines.

[0135] It is contemplated that the cells or cell lines described herein may be suitable for expressing modified HA proteins, e.g., for producing subunit vaccines containing such proteins. By another example, the cells or cell lines described herein are suitable for propagating influenza virus. Such cell lines may include MDCK cells derived from Madin-Darby canine kidney, Vero cells derived from African green monkey (Cercopithecus aethiops) kidney, or PER.C6 cells derived from human embryonic retinoblasts (Pau et al. (2001) Vaccine 19:2716-21). These cell lines are widely available, such as from the American Type Cell Culture (ATCC) collection, the Coriell Cell Repositories, and the European Collection of Cell Cultures (ECACC). Alternative cell lines may include avian cell lines, including cell lines derived from ducks (e.g., duck retinal cells) or chickens (e.g., chicken embryo fibroblasts (CEF)) (see, e.g., WO2003 / 076601, WO2005 / 042728, WO2003 / 043415). Examples include avian embryonic stem cells, including the EBx cell lines, EB45, EB14, EB14-074, and EB66, which are derived from chicken embryonic stem cells.

[0136] Preferably, the cells or cell line are MDCK cells derived from Madin-Darby canine kidney. The original MDCK cells are available from ATCC as CCL-34. Derivatives of MDCK cells can also be used. For example, MDCK cell lines can be adapted for growth in suspension culture (e.g., "MDCK33016," deposited under DSM ACC 2219). Similarly, WO 2001 / 064846 discloses an MDCK-derived cell line that grows in suspension in serum-free culture ("B-702," deposited under FERM BP-7449). WO2006 / 071563 discloses non-tumorigenic MDCK cells, including "MDCK-S" (ATCC PTA-6500), "MDCK-SF101" (ATCC PTA-6501), "MDCK-SF102" (ATCC PTA-6502), and "MDCK-SF103" (PTA-6503). WO2005 / 113758 discloses MDCK cell lines that are highly susceptible to infection, including "MDCK.5F1" cells (ATCC CRL-12042). Any MDCK cell line, including those provided herein, can be used in the methods of the present disclosure.

[0137] For viral growth or propagation in cell lines such as MDCK cells, influenza virus can be propagated in cells in suspension or adherent culture. Furthermore, the cells described herein can be cultured in various serum-free or substantially serum-free media, as known to those skilled in the art (e.g., Iscove's medium, Ultra CHO medium (BioWhittaker), EX-CELL (JRH Biosciences)). Alternatively, cells for replication can be cultured in serum-containing media (e.g., MEM or DMEM medium containing about 0.5% to about 10%, more specifically about 1.5% to about 5%, fetal bovine serum) or protein-free media (e.g., PF-CHO (JRH Biosciences)). Suitable culture vessels that can be used in the course of the methods described herein can be vessels known to those skilled in the art, such as spinner bottles, roller bottles, or fermenters.

[0138] In certain instances, cells are preferably grown in serum-free culture medium and / or protein-free medium, e.g., to support cell growth and / or influenza virus replication. A medium is referred to as serum-free medium in the context of the present disclosure if it is free of or substantially free of (e.g., less than 0.5%, 0.25%, or 0.1% by weight of) additives derived from serum of human or animal origin. Protein-free refers to a culture medium in which cell growth occurs excluding proteins, growth factors, other protein additives, and non-serum proteins, but can optionally include proteins such as trypsin or other proteases that may be necessary for viral growth. Cells grown in such a medium naturally contain proteins themselves.

[0139] Cell lines that support influenza virus replication are preferably cultured at temperatures below 37°C (e.g., about 30°C to about 36°C, or about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or any range therein) during virus replication. When the virus is grown in a cell line, the culture medium and the viral inoculum used to initiate the culture are preferably free of (e.g., tested negative for) contaminating viruses such as herpes simplex virus, respiratory syncytial virus, parainfluenza virus 3, SARS coronavirus, adenovirus, rhinovirus, reovirus, polyomavirus, birnavirus, circovirus, and / or parvovirus.

[0140] influenza virus In one aspect, the disclosure provides an isolated influenza virus comprising an HA virus segment encoding a modified HA protein, such as those described herein above, comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

[0141] Preferably, the HA viral segment has been modified, such as by one or more passages in cells and / or recombinant methods, to encode a modification to one or more amino acid residues in the trimer interface region. More specifically, the HA viral segment is preferably modified by recombinant methods prior to incorporation into an isolated influenza virus to encode a modified one or more amino acid residues of a modified HA protein. In particular examples, the HA viral segment comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, or 67, or a fragment, variant, or derivative thereof. In various examples, the HA viral segment encodes the amino acid sequence set forth in any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68, or a fragment, variant, or derivative thereof.

[0142] Influenza viruses are enveloped RNA viruses belonging to the family Orthomyxoviridae (Palese and Shaw (2007) Orthomyxoviridae: The Viruses and Their Replication, 5th ed. Fields' Virology, edited by BN Fields, DM Knipe, and PM Howley. Wolters Kluwer Health / Lippincott Williams & Wilkins, Philadelphia, USA, pp. 1647-1689). Influenza A and B viruses are major human pathogens, causing respiratory illnesses ranging in severity from asymptomatic infection to fatal primary viral pneumonia. The clinical effects of infection vary depending on the virulence of the influenza strain and the host's exposure, medical history, age, and immune status. Although the natural host of influenza viruses is primarily birds, influenza viruses, particularly influenza A viruses (including those of avian origin), can also infect and cause disease in humans and other animal hosts (bats, dogs, pigs, horses, marine mammals, and mustelids).

[0143] Influenza viruses referred to herein encompass any virus type, subtype, or strain, including, but not limited to, naturally occurring strains, variants or mutants, mutant viruses, reassortant viruses, and / or genetically modified viruses (e.g., those modified by reverse genetics or recombinant DNA techniques).

[0144] The influenza viruses of the present disclosure may be influenza A viruses or influenza B viruses. According to some examples, the influenza virus is an influenza A virus. In alternative examples, the influenza virus is an influenza B virus. The influenza A or B virus can be any viral strain. By way of example, the influenza A viruses provided herein can comprise an HA subtype selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16. In particular examples, the influenza A virus is of the H2 subtype (i.e., comprises a modified HA protein of the H2 subtype). Furthermore, such viruses can comprise an influenza A virus NA subtype N1, N2, N3, N4, N5, N6, N7, N8, or N9. In some examples, the NA protein is of the N1, N2, N3, or N5 subtype. More specifically, the NA protein can be of the N1, N2, or N3 subtype. In certain examples, the influenza A virus may be a strain selected from the group consisting of H1N1, H1N2, H2N1, H2N2, H2N3, H3N1, H3N2, H3N8, H5N1, H7N1, H7N2, H7N3, H7N7, H9N2, and H10N7. According to some examples, the influenza A virus is an H2N1 strain. In alternative examples, the influenza A virus is an H2N2 strain. In other examples, the influenza A virus is an H2N3 strain.

[0145] In view of the above, the influenza viruses referred to herein are preferably pandemic influenza virus strains. In this regard, the modified HA proteins provided herein may be derived at least in part from pandemic influenza virus strains. As used herein, the term "pandemic influenza virus strain" refers to a strain of influenza virus that is associated with or likely to be associated with influenza outbreaks. Influenza strains that confer the potential to cause a pandemic outbreak are generally characterized by the following: (a) containing a novel hemagglutinin compared to the hemagglutinins of currently circulating human strains, i.e., one that has not been evident in the human population in the past 10 years (e.g., H2) or one that has never been seen in the human population before (e.g., H5, H6, or H9, which are generally only seen in avian populations), and the human population is immunologically naive to the hemagglutinin of the strain; (b) capable of horizontal transmission within the human population; and (c) pathogenic to humans. Thus, modified HA proteins may be suitable for generating reassortant viruses for use in vaccines to protect against potential pandemic virus strains that may spread or have spread to humans from non-human animal populations. Thus, in certain examples, the term pandemic influenza virus strain refers to a strain of influenza A virus. Suitable pandemic strains include, but are not limited to, H5N1, H9N2, H7N7, H2N2, H2N3, H7N1, and H1N1. Other suitable pandemic strains in humans are H7N3, H10N7, and H5N2. In one example, a pandemic strain can be an influenza A H1 subtype other than the (H1N1)pdm09 strain.

[0146] Isolated recombinant influenza viruses are contemplated for the purposes of this disclosure. A "recombinant" virus is one that has been engineered in vitro by introducing changes into the viral genome, such as by using recombinant DNA techniques.

[0147] Preferably, the influenza virus is a reassortant virus, such as a recombinant reassortant virus. The term "reassortant virus" refers to a virus containing genetic material resulting from a combination of genetic material from at least two donor viruses (e.g., one or more gene segments from a first parental influenza virus strain (donor, backbone, or seed strain) and one or more gene segments from a second parental influenza virus strain (vaccine strain)). When a reassortant virus is used to prepare a vaccine composition, the genetic material typically includes at least the HA gene from a seasonal or pandemic influenza virus, while other genes (i.e., backbone genes) are derived from one or several other donor or seed viruses that have been selected for their ability to grow easily in the production substrate (e.g., the allantoic cavity of embryonated chicken eggs or permissive cell lines) used to make influenza vaccines and / or for their low or non-pathogenicity to humans. Examples of donor or seed viruses that serve as backbone gene donors include A / Puerto Rico / 8 / 1934 (PR8), A / Texas / 1 / 1977, A / New York / 55 / 2004, AJ Ann Arbor / 6 / 60, A / Leningrad / 134 / 17 / 57, B / Ann Arbor / 1 / 66, B / Florida / 4 / 2006, B / Panama / 45 / 1990, and B / Lee / 1940. Reassortant viruses can be produced by any method known in the art, including reverse genetics, classical reassortment, and hybrids thereof.

[0148] The influenza donor strain is typically the strain that provides the backbone segments in the reassortant influenza virus, although in some cases it may also provide the NA segment of the virus. The vaccine strain is the influenza strain that provides the HA and / or NA segment. Generally, both the HA and NA segments of the reassortant influenza virus are derived from the vaccine strain. The vaccine strain is usually an epidemic strain, such as a seasonal or pandemic influenza virus strain. Preferably, the vaccine strain is different or heterologous from the donor strain. The genome segments present in the reassortant virus can be described using a genotype ratio, which indicates the number of segments provided by each parent influenza virus strain. For example, if a reassortant virus contains genome segments from two parent influenza virus strains (e.g., a donor strain and a vaccine strain), it can have a genotype ratio of 1:7, 2:6, 3:5, 4:4, 5:3, 6:2, or 7:1.

