Modified influenza virus

Abstract

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

Description

[0001] The work described in this disclosure was performed 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

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 398,362, filed on August 16, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0004] 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 of making the same. Background Art

[0005] Influenza is a major respiratory disease in mammalian species, causing significant mortality, morbidity, and economic losses each year. There are three recognized types of influenza viruses, A, B, and C, which are defined by the absence of serological cross-reactivity between their internal proteins. Influenza A viruses are further divided into subtypes based on antigenic and genetic differences in their glycoproteins, hemagglutinin (HA), and neuraminidase (NA) proteins.

[0006] Newer vaccine production methods may involve generating and culturing reassortant influenza viruses in cell culture, such as in Madin Darby canine kidney (MDCK) cells. However, some influenza viruses appear to be less susceptible to growth and replication under such conditions than others, which may present challenges for vaccine manufacturing. Therefore, there remains a need to develop genetic modifications to the influenza virus genome that confer efficient growth and replication of vaccine virus candidates in cell culture. Summary of the invention

[0007] The present disclosure is based on the surprising discovery that modifications of 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 on 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 in cell culture.

[0008] Therefore, the present disclosure provides a modified hemagglutinin (HA) protein comprising an amino acid sequence in which one or more amino acid residues in its trimer interface region are modified, wherein an influenza virus expressing the modified HA protein is capable of growing in a cell.

[0009] Relatedly, the present disclosure also provides a modified H2 subtype HA protein comprising an amino acid sequence in which one or more amino acid residues thereof 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 of the full-length H2 amino acid sequence, and any combination thereof.

[0010] In addition, the present disclosure provides a modified H2 subtype HA protein comprising an amino acid sequence wherein one or more amino acid residues thereof in the trimer interface region are modified, and wherein an influenza virus expressing the modified HA protein is capable of growing in a cell.

[0011] The present disclosure also provides:

[0012] An isolated nucleic acid comprising a nucleotide sequence encoding a modified HA protein disclosed herein or a nucleotide sequence complementary thereto;

[0013] A genetic construct comprising: (i) an isolated nucleic acid of the present disclosure; or (ii) a nucleotide sequence complementary thereto; operably linked or linked to one or more regulatory sequences; and

[0014] A host cell transformed with an isolated nucleic acid of the disclosure or a genetic construct of the disclosure.

[0015] In addition, the present disclosure provides a method for producing the modified HA protein of the present disclosure, the method comprising the steps of: (i) culturing the previously transformed host cell of the present disclosure; and (ii) isolating the modified HA protein from the host cell cultured in step (i).

[0016] In addition, the present disclosure provides an isolated influenza virus comprising an HA viral segment, wherein the HA viral segment encodes a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region thereof are modified.

[0017] In addition, the present disclosure provides a method for preparing influenza virus in a cell, the method 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.

[0018] The present disclosure also 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.

[0019] Relatedly, the present disclosure provides a method for preparing a vaccine composition, the method comprising the steps of:

[0020] (a) providing an isolated influenza virus of the present disclosure and / or a modified HA protein of the present disclosure; and

[0021] (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 the virus.

[0022] Therefore, the present disclosure also provides vaccine compositions produced according to the methods for preparing vaccine compositions disclosed herein.

[0023] Similarly, the present disclosure provides a vaccine composition, wherein the vaccine composition comprises:

[0024] (a) an isolated influenza virus of the present disclosure and a pharmaceutically acceptable carrier, diluent or excipient; or

[0025] (b) the modified HA protein of the present disclosure and a pharmaceutically acceptable carrier, diluent or excipient.

[0026] In addition, the present disclosure provides a method of eliciting an immune response in