[0149] Generally, the majority of the gene segments of a reassortant virus are derived from a donor strain because it is desirable to take advantage of the properties of the donor strain (e.g., improved replication and / or yield in cell culture) by reassorting segments of the donor strain with segments from the vaccine strain. In particular examples, the reassortant influenza viruses produced by the methods provided herein have a genotype ratio of 5:3, 6:2, or 7:1, where the first number in the ratio indicates the number of segments from the donor strain and the second number in the ratio indicates the number of segments from the vaccine strain.

[0150] In particular examples, the donor strain is a strain that has regulatory approval for use in vaccine production. Using a donor strain that has regulatory approval is advantageous, as the reassortant viruses produced by the methods provided herein may be used to prepare vaccines, which may be more easily commercialized than if the donor strain did not have existing regulatory approval.

[0151] In certain examples, the reassortant influenza viruses have a 6:2 genogroup ratio. In these examples, the reassortant influenza viruses include six backbone segments (i.e., PB1, PB2, PA, NP, M, and NS) from the donor strain and two segments (i.e., HA and NA) from the vaccine strain. In such examples, the HA viral segment encodes a modified HA protein provided herein.

[0152] In other examples, the reassortant influenza virus has a 7:1 genotype ratio. In such examples, the reassortant influenza virus can include six backbone segments from the donor strain, an HA segment from the vaccine strain, and an NA segment from the donor strain. In other words, the reassortant influenza virus includes an HA segment from the vaccine strain, and the remaining seven segments are from the donor strain. In an alternative example, the reassortant influenza virus includes six backbone segments and an HA segment from the donor strain, and an NA segment from the vaccine strain. In other words, the reassortant influenza virus includes an NA segment from the vaccine strain and the remaining seven segments from the donor strain.

[0153] In further examples, the reassortant influenza virus has a 5:3 genogroup ratio. In these examples, the reassortant virus may include five backbone segments from the donor strain (i.e., five segments selected from the group consisting of PB1, PB2, PA, NP, M, and NS) and three segments from the vaccine strain. In such examples, the three segments from the vaccine strain are typically HA, NA, and one backbone segment (i.e., one segment selected from the group consisting of PB1, PB2, PA, NP, M, and NS). In a particular example, the three segments from the vaccine strain are HA, NA, and PB1, and the remaining five backbone segments (i.e., PB2, PA, NP, M, and NS) are from the donor strain.

[0154] According to particular examples, isolated reassortant influenza viruses include: (a) one or more PA, PB1, PB2, NP, NS, and M viral segments derived from a first influenza virus isolate (e.g., a donor virus); (b) an HA viral segment (including chimeric forms thereof) derived from a second influenza virus isolate (e.g., vaccine virus), the HA viral segment being modified to encode a modification to one or more amino acid residues in the trimer interface region thereof; and (c) optionally, an NA virus segment (including chimeric forms thereof) derived from a first influenza virus isolate, a second influenza virus isolate, or a third influenza virus isolate.

[0155] For this purpose, the NA viral segment and the HA viral segment encoding the modified HA protein can be derived from the same influenza virus isolate. Alternatively, the NA viral segment can be derived from the same influenza virus isolate (e.g., donor virus) as the backbone viral segment. Furthermore, the NA viral segment can be derived from a different influenza virus isolate from which the backbone and HA viral segments are derived.

[0156] Preferably, the isolated influenza viruses provided herein can grow or replicate in cells, more particularly, mammalian cells such as MDCK cells. To this end, the isolated influenza viruses can be capable of forming virions having a stabilized or more stable HA trimer (e.g., compared to an isolated influenza virus having an unmodified HA viral segment encoding an unmodified HA protein) when cultured in cells. In certain examples, the isolated influenza viruses provided herein can have enhanced or improved growth or replication in cells such as MDCK cells when compared to an isolated influenza virus having an unmodified HA viral segment encoding an unmodified HA protein.

[0157] Thus, in another aspect of the disclosure, there is provided a method for improving the growth of influenza virus in a cell, the method comprising modifying an influenza virus to express a modified HA protein, such as those described herein, comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

[0158] It is contemplated that the methods of the present invention may include the further step of passing the influenza virus expressing the modified HA protein in cells one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) As demonstrated in the passaging experiments in Example 1, this may serve to induce additional mutations (i.e., in addition to the mutations originally contained in the modified HA protein) or combinations of mutations in the trimer interface region of the modified HA protein that are particularly beneficial for promoting or enhancing the growth of influenza viruses, more particularly influenza viruses of the H2 subtype.

[0159] Therefore, for vaccine viruses grown or passaged in cells such as MDCK cells, modified HA proteins can be encoded by modifying one or more residues in the trimer interface region in HA (e.g., V39, L219, V233, V320, K383, I388, N390, K391, V392, S394, A405, R416, D430, F450 of an H2 subtype HA protein, or any combination thereof), for example, by modifying or substituting specific amino acid residues in HA. Mutations / substitutions or selection of the HA viral segment involved (e.g., V39I, L219P, V233A, V320A, V320I, K383E, I388T, N390I, K391R, K391N, V392A, S394Y, A405T, R416G, D430N, F450S, or any combination thereof (numbering is based on full-length HA) can result in increased stability or stabilization of HA trimer formation and / or increased virus titer at the end of infection.

[0160] In particular examples, the disclosure provides an isolated influenza virus comprising PA, PB1, PB2, NP, NS, M, and NA viral segments (including heterologous or chimeric forms thereof) and an HA viral segment (including heterologous or chimeric forms thereof) encoding an HA selected to encode one or more modified amino acid residues (numbering based on full-length H2) in H2, such as at V39, L219, V233, V320, K383, 1388, N390, K391, V392, S394, A405, R416, D430, F450, or any combination thereof, wherein the recombinant influenza virus is capable of growth in cell culture, has enhanced growth / replication in cell culture, and / or can form HA trimers with enhanced stability, e.g., during vaccine production. More specifically, the isolated influenza virus can include an HA viral segment encoding an HA protein comprising one or more modified amino acid residues in the lower region of its stalk domain (e.g., modified at one or more of V39, K383, I388, N390, K391, V392, S394, and F450 of the full-length H2 HA protein, or any combination thereof).

[0161] As used herein, a "heterologous" influenza virus gene or viral segment is derived from a different influenza virus source or isolate than the majority of other influenza virus genes or gene segments in a recombinant influenza virus, including a reassortant influenza virus. Thus, the heterologous NA viral segment is preferably derived from a different influenza virus source or isolate than the influenza virus source or isolate from which one or more of the PA, PB1, PB2, NP, NS, and M viral segments are derived. In such an example, the heterologous NA viral segment can be derived from the same or a different influenza virus source or isolate from which the HA viral segment (e.g., one encoding a modified HA protein described herein) is derived.

[0162] Methods for preparing influenza viruses Also provided herein are methods for preparing or producing influenza virus in cells. Such methods preferably include contacting cells with a genetic construct comprising a nucleic acid encoding a modified HA protein, where the modified HA protein comprises an amino acid sequence in which one or more amino acid residues in the trimer interface region have been modified. It is also contemplated by the present disclosure that classical reassortment methods (or portions thereof) can be used to prepare or produce influenza virus in cells. By way of example, such methods can include contacting cells with an influenza virus isolate that expresses a modified HA protein (e.g., the influenza virus isolate comprises an HA virus segment encoding a modified HA protein), where the modified HA protein comprises an amino acid sequence in which one or more amino acid residues in the trimer interface region have been modified.

[0163] In view of the foregoing, the present methods can be utilized to prepare or generate modified or variant strains of influenza virus, such as those provided herein, that include a modified HA viral segment encoding a modified HA protein as described herein. To this end, all eight viral segments (i.e., PA, PB1, PB2, NP, NS, M, NA, and HA) can be derived from a single influenza virus isolate. In such instances, the influenza virus isolate is preferably a pandemic influenza virus isolate, such as those previously described herein. Furthermore, such modified or variant strains of influenza virus are preferably not considered to be reassortant influenza viruses.

[0164] Alternatively, the methods can be utilized to prepare or generate reassortant influenza viruses, such as those provided herein, that include an HA viral segment encoding a modified HA protein as described herein.

[0165] Preferably, the method can be, or at least partially involve, a reverse genetics method for generating reassortant viruses. In reverse genetics, the genetic information necessary to generate the desired influenza virus is delivered to cells, which can then produce influenza virus. Reverse genetics initially required the in vitro assembly of viral ribonucleoproteins (RNPs) and transfection into cells infected with a helper virus (Luytjes et al. (1989) Cell 59(6):1107-1113, Enami et al. (1990) PNAS 87(10):3802-3805). Subsequent techniques required the transfection of RNA polymerase I plasmids encoding all viral RNAs (vRNAs) along with protein expression constructs for the polymerase and NP genes (Fodor et al. (1999) J Virol. 73(11):9679-9682). More recently, reverse genetics techniques have included the use of modified RNA polymerase I systems that allow expression of both negative-sense vRNA and positive-sense mRNA from the same template (Hoffmann et al. (2000) PNAS 97(11):6108-6113). In such methods, each of the desired genes is cloned into the pHW2000 plasmid, which consists of viral cDNA inserted between an RNA polymerase I promoter and termination sequence and flanked by a CMV promoter and polyadenylation signal. After transfection of the eight plasmids into cells, synthesis of both vRNA and mRNA occurs, and virus is produced. Further refinements have led to the development of a system in which linear DNA expression constructs are used instead of plasmids (WO2009 / 000891) and the use of a single expression construct (WO2011 / 012999).

[0166] Thus, the method may further comprise contacting the cell with one or more additional genetic constructs, wherein the one or more additional genetic constructs comprise one or more additional nucleic acids encoding one or more of a PA protein, a PB1 protein, a PB1-F2 protein, a PB2 protein, an NP protein, an NS1 protein, an NEP protein, an M1 protein, an M2 protein, and an NA protein. In particular examples, the method comprises one or more of the following steps: (a) contacting the cell with one or more expression constructs, such as those described herein above, comprising one or more nucleic acid molecules that include and / or encode one or more of the following viral segments: a PA viral segment, a PB1 viral segment, a PB2 viral segment, a NP viral segment, a M viral segment, and a NS viral segment derived from a first influenza virus (e.g., a donor influenza virus); (b) contacting the cells with one or more expression constructs, such as those described herein above, comprising one or more nucleic acid molecules comprising or encoding an HA viral segment and optionally an NA viral segment, derived from a second influenza virus (e.g., a vaccine influenza virus), wherein the HA viral segment encodes a modified HA protein; (c) culturing the cells to produce one or more reassortant viruses; and (d) selecting a reassortant virus comprising an HA viral segment and optionally an NA viral segment from a second influenza virus.

[0167] In other examples, the method may include a hybrid of classical gene reassortment and reverse genetics, such as a method in which a host cell is infected with a first influenza strain (e.g., a donor strain) and transfected (e.g., before, after, or simultaneously) with one or more expression constructs encoding at least one viral segment from a second influenza strain (e.g., an HA and optionally an NA viral segment from a vaccine strain). Examples of such methods are outlined in WO2021099419, which is incorporated herein by reference.

[0168] Thus, in certain examples, the method includes one or more of the following steps: (a) contacting a cell with a donor influenza virus strain comprising a first HA viral segment and a first NA viral segment; (b) contacting the cells with one or more expression constructs, such as those described herein above, comprising one or more nucleic acid molecules comprising or encoding a second HA viral segment and optionally a second NA viral segment derived from a vaccine influenza virus, wherein the second HA viral segment encodes a modified HA protein; (c) culturing the cells to produce one or more reassortant viruses; and (d) selecting a reassortant virus comprising a second HA viral segment and optionally a second NA viral segment.

[0169] As used herein, the term "vaccine influenza virus strain" refers to an influenza virus strain suitable for use in an immunogenic composition or immunogenic virus (e.g., a reassortant virus). Vaccine influenza virus strains can include, but are not necessarily limited to, pathogenic strains, non-pathogenic or relatively non-pathogenic strains, killed strains, and / or attenuated strains. In a particular example, the vaccine influenza virus strain is a pandemic influenza virus strain.

[0170] The vectors or expression constructs utilized in the present methods can be similar to those known in the art, including those described above. Accordingly, the present disclosure contemplates the use of isolated and purified vectors or plasmids that express or encode influenza virus proteins or that express or encode influenza vRNA, both natural and recombinant vRNA. The vectors can contain influenza cDNA (see, e.g., Fields Virology (Fields et al. (eds.), Lippincott, Williams and Wickens (2013)), incorporated herein by reference). Any suitable promoter or transcription termination sequence can be used to express viral proteins, such as proteins or peptides, e.g., modified HA proteins described herein. By way of example, one or more of the expression constructs can be (a) adapted for vRNA production and include a promoter operably linked to an influenza virus DNA molecule linked to a transcription termination sequence, and / or (b) adapted for mRNA production and include a promoter operably linked to a DNA segment encoding an influenza virus segment.

[0171] As noted above, an additional selection step to enhance production of reassortant viruses containing modified HA viral segments (i.e., HA viral segments of a vaccine strain that have been modified to encode a modified HA protein) is also contemplated by the present disclosure. The selection step can include any method that enhances selection of reassortant viruses containing HA viral segments encoding a modified HA protein derived from a vaccine strain. Preferably, the selection step is performed after culturing the host cells to produce reassortant influenza viruses capable of expressing a modified HA protein.

[0172] Preferably, the methods provided herein include a step of separating the reassortant viruses from the host cells prior to the selection step. Illustratively, a cell culture supernatant containing the reassortant viruses is separated from the host cells, and the selection step is performed on the supernatant containing the reassortant viruses.

[0173] In some examples, the selection step includes negative selection against reassortant viruses containing an HA protein from the donor strain. Negative selection can include, for example, contacting the host cells, the reassortant viruses isolated from the host cells, and / or the cell culture supernatant with one or more antibodies that specifically bind to or are directed against the HA protein from the donor strain. Negative selection can also include exposing the host cells to an inhibitor (e.g., small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), or small interfering DNA (siDNA)) that preferentially or specifically reduces transcription and / or translation of the HA gene or protein of the donor strain compared to the HA gene or protein of the vaccine strain. In addition to the above, the selection step can include negative selection against reassortant viruses containing an NA protein from the donor strain, such as by utilizing one or more antibodies that specifically bind to or are directed against the NA protein from the donor strain.

[0174] In other examples, the selection step is or includes a positive selection step. The positive selection step can include contacting the host cells, the reassortant viruses isolated from the host cells, and / or the cell culture supernatant with one or more antibodies specific for the modified HA protein derived from the vaccine virus isolate or strain. In this way, reassortant viruses containing modified HA viral segments can be positively selected from the host cells or cell culture supernatant. Preferably, the one or more antibodies used for positive selection are labeled (e.g., with magnetic beads). The labeling assists in the subsequent isolation of reassortant viruses containing the HA gene encoding the modified HA protein, such as by affinity chromatography.

[0175] The methods described herein can include one or more positive and / or negative selection steps. For example, the reassortant viruses can be passaged multiple times in the presence of one or more of the antibodies described above for positive and / or negative selection. Multiple selection steps can be performed to enhance the selection of reassortant influenza viruses containing HA viral segments encoding modified HA proteins.

[0176] The method advantageously produces a pool of reassortant viruses from which specific classes of reassortant viruses can be isolated. For example, reassortant viruses with high growth properties and containing modified HA proteins and optionally NA proteins of seasonal or pandemic influenza strains can be isolated for use in vaccine production. Thus, the above method can further include the step of isolating reassortant influenza viruses containing HA viral segments encoding modified HA proteins.

[0177] To this end, the method may include harvesting or isolating intact or whole virions from the cell culture medium. Alternatively, or in addition, the method may include harvesting or isolating split virions from the culture medium. In such instances, the harvesting or isolating step preferably includes contacting the influenza virus with a splitting agent, such as a detergent. Alternatively, or in addition, the method may include harvesting or isolating one or more specific influenza virus proteins, such as a modified HA protein, from the culture medium or from the harvested influenza virus, such as by affinity chromatography.

[0178] During collection or isolation of influenza virus and / or influenza virus proteins, cells can be separated from the culture medium by standard methods such as centrifugation, separation, filtration, or ultrafiltration. The influenza virus or viral proteins produced therefrom can then be concentrated and / or purified according to methods known to those skilled in the art, such as gradient centrifugation (e.g., gradient ultracentrifugation (GUC)), filtration, precipitation, chromatography, and any combination thereof. In certain examples, influenza virus and / or one or more proteins produced therefrom are isolated or collected from the culture medium by gradient ultracentrifugation. Preferably, influenza virus is inactivated during or after purification. Viral inactivation can occur, for example, by contacting the influenza virus with an inactivating agent (e.g., adding b-propiolactone or formaldehyde) at any time during the purification process.

[0179] Preferably, the method may further include selecting an influenza virus, such as a reassortant influenza virus, that can be propagated or rescued in cells, more particularly, mammalian cells such as MDCK cells. In particular examples, the method includes determining whether the influenza virus (e.g., the reassortant virus) forms virions that can form HA trimers (or stable HA trimers) when cultured in cells. Thus, in one broad aspect, the present disclosure provides methods for preparing influenza viruses that can form stable HA trimers and / or propagate in cells.

[0180] Based on the foregoing, the present disclosure further provides an isolated influenza virus prepared by the methods described herein.

[0181] In a related aspect, the disclosure also provides modified HA proteins prepared by the methods described herein.

[0182] vaccine The present disclosure contemplates that influenza viruses and / or modified HA proteins derived from influenza viruses produced according to the methods described herein may be utilized in vaccine compositions.

[0183] Thus, in one aspect, the present disclosure provides a method of making a vaccine composition, comprising the steps of: (a) providing an isolated influenza virus and / or modified HA protein provided herein; and (b) combining the isolated influenza virus and / or the modified HA protein with an adjuvant and / or treating the isolated influenza virus with an agent that inactivates or attenuates the virus.

[0184] In a related aspect, the present disclosure relates to a vaccine composition, wherein the vaccine composition is produced according to the methods provided herein.

[0185] In another related aspect, the disclosure relates to a vaccine composition, wherein the vaccine composition comprises: (a) comprising an isolated influenza virus as described herein and a pharmaceutically acceptable carrier, diluent, or excipient; or (b) a modified HA protein described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0186] Influenza vaccines are generally based on either live attenuated or inactivated viruses. Inactivated vaccines can be based on whole virions, "split" virions, or purified surface antigens. Viral antigens can also be provided in the form of virosomes. The methods of the present invention can be used to produce any of these types of vaccines. When inactivated influenza viruses are used, the vaccines can contain whole virions, split virions, or purified surface antigens (e.g., hemagglutinin and optionally neuraminidase). Chemical means for virus inactivation include treatment with an effective amount of one or more of the following inactivating agents: detergent, formaldehyde, b-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), binary ethylamine, acetylethyleneimine, or combinations thereof. Non-chemical methods of virus inactivation are also known in the art, such as UV light or gamma irradiation. Subunit vaccines containing the modified HA proteins described herein are also contemplated.

[0187] Virions can be collected from virus-containing fluids, such as cell culture supernatants, by various methods. For example, the purification process can include zonal centrifugation using a linear sucrose gradient solution (optionally containing a detergent to disrupt virions) or affinity chromatography. Then, optionally after dilution, the antigen can be purified by diafiltration.

[0188] The vaccine composition may contain a pharmaceutically acceptable carrier, diluent, or excipient. "Pharmaceutically acceptable carrier, diluent, or excipient" refers to a solid or liquid filler, diluent, or encapsulating substance that can be safely used in systemic administration. Depending on the specific route of administration, various carriers, diluents, and excipients known in the art may be used. These may be selected from the group consisting of sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions, emulsifying substances, isotonic saline solutions, and salts (such as mineral acid salts, including hydrochlorides, bromides, and sulfates), organic acids (such as acetates, propionates, and malonates), water, and pyrogen-free water.

[0189] A useful reference describing acceptable carriers, diluents, and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991), incorporated herein by reference.

[0190] Suitably, for purposes of eliciting an immune response, certain immunological or immunogenic agents may be used in combination with the immunogenic proteins described herein. The term "immunogenic agent" includes within its scope carriers, delivery agents, immunostimulants, and / or adjuvants well known in the art. As understood in the art, immunostimulants and adjuvants refer to or include one or more substances that enhance the immunogenicity and / or efficacy of a composition. Non-limiting examples of suitable immune stimulants and adjuvants include squalane and squalene (or other oils of plant or animal origin), including oil-in-water emulsions of squalene (e.g., MF59, AS03, and AF03); block copolymers; TLR agonists, including pathogen-derived compounds, lipopeptides, glycolipids, nucleotides, small molecule inhibitors, and bacterial-derived compounds, such as flagellin; detergents (Tween® 80); Quil® A, mineral oils (such as Drakeol or Marcol), vegetable oils (such as peanut oil); adjuvants derived from Corynebacterium spp. (such as Corynebacterium parvum); adjuvants derived from Propionibacterium spp. (such as Propionibacterium acne); Mycobacterium bovis (Bacille Calmette Guerin or BCG); Bordetella pertussis antigens; tetanus toxoid; diphtheria toxoid; surfactants (such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dicoctadecyl-N',N'-bis(2-hydroxyethyl-propanediamine), methoxyhexadecylglycerol, and Pluronic polyols); polyamines (such as pyran, dextran sulfate, poly IC carbopol); peptides (such as muramyl dipeptide and derivatives, dimethylglycine, tuftsin); oil emulsions; and mineral gels (such as aluminum phosphate, aluminum hydroxide, or alum); interleukins (such as interleukin-2 and interleukin-12); monokines (such as interleukin-1); tumor necrosis factors;Interferons (such as gamma interferon); immunostimulatory DNA (such as CpG DNA), combinations (such as saponin aluminum hydroxide or Quil A aluminum hydroxide); saponins (such as Matrix-M); liposomes; ISCOM® adjuvants and ISCOMATRIX® adjuvants; mycobacterial cell wall extracts; synthetic glycopeptides (such as muramyl dipeptide or other derivatives); avridine; lipid A derivatives; dextran sulfate; DEAE-dextran alone or with aluminum phosphate; carboxypolymethylene (such as Carbopol'EMA); acrylic copolymer emulsions (such as Neocryl A640) (e.g., U.S. Pat. No. 5,047,238); water-in-oil emulsifying agents (such as Montanide ISA 720); poliovirus, cowpox, or animal poxvirus proteins; or mixtures thereof;

[0191] Immunogenic agents may include carriers such as thyroglobulin; albumin (such as human serum albumin); toxins, toxoids, or any mutant cross-reactive material (CRM) of toxins from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus; polyamino acids (such as poly(lysine:glutamic acid)); influenza; rotavirus VP6, parvovirus VP1 and VP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine, and the like. Alternatively, fragments or epitopes of carrier proteins or other immunogenic proteins may be used. For example, T cell epitopes of bacterial toxins, toxoids, or CRMs may be used. In this regard, reference may be made to U.S. Pat. No. 5,785,973 (incorporated herein by reference). It is contemplated that the carrier protein or other immunogenic protein may be directly linked to the modified HA proteins described herein, or may be indirectly linked (e.g., via a linker well known in the art).

[0192] Oil-in-water emulsions have been found to be particularly suitable for use in adjuvanting influenza virus vaccines. A variety of such emulsions are known, and they typically contain at least one oil and at least one surfactant, where the oil(s) and surfactant(s) are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion generally have a diameter of less than 5 μm, and may even have a submicron diameter; these small sizes are achieved with a microfluidizer to provide a stable emulsion. Droplets with an average size of less than 220 nm are preferred because they can be subjected to filtration sterilization.

[0193] In various examples, the oil-in-water emulsion is homogeneous. A homogeneous emulsion is characterized by a majority of the droplets (particles) dispersed therein falling within a specified size range (e.g., diameter). Suitable specified size ranges can be, for example, about 50-220 nm (e.g., about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 nm, or any range therebetween), about 50-180 nm, about 80-180 nm, about 100-175 nm, about 120-185 nm, about 130-190 nm, about 135-175 nm, or about 150-175 nm. In some examples, a homogeneous emulsion contains less than 10% of the droplets (particles) falling outside the specified diameter range. In certain instances, the mean particle size of oil droplets in an oil-in-water emulsion preparation is about 135-175 nm, e.g., about 155 nm±20 nm, as measured by dynamic light scattering, and such preparations have a mean particle size of 1×10 per mL of preparation, as measured by optical particle counting. 7This includes large particles having a diameter of 1.2 μm or less. As used herein, "large particles" refers to particles having a diameter of 1.2 μm or more, typically about 1.2 to 400 μm. In specific examples, a uniform emulsion contains less than 10%, less than 5%, or less than 3% of droplets outside the preferred size range. In some examples, the average droplet size of particles in an oil-in-water emulsion preparation is about 125 to 185 nm, e.g., about 130 nm, about 140 nm, about 150 nm, about 155 nm, about 160 nm, about 170 nm, or about 180 nm, and the oil-in-water emulsion is uniform in that less than 5% of the droplets in the preparation are outside the range of about 125 to 185 nm.

[0194] The vaccine compositions described herein can be used with oils, such as those derived from animal (e.g., fish) or plant sources. Sources of vegetable oils include nuts, seeds, and grains. Peanut oil, soybean oil, coconut oil, and olive oil are the most commonly available examples of nut oils. Jojoba oil, obtained from jojoba beans, can also be used. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, and sesame seed oil. Corn oil is the most readily available among the grains, but oils from other grains, such as wheat, oats, rye, rice, teff, and triticale, can also be used. Fatty acid esters of 6-10 carbon atoms with glycerol and 1,2-propanediol do not naturally occur in seed oils but can be prepared by hydrolysis, separation, and esterification of appropriate materials starting from nut and seed oils. Fats and oils derived from mammalian milk are metabolizable and, therefore, can be used in the vaccine compositions described herein. The procedures for separation, purification, saponification, and other means necessary to obtain pure oils from animal sources are well known in the art. Most fish contain metabolizable oils that can be easily recovered. For example, cod liver oil, shark liver oil, and whale oil, such as spermaceti, exemplify some fish oils that can be used herein.

[0195] Many branched-chain oils are biochemically synthesized from five-carbon isoprene units and are commonly referred to as terpenoids. Shark liver oil contains a branched, unsaturated terpenoid known as squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene. Squalane, a saturated analog of squalene, can also be used in the vaccine composition. Fish oils containing squalene and squalane are readily available from commercial sources or can be obtained by methods known in the art. Another suitable oil is tocopherol. Mixtures of oils are also contemplated.

[0196] Surfactants can be classified by their "HLB" (hydrophilic / lipophilic balance). Suitably, the surfactants described herein have an HLB of at least 10, more particularly at least 15, and even more particularly at least 16. The vaccine composition may comprise one or more surfactants, including, but not limited to: polyoxyethylene sorbitan ester surfactants (commonly referred to as Tweens), particularly polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), such as linear EO / PO block copolymers, sold under the trade name DOWFAX™; octoxynols, such as octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol), which vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups; (octylphenoxy)polyethoxyethanol (IGEPAL); CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty acid ethers derived from lauryl, cetyl, stearyl, and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Nonionic surfactants are preferred. Exemplary surfactants for inclusion in emulsions are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin, and Triton X-100.

[0197] A mixture of surfactants can also be used (e.g., a Tween 80 / Span 85 mixture). A combination of a polyoxyethylene sorbitan ester, such as polyoxyethylene sorbitan monooleate (Tween 80), and an octoxynol, such as t-octylphenoxypolyethoxyethanol (Triton X-100), is also suitable. Another possible combination includes laureth 9 plus a polyoxyethylene sorbitan ester and / or an octoxynol.

[0198] Exemplary amounts (by weight) of surfactants are as follows: polyoxyethylene sorbitan esters (such as Tween 80) from 0.01% to 1%, specifically about 0.1%; octyl- or nonylphenoxypolyoxyethanols (such as Triton X-100, or other detergents in the Triton series) from 0.001% to 0.1%, specifically 0.005% to 0.02%; polyoxyethylene ethers (such as Laureth 9) from 0.1% to 20%, more specifically 0.1% to 10%, even more specifically 0.1% to 1%, or about 0.5%.

[0199] In particular examples, the oil-in-water emulsion is a squalene-in-water emulsion, more particularly a submicron squalene-in-water emulsion. According to some examples, the vaccine composition comprises MF59.

[0200] Any suitable procedure is contemplated for the production of vaccine compositions. Exemplary procedures include, for example, those described in New Generation Vaccines (1997, Levine et al., Marcel Dekker, Inc. New York, Basel, Hong Kong), which is incorporated herein by reference.

[0201] Any safe route of administration may be used, including, but not limited to, oral, rectal, parenteral, sublingual, buccal, intravenous, intraarticular, intramuscular, intradermal, subcutaneous, by inhalation, intranasal, intraocular, intraperitoneal, intracerebroventricular, topical, mucosal, and transdermal administration.

[0202] Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, lozenges, capsules, nasal sprays, suppositories, aerosols, transdermal patches, and the like. These dosage forms may also include injections or implants of controlled-release devices specifically designed for this purpose, or other forms of implants modified to additionally act in this manner. Controlled release may be achieved by coating with hydrophobic polymers, including acrylic resins, waxes, higher aliphatic alcohols, polylactic acid, polyglycolic acid, and certain cellulose derivatives such as hydroxypropylmethylcellulose. In addition, controlled release may be achieved by the use of other polymer matrices, liposomes, and / or microspheres.

[0203] The compositions may be presented as discrete units (such as capsules, sachets, functional foods / feeds, or tablets, each containing a predetermined amount of one or more therapeutic agents of the present disclosure), as a powder or granules, or as a solution or suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy, but all methods include the step of bringing into association one or more agents described above with a carrier, which may constitute one or more necessary ingredients. Generally, the compositions may be prepared by uniformly and intimately admixing an agent of the present disclosure with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired presentation.

[0204] The compositions can be administered in a manner compatible with the dosage formulation and in an effective amount. The dose administered to a subject, in the context of the present disclosure, should be sufficient to effect a beneficial response in the subject (e.g., to generate a protective immune response) over a reasonable period of time. The amount of agent(s) administered can depend on the subject being treated, including age, sex, weight, and their general health condition, factors that will depend on the judgment of a physician.

[0205] Also disclosed herein is a container containing the immunogenic or vaccine composition disclosed herein. Any suitable container known in the art can be used. For example, the container can be selected from the group consisting of a vial, a syringe, an ampoule, a flask, a fermenter, a bioreactor, a bag, a jar, an ampoule, a cartridge, and a disposable pen. In one example, the container is a vial, an ampoule, or a syringe.

[0206] The container may be made of glass, metal (e.g., steel, stainless steel, aluminum, etc.) and / or polymer (e.g., thermoplastic, elastomer, thermoplastic elastomer). The container may be at least partially siliconized.

[0207] The vaccine compositions disclosed herein may further comprise a buffer. The buffer may be any suitable buffer known in the art. For example, the buffer may be TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, and triethanolamine buffer, and / or phosphate buffer. In one example, the buffer is a phosphate buffer. In another example, the buffer is a succinate buffer. In another example, the buffer is a histidine buffer. In another example, the buffer is a citrate buffer.

[0208] The buffer solution may be selected from USP-compliant buffer solutions for parenteral use, especially when the pharmaceutical preparation is for parenteral use. For example, the buffer solution may be selected from the group consisting of monobasic acids, such as acetic acid, benzoic acid, gluconic acid, glyceric acid, and lactic acid; dibasic acids, such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, and tartaric acid; polybasic acids, such as citric acid and phosphoric acid; and bases, such as ammonia, diethanolamine, glycine, triethanolamine, and TRIS.

[0209] Methods for inducing immune responses and treatments The influenza virus proteins and vaccine compositions described herein may be suitable for administration to a human or non-human animal subject, such that the present disclosure provides methods of generating an immune response and / or preventing and / or treating an influenza-related disease, disorder, or condition in a subject. The present disclosure also provides compositions for use as a medicament, and provides use of a composition of the present disclosure for the manufacture of a medicament for generating an immune response and / or preventing and / or treating an influenza-related disease, disorder, or condition in a subject.

[0210] Thus, in one aspect, the present disclosure provides a method of inducing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of an isolated influenza virus, modified HA protein, or vaccine composition provided herein, thereby inducing an immune response in the subject.

[0211] In a related aspect, the present disclosure provides a method for preventing and / or treating an influenza-associated disease, disorder or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of an isolated influenza virus, modified HA protein, or vaccine composition described herein, thereby preventing and / or treating the influenza-associated disease, disorder, or condition.

[0212] With respect to the embodiments described herein, the terms "subject," "patient," and "individual" include, but are not limited to, mammals, including humans, performance animals (such as horses, camels, greyhounds), livestock (such as cattle, sheep, horses, pigs, chickens, ducks), and companion animals (such as cats and dogs). Preferably, the subject is a human.

[0213] "Eliciting an immune response" means generating or stimulating the production or activity of one or more elements of the immune system, including the cellular immune system, the humoral immune system (i.e., antibodies), and / or the innate immune system. Suitably, the immune response described herein includes one or more elements of the immune system, such as T lymphocytes, B lymphocytes, antibodies, neutrophils, dendritic cells, including plasmacytoid dendritic cells, cytokines, and / or chemokines. Non-limiting examples of cytokines include inflammatory cytokines, such as TNF-α, IL-2, IL-6, IL-8, IL-17A, and IL-1 (e.g., IL-1β). A non-limiting example of a chemokine is IL-8, a chemotactic factor for neutrophils. In certain instances, the immune response elicited by the vaccine compositions described herein is protective.

[0214] As generally used herein, the terms "immunize," "vaccine," and "vaccine" refer to methods and / or compositions that induce a protective immune response against influenza virus such that subsequent infection with influenza virus or related serotypes, strains, or variants is at least partially prevented or minimized.

[0215] "Protective immunity" means a level of immunity in which responsiveness to an antigen or antigens leads to rapid binding and / or clearance of the antigens, thereby sufficient to at least partially ameliorate or prevent subsequent influenza virus infection in a subject.

[0216] By "protective immune response" is meant a level of immune response sufficient to prevent or reduce the severity, symptoms, aspects, or characteristics of current and / or influenza virus infection in a subject.

[0217] As used herein, the terms "treating," "treat," or "treatment" refer to a therapeutic intervention that at least partially improves, eliminates, or alleviates the post-onset symptoms or pathological signs of an influenza-related disease, disorder, or condition, e.g., influenza infection. Treatment need not be absolute to be beneficial to the subject. A beneficial effect may be determined using any method or criteria known to one of skill in the art.

[0218] As used herein, "preventing," "prevent," or "prevention" refers to a course of action initiated prior to infection or exposure to an influenza virus or molecular components thereof and / or prior to the onset of symptoms or pathological signs of a disease, disorder, or condition in order to prevent infection and / or alleviate symptoms or pathological signs. It is understood that such prevention need not be absolute to be beneficial to the subject. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit symptoms, or who exhibits only early signs, of a disease, disorder, or condition for the purpose of reducing the risk of developing symptoms or pathological signs of the disease, disorder, or condition.

[0219] The vaccines described herein can be used to treat both children and adults. Influenza vaccines are currently recommended for use in vaccinating children and adults from 6 months of age. Thus, human subjects can be under 1 year of age, 1-5 years of age, 5-15 years of age, 15-55 years of age, or at least 55 years of age. Preferred subjects for receiving the vaccine include the elderly (e.g., 50 years of age or older, 60 years of age or older, and preferably 65 years of age or older), young people (e.g., 5 years of age or younger), hospitalized subjects, healthcare workers, military and civilian personnel, pregnant women, chronically immunocompromised subjects, subjects who have taken antiviral compounds within 7 days prior to receiving the vaccine, individuals with egg allergies, and individuals traveling abroad. However, the vaccine is not exclusively suitable for these groups and may be used more generally in the population. In the case of pandemic strains, administration to all age groups is preferred.

[0220] Treatment can be by a single-dose schedule or a multiple-dose schedule. Multiple doses may be used in a primary immunization schedule and / or a booster immunization schedule. In a multiple-dose schedule, various doses can be administered by the same or different routes (e.g., parenteral prime and mucosal boost, mucosal prime and parenteral boost). Multiple (usually two) administrations are particularly useful in immunologically naive patients (e.g., subjects who have not previously received an influenza vaccine) or for vaccination against new HA subtypes (e.g., during a pandemic outbreak). Multiple doses are usually administered at least one week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

[0221] Screening Method The present disclosure further relates to methods that involve testing or screening for modifications of HA proteins, such as those of the H2 subtype, that can confer favorable growth characteristics to influenza viruses when grown in cells, such as MDCK cells.

[0222] Thus, in one aspect, the disclosure provides a method for identifying modifications of an HA protein (e.g., of the H2 subtype) that promote or improve the growth of influenza virus in a cell, the method comprising the steps of: (a) performing one or more passages of one or more candidate influenza viruses expressing an HA protein in cells; (b) selecting candidate influenza viruses that are able to grow or show improved growth in the cells; (c) screening the candidate influenza viruses selected in step (b) for one or more modifications to the HA protein, for example, the trimerization region or the lower region of its stalk domain.

[0223] Suitably, the candidate influenza virus is unable to grow, or is only capable of limited growth, in cells when expressing a wild-type or unmodified form of the HA protein.

[0224] In another aspect, the disclosure provides a method for identifying or screening for modifications of an HA protein (e.g., of the H2 subtype) that promote or improve the growth of influenza virus in a cell, the method comprising the steps of: (a) modifying an influenza virus to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified; and (b) testing the ability of the modified influenza virus to grow in cells.

[0225] Preferably, the method further comprises selecting a modification to the HA protein or a modified HA protein that enhances or improves the growth of influenza viruses in cells. Thus, one or more modifications in a modified HA protein selected based on their ability to enhance or improve the growth of influenza viruses in cell culture can be identified as a result of the method. Such modifications can then be introduced into the HA proteins of wild-type, recombinant, or reassortant viruses, such as by the methods described herein, so as to enhance or improve the growth of these influenza viruses in cell culture.

[0226] Preferably, the modified HA protein comprises an amino acid sequence in which one or more amino acid residues in the lower region of the stalk domain of the trimer interface region have been modified. As described herein, the lower region of the stalk domain can comprise amino acid residues N26-D46, L325-P335, D377-E397, and L439-D452 of a full-length HA protein of the H2 subtype. In another example, the lower region of the stalk domain comprises amino acid residues N26-D46, D377-E397, and L439-D452 of a full-length HA protein of the H2 subtype.

[0227] To cite some examples, the one or more modified amino acid residues are located at positions selected from the group consisting of 39, 219, 233, 320, 383, 388, 390, 391, 392, 394, 405, 416, 430, 450, and any combination thereof in the full-length H2 amino acid sequence. More specifically, the one or more modified amino acid residues can be selected from the group consisting of V39, L219, V233, V320, K383, I388, N390, K391, V392, S394, A405, R416, D430, F450, and any combination thereof in the full-length H2 amino acid sequence.

[0228] In a related aspect, the disclosure provides a method for identifying modifications of an HA protein (e.g., of the H2 subtype) that promote or improve the growth of influenza virus in a cell, the method comprising the steps of: (a) modifying an influenza virus to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified; and (b) Passaging the modified influenza virus expressing the modified HA protein one or more times in the cells.

[0229] Preferably, the method further comprises screening the modified influenza virus after one or more passages in the cell for one or more additional modifications to the modified HA protein. It is contemplated that such screening may be performed by any method known in the art, such as DNA and protein sequencing methods, including Sanger sequencing, chain termination sequencing, dye terminator sequencing, pyrosequencing, and mass spectrometry.

[0230] According to a specific example of this method, the modified HA protein is as described herein above.

[0231] So that preferred embodiments of the present disclosure can be fully understood and practiced, reference is made to the following non-limiting examples. [Example]

[0232] Example 1 The objective of this example was to rescue synthetic seed viruses based on influenza A H2N3 pre-pandemic strains A / Chicken / ohio / 494832 / 2007 and A / Swine / Missouri / 2124514 / 2006.

[0233] Methods and Results However, we found that most attempts to rescue synthetic viruses using WT H2N3 alleles derived from A / Chicken / Ohio / 494832 / 2007 failed to produce viruses. Those that did produce viruses with mutations or variants in the HA viral segment. All variants were identified by visual analysis of Sanger sequencing results. A variant was identified if multiple peaks were observed at a given nucleotide base in all reads, with a minimum read depth of 2 reads. In this example, only variants that resulted in amino acid changes were further tested.

[0234] HA mutations / variants were identified by one of four different pathways. 1. Passage of WT A / Chicken / Ohio / 494832 / 2007. Sequencing of this virus revealed four identifiable variants (1x). a. A / chicken / ohio / 484832 / 2007 i. V129I variant - This variant is not at or near the trimer interface. It is outside the head domain and is unlikely to contribute to trimer stability. ii. V320I variant iii. N390I variant iv. K391R variant 2. Rescue of A / Swine / Missouri / 2124514 / 2006 (2x independent rescues). a. RG4 A / swine / Missouri / 2124514 / 2006 i. K383E variant b. RG5 A / swine / Missouri / 2124514 / 2006 i. L219P mutation 3. Rescue of A / Chicken / Ohio / 494832 / 2007 a. GDE 80.4AA / chicken / ohio / 484832 / 2007 i. V233A variant ii. V320A variant b. GDE 80.4BA / chicken / ohio / 484832 / 2007 i. V39I mutation c. GDE 80.7BA / chicken / ohio / 484832 / 2007 i. F450S d. HS_Sys_14 A / chicken / ohio / 484832 / 2007 i. S394Y variant ii. R416G variant e. HS_Sys_15 A / chicken / ohio / 484832 / 2007 i. A405T variant

[0235] Synthetic Seed Process and Procedures Recombinant assembly of the HA and NA sequences of A / Chicken / Ohio / 494832 / 2007 into an expression construct according to the method described in Dormitzer et al. (Sci Transl Med. 2013 May 15;5(185)) (see, e.g., Figure 1 of Dormitzer et al.). MDCK cells are then transfected with expression constructs for the HA and NA sequences of A / Chicken / Ohio / 494832 / 2007 and the PA, PB1, PB2, NP, NS, and M backbone viral segments from a high-growth parent strain (e.g., A / Puerto Rico / 8 / 1934 or its cell-adapted form, such as PR8X). The reassortant viruses are then rescued and characterized. A schematic diagram of the transfection and rescue procedure is shown in Figure 1. The first two rescue attempts failed to produce rescued isolates of the H2N3 strain. Amplification of viral RNA from the supernatant of transfected cells confirmed the absence of viral RNA amplification. Additional transfection and rescue experiments were performed, yielding rescued virus isolates in 5 of a total of 28 experiments.

[0236] Sequence analysis · Sequencing of eight rescued or serially passaged isolates of H2N3 strains showed that all contained variant nucleotide bases in the HA viral segment rather than encoding modified amino acid residues in the HA protein (see Figure 2). No other modifications were observed in any of the other seven viral segments of these strains.

[0237] Mutation mapping ·A / Swine / Missouri / 2124514 / 2006 The structure of the HA protein has been determined. When these rescue-identified variants were mapped onto the structure, they all resided at the interface between adjacent monomers in the HA trimer structure, as seen in Figure 3. Figure 4 further demonstrates that the A405T mutation in the HS Sys15 isolate causes this residue to associate more closely with the K423 residue on the adjacent HA monomer when in a homotrimeric configuration, which may function to stabilize the trimer structure. Without being bound by any theory, and based on the locations of the variants and the fact that we were unable to rescue WT virus in the absence of at least one variant location, it is hypothesized that the wild-type H2 monomer is unable to form functional trimers when grown in cells, thereby preventing or limiting growth of viruses expressing this HA in cell culture.

[0238] Isolation of clones · Clonal isolation experiments were performed to determine the stability of the A405T mutation in HS Sys15 isolates. Several 10-fold limiting dilutions were performed. > 10x plates (-4 to -9 serial dilutions), 60 plates in total > Perform titer check on day 3 Ideally, the fewer colonies a plate contains, the more likely they are clonal. > The more clonal isolations there are, the more likely an isolate is clonal. 28 clones were isolated from 60 plates. >12 clones from the -5 dilution plate >16 clones from -4 dilution plate All 28 clones were sequenced, and the majority (25 of 28) showed a clean population of viruses with the A405T mutation for each of the clones, while the remaining 3 clones showed evidence of a mixture of A405T mutant virus and WT virus, with the WT virus being only a minor variant. The data indicate that the A405T mutation in HS Sys15 isolates is stable. Like the V129I variant, the E184K variant is not at or near the trimer interface, and it is located outside the head domain, making it unlikely to contribute to trimer stability.

[0239] Further passages We took five of the H2N3 isolates and performed three additional passages (duplicates) to determine the fidelity of these variants. Sanger sequencing of this passage material revealed additional variants, as outlined in Table 1. In HS_Sys_15, the A405T variant was the dominant allele and no additional mutations were acquired. Both variants found in HS_Sys_14 were stable throughout passage and did not acquire additional mutations. Many of the additional variants that have emerged have been present multiple times, so there appears to be some convergence in the HA virus segments that acquire specific mutations. Residues 39, 383, 388, 390, 391, 392, 394, and 450 all map to the lower region of the stalk (i.e., the lower stalk cluster). [Table 2]

[0240] conclusion In this example, we identified multiple mutations that can support the rescue or propagation of H2 influenza viruses in MDCK cells. These mutations appear along the length of the molecule and consistently in the trimer interface region between HA monomers. While specific regions of the lower stalk appear to be hotspots for mutations that support virus rescue, mutations were also identified elsewhere in the HA molecule. While the passaging data suggest that some mutations are more stable over time than others, this example suggests that at least all of these mutations at the trimer interface contribute to the virus's viability when grown in cell culture. To this end, this example suggests that wild-type or unmodified H2 HA proteins are close to being able to support the propagation of H2 influenza viruses in cells. Furthermore, these data support the notion that a dynamic range of mutations throughout the trimer interface region can facilitate the rescue and propagation of H2 influenza viruses in cell culture, and that the virus can further convert and alter these modifications with passaging to further support its stability or propagation in cells.

[0241] Listing of embodiments by bullet points

[0242] 1. A modified hemagglutinin (HA) protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified, wherein an influenza virus expressing the modified HA protein can grow in a cell.

[0243] 2. The modified HA protein of claim 1, which is capable of forming HA trimers when expressed by influenza viruses propagated in cells.

[0244] 3. The modified HA protein of claim 1 or claim 2, wherein the modification to the one or more amino acid residues in the trimer interface region increases the stability of an HA trimer formed from the modified HA protein compared to an HA trimer formed from a corresponding unmodified HA protein.

[0245] 4. The modified HA protein of any one of the preceding claims, wherein the modified HA protein is of the H2, H1, H5, H3, H7, or H9 subtype.

[0246] 5. The modified HA protein of claim 4, wherein the modified HA protein is of the H2, H1, or H5 subtype.

[0247] 6. The modified HA protein of claim 4 or claim 5, wherein the modified HA protein is of the H2 subtype.

[0248] 7. The modified HA protein of claim 6, wherein the one or more modified amino acid residues are selected from the group consisting of V39, L219, V233, V320, K383, I388, N390, K391, V392, S394, A405, R416, D430, F450 of the full-length H2 amino acid sequence, and any combination thereof.

[0249] 8. The one or more amino acid residues to be modified are (a) V39, (b) L219, (c)V233, (d) V320, (e) K383, (f)I388, (g) N390, (h)K391, (i) V392, (j)S394, (k) A405, (l) R416, (m)D430, (n)F450, (o) S394 and R416, (p)V233 and V320, (q) V320, N390 and K391, (r) V320 and N390, (s) V320 and K391, (t) N390 and K391, (u) F450 and K391, (v) V233, F450 and K391, (w) V233 and F450, (x) V233 and K391, (y) V39 and D430, (z) V39, I388 and V392, (aa) V39 and I388, (ab) V39 and V392, (ac)I388 and V392, (ad)V39, K391 and D430, (ae) V39 and K391, or (af) K391 and D430, 8. The modified HA protein of claim 7, comprising:

[0250] 9. The modified HA protein of claim 7 or claim 8, wherein the modifications to the one or more amino acid residues are selected from the group consisting of V39I, L219P, V233A, V320A, V320I, K383E, I388T, N390I, K391R, K391N, V392A, S394Y, A405T, R416G, D430N, F450S, and any combination thereof of the full-length H2 amino acid sequence.

[0251] 10. The modification of the one or more amino acid residues is (a) V39I, (b) L219P, (c) V233A, (d) V320A, (e) V320I, (f)K383E, (g)I388T, (h)N390I, (i) K391R, (j)K391N, (k)V392A, (l)S394Y, (m)A405T, (n)R416G, (o)D430N, (p)F450S, (q) S394Y and R416G, (r) V233A and V320A, (s) V320I, N390I and K391R, (t) V320I and N390I, (u) V320I and K391R, (v) N390I and K391R, (w)F450S and K391N, (x) V233A, F450S and K391N, (y) V233A and F450S, (z) V233A and K391N, (aa) V39I and D430N, (ab) V39I, I388T and V392A, (ac)V39I and I388T, (ad)V39I and V392A, (ae) I388T and V392A, (af)V39I, K391N and D430N, (ag) V39I and K391N, or (ah) K391N and D430N, 10. The modified HA protein of claim 9, comprising:

[0252] 11. The modified HA protein of any one of the preceding claims, wherein one or more of the modified amino acid residues are present in the lower region of the stalk domain.

[0253] 12. The modified HA protein of claim 11, wherein the lower region of the stalk domain comprises, consists of, or consists essentially of amino acid residues 377-397, 439-452, 26-46, and optionally 325-335 of a full-length HA protein of the H2 subtype.

[0254] 13. The modified HA protein of claim 11, wherein the lower region of the stalk domain comprises, consists of, or consists essentially of amino acid residues 383-394, 439-452, 31-40, and optionally 325-335 of a full-length HA protein of the H2 subtype.

[0255] 14. The modified HA protein of claim 11, wherein the lower region of the stalk domain comprises, consists of, or consists essentially of amino acid residues N26 to D46, L325 to P335, D377 to E397, and L439 to D452 of a full-length HA protein of the H2 subtype.

[0256] 15. The modified HA protein of claim 14, wherein the lower region of the stalk domain comprises amino acid residues N26 to D46, D377 to E397, and L439 to D452 of a full-length HA protein of the H2 subtype.

[0257] 16. The modified HA protein of any one of claims 11 to 15, wherein the one or more modified amino acid residues are selected from the group consisting of V39, K383, I388, N390, K391, V392, S394, F450 of the full-length H2 amino acid sequence, and any combination thereof.

[0258] 17. The modified HA protein of any one of claims 11 to 16, wherein the modifications to the one or more amino acid residues are selected from the group consisting of V39I, K383E, I388T, N390I, K391R, K391N, V392A, S394Y, F450S of the full-length H2 amino acid sequence, and any combination thereof.

[0259] 18. A modified HA protein according to any one of the preceding claims, comprising, consisting of or consisting essentially of an amino acid sequence set out in any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68, or a fragment, variant or derivative thereof.

[0260] 19. The modified HA protein of any one of the preceding claims, wherein the modified HA protein has been modified by (a) one or more passages of an influenza virus isolate in cells and / or eggs, and / or (b) recombinant methods.

[0261] 20. A modified HA protein of the H2 subtype, comprising an amino acid sequence in which one or more amino acid residues have been modified at a position selected from the group consisting of V39, L219, V233, V320, K383, I388, N390, K391, V392, S394, A405, R416, D430, F450, and any combination thereof, of the full-length H2 amino acid sequence.

[0262] 21. The modified HA protein of claim 20, wherein an influenza virus expressing the modified HA protein is capable of growing intracellularly.

[0263] 22. The modified HA protein of claim 21, wherein the cell is an MDCK cell.

[0264] 23. A modified HA protein of the H2 subtype, comprising an amino acid sequence in which one or more amino acid residues are modified in the trimer interface region, wherein an influenza virus expressing the modified HA protein can grow in a cell.

[0265] 24. An isolated nucleic acid comprising a nucleotide sequence encoding a modified HA protein according to any one of claims 1 to 23 or a nucleotide sequence complementary thereto.

[0266] 25. The isolated nucleic acid of claim 24, comprising, consisting of, or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, or 67, or a fragment, variant, or derivative thereof, or a nucleotide sequence complementary thereto.

[0267] 26. A genetic construct comprising (i) the isolated nucleic acid of claim 24 or claim 25, or (ii) a nucleotide sequence complementary thereto, operably linked or connected to one or more regulatory sequences.

[0268] 27. A host cell transformed with an isolated nucleic acid according to claim 24 or claim 25, or with a genetic construct according to claim 26.

[0269] 28. A method for producing a modified HA protein according to any one of claims 1 to 23, comprising the steps of: (i) culturing a pre-transformed host cell according to claim 27; and (ii) isolating the modified HA protein from the host cell cultured in step (i).

[0270] 29. An isolated influenza virus comprising an HA viral segment encoding a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

[0271] 30. The isolated influenza virus of claim 29, wherein the modified HA protein is as defined in any one of claims 1 to 23.

[0272] 31. The isolated influenza virus of claim 29 or claim 30, wherein the HA viral segment comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, or 67, or a fragment, variant, or derivative thereof.

[0273] 32. The isolated influenza virus of any one of claims 29 to 31, which is a recombinant influenza virus.

[0274] 33. The isolated influenza virus of any one of claims 29 to 32, which is a reassortant influenza virus.

[0275] 34. The isolated influenza virus of any one of claims 29 to 33, which is capable of growing in a cell.

[0276] 35. The isolated influenza virus of claim 34, which is capable of growing in MDCK cells.

[0277] 36. The isolated influenza virus of any one of claims 29 to 35, which, when propagated in a cell, is capable of forming an HA trimer comprising the modified HA protein.

[0278] 37.Furthermore, (a) one or more of the PA, PB1, PB2, NP, NS, and M viral segments from a donor influenza virus; and 37. The isolated influenza virus of any one of claims 29 to 36, comprising (b) a heterologous or chimeric NA virus segment.

[0279] 38. A method for preparing influenza virus in a cell, comprising contacting the cell with a genetic construct comprising a nucleic acid encoding a modified HA protein, wherein the modified HA protein comprises an amino acid sequence in which one or more amino acid residues in its trimer interface region have been modified.

[0280] 39. The method of claim 38, wherein the modified HA protein is one of claims 1 to 23, the nucleic acid is an isolated nucleic acid of claim 24 or claim 25, and / or the genetic construct is one of claim 26.

[0281] 40. The method of claim 38 or claim 39, further comprising contacting the cell with one or more additional genetic constructs, wherein the one or more additional genetic constructs comprise one or more additional nucleic acids encoding one or more of a PA protein, a PB1 protein, a PB1-F2 protein, a PB2 protein, an NP protein, an NS1 protein, an NEP protein, an M1 protein, an M2 protein, and an NA protein.

[0282] 41. The method of claim 40, wherein the NA protein is of the N1, N2, or N3 subtype.

[0283] 42. The method of any one of claims 38 to 41, wherein a modified pandemic influenza virus is prepared comprising said nucleic acid encoding said modified HA protein.

[0284] 43. The method of any one of claims 38 to 42, further comprising the step of isolating or harvesting the influenza virus and / or the modified HA protein from the cells.

[0285] 44. An isolated influenza virus prepared by the method of any one of claims 38 to 43.

[0286] 45. A modified HA protein prepared by the method of any one of claims 28 or 38 to 43.

[0287] 46. ​​A method of making a vaccine composition, comprising: (a) providing an isolated influenza virus according to any one of claims 29-37 or 44 and / or a modified HA protein according to any one of claims 1-23 or 45; and (b) combining the isolated influenza virus and / or the modified HA protein with an adjuvant and / or treating the isolated influenza virus with an agent that inactivates or attenuates the virus.

[0288] 47. The method of claim 46, wherein the adjuvant comprises an immunostimulatory DNA sequence, a bacterially derived component, aluminum salts (alum), or a squalene oil-in-water emulsion system.

[0289] 48. A vaccine composition produced according to the method of claim 46 or claim 47.

[0290] 49. A vaccine composition comprising: (a) an isolated influenza virus according to any one of claims 29-37 or 44, and a pharmaceutically acceptable carrier, diluent, or excipient; or (b) The vaccine composition, comprising the modified HA protein of any one of claims 1 to 23 or 45, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0291] 50. A method for inducing an immune response in a subject, comprising administering to the subject a therapeutically effective amount of an isolated influenza virus of any one of claims 29-37 or 44, a modified HA protein of any one of claims 1-23 or 45, or a vaccine composition of claim 48 or claim 49, thereby inducing the immune response in the subject.

[0292] 51. A method for preventing and / or treating an influenza-associated disease, disorder or condition in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of an isolated influenza virus of any one of claims 29-37 or 44, a modified HA protein of any one of claims 1-23 or 45, or a vaccine composition of claim 48 or claim 49, thereby preventing and / or treating the influenza-associated disease, disorder or condition.

[0293] 52. A method for identifying or screening for modifications of an HA protein that promote or improve the growth of influenza virus in a cell, comprising: (a) modifying the influenza virus to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified; and (b) testing the ability of the modified influenza virus to grow in a cell.

[0294] 53. The method of claim 52, further comprising selecting the modified HA protein that promotes or improves growth of the influenza virus in a cell.

[0295] 54. The method of claim 52 or claim 53, wherein the modified HA protein comprises an amino acid sequence in which one or more amino acid residues in the lower stalk region of its trimer interface region have been modified.

[0296] 55. A method for identifying modifications to an HA protein that promote or improve the growth of influenza virus in a cell, comprising: (a) modifying the influenza virus to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified; and (b) performing one or more passages of the modified influenza virus expressing the modified HA protein in a cell.

[0297] 56. The method of claim 55, further comprising screening the modified influenza virus for one or more additional modifications to the modified HA protein after one or more passages in the cells.

[0298] 57. A method for improving the growth of an influenza virus in a cell, the method comprising modifying the influenza virus to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

[0299] 58. The method of claim 57, further comprising one or more passages of the influenza virus expressing the modified HA protein in cells.

[0300] 59. The method of any one of claims 55 to 58, wherein the modified HA protein is as defined in any one of claims 1 to 23.

[0301] 60. A method for improving the stability of an influenza virus strain in a cell, the method comprising modifying the influenza virus strain to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

[0302] 61. The method of claim 60, further comprising one or more passages of the influenza virus expressing the modified HA protein in cells.

[0303] 62. The method of any one of claims 60 to 61, wherein the modified HA protein is as defined in any one of claims 1 to 23.

[0304] 63. A method for improving the production of an influenza virus strain, the method comprising modifying the influenza virus strain to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

[0305] 64. The method of claim 63, wherein the influenza virus strain is unable to grow in culture when expressing a wild-type or unmodified HA protein.

[0306] 65. The method of claim 63 or 64, wherein the production of the influenza virus strain occurs intracellularly.

[0307] 66. The method of any one of claims 60-62 or 65, wherein the cell is selected from the group consisting of mammalian, avian, yeast, and plant cells.

[0308] 67. The method of claim 66, wherein the cell is a mammalian cell.

[0309] 68. The method of claim 67, wherein the cells are canine kidney cells.

[0310] 69. The method of claim 68, wherein the canine kidney cells are Madin-Darby canine kidney (MDCK) cells.

[0311] 70. The method of any one of claims 60-69, wherein the influenza virus strain is characterized by at least one of the following: (a) expresses an HA protein that is not present in currently circulating human strains or has not previously been detected in the human population, and the human population is immunologically naive to the HA protein of the influenza virus strain; (b) is capable of horizontal transmission within the human population; and (c) is pathogenic to humans.

[0312] Sequence Listing [Table 3-1]

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Table 3-22

Table 3-23

Table 3-24

Table 3-25

Table 3-26

Table 3-27

Table 3-28

Table 3-29

Table 3-30

Table 3-31

Table 3-32

Table 3-33

Table 3-34

Table 3-35

Table 3-36

Table 3-37

Table 3-38

Table 3-39

Table 3-40

Table 3-41

Table 3-42

Table 3-43

Table 3-44

Table 3-45

Table 3-46

Table 3-47

Table 3-48

Table 3-49

Table 3-50

Claims

1. A modified hemagglutinin (HA) protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified, wherein an influenza virus expressing the modified HA protein can propagate in a cell.

2. 2. The modified HA protein of claim 1, which is capable of forming HA trimers when expressed by influenza viruses propagated in cells.

3. The modified HA protein of claim 1, wherein the modification to the one or more amino acid residues in the trimer interface region increases the stability of the HA trimer formed from the modified HA protein compared to the HA trimer formed from the corresponding unmodified HA protein.

4. 2. The modified HA protein of claim 1, wherein the modified HA protein is of the H2, H1, H5, H3, H7, or H9 subtype.

5. The modified HA protein of claim 4, wherein the modified HA protein is of the H2, H1, or H5 subtype.

6. The modified HA protein of claim 4, wherein the modified HA protein is of the H2 subtype.

7. The modified HA protein of claim 6, wherein the one or more modified amino acid residues are selected from the group consisting of V39, L219, V233, V320, K383, I388, N390, K391, V392, S394, A405, R416, D430, F450 of the full-length H2 amino acid sequence, and any combination thereof.

8. The modified one or more amino acid residues are (a) V39, (b) L219, (c) V233, (d) V320, (e) K383, (f) I388, (g) N390, (h) K391, (i) V392, (j) S394, (k) A405, (l) R416, (m) D430, (n) F450, (o) S394 and R416, (p) V233 and V320, (q) V320, N390 and K391; (r) V320 and N390; (s) V320 and K391, (t) N390 and K391, (u) F450 and K391; (v) V233, F450 and K391; (w) V233 and F450, (x) V233 and K391, (y) V39 and D430, (z) V39, I388 and V392, (aa) V39 and I388, (ab) V39 and V392, (ac) I388 and V392, (ad) V39, K391 and D430, (ae) V39 and K391, or (af) K391 and D430, The modified HA protein of claim 7, comprising:

9. 8. The modified HA protein of claim 7, wherein the modifications to the one or more amino acid residues are selected from the group consisting of V39I, L219P, V233A, V320A, V320I, K383E, I388T, N390I, K391R, K391N, V392A, S394Y, A405T, R416G, D430N, F450S of the full-length H2 amino acid sequence, and any combination thereof.

10. The modification to the one or more amino acid residues is (a) V39I, (b) L219P, (c) V233A, (d) V320A, (e) V320I, (f) K383E, (g) I388T, (h) N390I, (i) K391R, (j) K391N, (k) V392A, (l) S394Y, (m) A405T, (n) R416G, (o) D430N, (p) F450S, (q) S394Y and R416G, (r) V233A and V320A, (s) V320I, N390I and K391R, (t) V320I and N390I, (u) V320I and K391R; (v) N390I and K391R, (w) F450S and K391N, (x) V233A, F450S and K391N, (y) V233A and F450S, (z) V233A and K391N, (aa) V39I and D430N, (ab) V39I, I388T and V392A, (ac) V39I and I388T, (ad) V39I and V392A, (ae) I388T and V392A, (af) V39I, K391N and D430N, (ag) V39I and K391N, or (ah) K391N and D430N, The modified HA protein of claim 9, comprising:

11. 2. The modified HA protein of claim 1, wherein one or more of the modified amino acid residues are present in the lower region of the stalk domain.

12. 12. The modified HA protein of claim 11, wherein the lower region of the stalk domain comprises, consists of, or consists essentially of amino acid residues 377-397, 439-452, 26-46, and optionally 325-335 of a full-length HA protein of the H2 subtype.

13. 12. The modified HA protein of claim 11, wherein the lower region of the stalk domain comprises, consists of, or consists essentially of amino acid residues 383-394, 439-452, 31-40, and optionally 325-335 of a full-length HA protein of the H2 subtype.

14. 12. The modified HA protein of claim 11, wherein the lower region of the stalk domain comprises, consists of, or consists essentially of amino acid residues N26 to D46, L325 to P335, D377 to E397, and L439 to D452 of a full-length HA protein of the H2 subtype.

15. 15. The modified HA protein of claim 14, wherein the lower region of the stalk domain comprises amino acid residues N26 to D46, D377 to E397, and L439 to D452 of a full-length HA protein of the H2 subtype.

16. The modified HA protein of claim 11, wherein the one or more modified amino acid residues are selected from the group consisting of V39, K383, I388, N390, K391, V392, S394, F450 of the full-length H2 amino acid sequence, and any combination thereof.

17. 12. The modified HA protein of claim 11, wherein the modifications to the one or more amino acid residues are selected from the group consisting of V39I, K383E, I388T, N390I, K391R, K391N, V392A, S394Y, F450S of the full-length H2 amino acid sequence, and any combination thereof.

18. 2. The modified HA protein of claim 1, comprising, consisting of, or consisting essentially of an amino acid sequence set forth in any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68, or a fragment, variant, or derivative thereof.

19. 2. The modified HA protein of claim 1, wherein the modified HA protein is modified by (a) one or more passages of an influenza virus isolate in cells and / or eggs, and / or (b) recombinant methods.

20. A modified HA protein of the H2 subtype, comprising an amino acid sequence in which one or more amino acid residues are modified at a position selected from the group consisting of V39, L219, V233, V320, K383, I388, N390, K391, V392, S394, A405, R416, D430, F450, and any combination thereof, of the full-length H2 amino acid sequence.

21. 21. The modified HA protein of claim 20, wherein an influenza virus expressing the modified HA protein is capable of growing intracellularly.

22. 22. The modified HA protein of claim 21, wherein the cell is an MDCK cell.

23. A modified HA protein of the H2 subtype, comprising an amino acid sequence in which one or more amino acid residues are modified in the trimer interface region, wherein an influenza virus expressing the modified HA protein can grow in a cell.

24. An isolated nucleic acid comprising a nucleotide sequence encoding a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified, wherein the nucleotide sequence or a complementary nucleotide sequence thereof enables an influenza virus expressing the modified HA protein to grow in a cell.

25. 25. The isolated nucleic acid of claim 24, comprising, consisting of, or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, or 67, or a fragment, variant, or derivative thereof, or a complementary nucleotide sequence thereto.

26. A genetic construct comprising (i) an isolated nucleic acid comprising a nucleotide sequence encoding a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified, operably linked or connected to one or more regulatory sequences, wherein the isolated nucleic acid enables influenza viruses expressing the modified HA protein to propagate in cells, or (ii) a nucleotide sequence complementary thereto.

27. A host cell transformed with an isolated nucleic acid encoding a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified, wherein an influenza virus expressing the modified HA protein can propagate in the cell, or a genetic construct comprising (i) the isolated nucleic acid, or (ii) a nucleotide sequence complementary thereto, operably linked or connected to one or more regulatory sequences.

28. A method for producing a modified HA protein, comprising: (i) culturing a host cell transformed with an isolated nucleic acid encoding a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified, wherein the isolated nucleic acid or a complementary nucleotide sequence thereof, or a genetic construct comprising (i) the isolated nucleic acid or (ii) a complementary nucleotide sequence thereof, operably linked or connected to one or more control sequences, such that an influenza virus expressing the modified HA protein can propagate in the cell; and (ii) isolating the modified HA protein from the host cell cultured in step (i).

29. An isolated influenza virus comprising an HA virus segment encoding a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

30. 30. The isolated influenza virus of claim 29, wherein the modified HA protein is of the H2 subtype.

31. 30. The isolated influenza virus of claim 29, wherein the HA viral segment comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, or 67, or a fragment, variant, or derivative thereof.

32. 30. The isolated influenza virus of claim 29, which is a recombinant influenza virus.

33. 30. The isolated influenza virus of claim 29, which is a reassortant influenza virus.

34. 30. The isolated influenza virus of claim 29, which is capable of growing in a cell.

35. 35. The isolated influenza virus of claim 34, which is capable of growing in MDCK cells.

36. 30. The isolated influenza virus of claim 29, which, when propagated in a cell, is capable of forming an HA trimer comprising the modified HA protein.

37. moreover, (a) one or more of the PA, PB1, PB2, NP, NS, and M viral segments from a donor influenza virus; and (b) heterologous or chimeric NA virus segments; 30. The isolated influenza virus of claim 29, comprising:

38. A method for preparing influenza virus in a cell, comprising the step of contacting the cell with a genetic construct comprising a nucleic acid encoding a modified HA protein, wherein the modified HA protein comprises an amino acid sequence in which one or more amino acid residues in its trimer interface region are modified.

39. 39. The method of claim 38, wherein the modified HA protein is of the H2 subtype of claim 1.

40. 39. The method of claim 38, further comprising contacting the cell with one or more additional genetic constructs, wherein the one or more additional genetic constructs comprise one or more additional nucleic acids encoding one or more of a PA protein, a PB1 protein, a PB1-F2 protein, a PB2 protein, an NP protein, an NS1 protein, a NEP protein, an M1 protein, an M2 protein, and an NA protein.

41. 41. The method of claim 40, wherein the NA protein is of the N1, N2, or N3 subtype.

42. 39. The method of claim 38, wherein a modified pandemic influenza virus is prepared comprising the nucleic acid encoding the modified HA protein.

43. 39. The method of claim 38, further comprising isolating or harvesting the influenza virus and / or the modified HA protein from the cells.

44. 39. An isolated influenza virus prepared by the method of claim 38.

45. 29. A modified HA protein prepared by the method of claim 28.

46. 1. A method of making a vaccine composition, comprising: (a) providing an isolated influenza virus comprising an HA virus segment encoding a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in a trimer interface region are modified, and / or the modified HA protein; and (b) combining the isolated influenza virus and / or the modified HA protein with an adjuvant and / or treating the isolated influenza virus with an agent that inactivates or attenuates the virus; The method comprising:

47. 47. The method of claim 46, wherein the adjuvant comprises an immunostimulatory DNA sequence, a bacterially derived component, aluminum salts (alum), or a squalene oil-in-water emulsion system.

48. 47. A vaccine composition produced according to the method of claim 46.

49. 1. A vaccine composition comprising: (a) an isolated influenza virus comprising an HA viral segment encoding a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified, and a pharmaceutically acceptable carrier, diluent, or excipient; or (b) a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified, and a pharmaceutically acceptable carrier, diluent, or excipient; The vaccine composition comprising:

50. A method for inducing an immune response in a subject, comprising the step of administering to the subject a therapeutically effective amount of an isolated influenza virus comprising an HA virus segment encoding a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified, the modified HA protein, or a vaccine composition comprising the isolated influenza virus or the modified HA protein, thereby inducing the immune response in the subject.

51. A method for preventing and / or treating an influenza-associated disease, disorder or condition in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of an isolated influenza virus comprising an HA virus segment encoding a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified, the modified HA protein, or a vaccine composition comprising the isolated influenza virus or the modified HA protein, thereby preventing and / or treating the influenza-associated disease, disorder or condition.

52. 1. A method for identifying or screening for modifications of an HA protein that enhance or improve the growth of influenza virus in a cell, comprising: (a) modifying the influenza virus to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified; and (b) testing the ability of the modified influenza virus to grow in cells; The method comprising:

53. 53. The method of claim 52, further comprising selecting the modified HA protein that promotes or improves growth of the influenza virus in a cell.

54. 53. The method of claim 52, wherein the modified HA protein comprises an amino acid sequence in which one or more amino acid residues in the lower stalk region of its trimer interface region have been modified.

55. 1. A method for identifying modifications to an HA protein that enhance or improve influenza virus growth in a cell, comprising: (a) modifying the influenza virus to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified; and (b) passaging the modified influenza virus expressing the modified HA protein one or more times in a cell; The method comprising:

56. 56. The method of claim 55, further comprising screening the modified influenza virus for one or more additional modifications to the modified HA protein after one or more passages in the cell.

57. A method for improving the growth of an influenza virus in a cell, the method comprising modifying the influenza virus to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

58. 58. The method of claim 57, further comprising one or more passages of the influenza virus expressing the modified HA protein in cells.

59. 56. The method of claim 55, wherein the modified HA protein is of the H2 subtype.

60. 58. The method of claim 57, wherein the modified HA protein is of the H2 subtype.

61. A method for improving the stability of an influenza virus strain in a cell, the method comprising modifying the influenza virus strain to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

62. 62. The method of claim 61, further comprising one or more passages of the influenza virus expressing the modified HA protein in cells.

63. 62. The method of claim 61, wherein the modified HA protein is of the H2 subtype.

64. 1. A method for improving the production of an influenza virus strain, the method comprising modifying the influenza virus strain to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

65. 65. The method of claim 64, wherein the influenza virus strain is unable to grow in culture when expressing a wild-type or unmodified HA protein.

66. 65. The method of claim 64, wherein the production of the influenza virus strain occurs intracellularly.

67. 62. The method of claim 61, wherein the cell is selected from the group consisting of mammalian, avian, yeast, and plant cells.

68. 68. The method of claim 67, wherein the cell is a mammalian cell.

69. 69. The method of claim 68, wherein the cells are canine kidney cells.

70. 70. The method of claim 69, wherein the canine kidney cells are Madin-Darby canine kidney (MDCK) cells.

71. 62. The method of claim 61, wherein the influenza virus strain is characterized by at least one of the following: (a) expresses an HA protein that is not present in currently circulating human strains or has not been previously detected in the human population, and the human population is immunologically naive to the HA protein of the influenza virus strain; (b) is capable of horizontal transmission within the human population; and (c) is pathogenic to humans.

72. 65. The method of claim 64, wherein the cell is selected from the group consisting of mammalian, avian, yeast, and plant cells.

73. 73. The method of claim 72, wherein the cell is a mammalian cell.

74. 74. The method of claim 73, wherein the cells are canine kidney cells.

75. 75. The method of claim 74, wherein the canine kidney cells are Madin-Darby canine kidney (MDCK) cells.

76. 65. The method of claim 64, wherein the influenza virus strain is characterized by at least one of the following: (a) expresses an HA protein that is not present in currently circulating human strains or has not been previously detected in the human population, and the human population is immunologically naive to the HA protein of the influenza virus strain; (b) is capable of horizontal transmission within the human population; and (c) is pathogenic to humans.

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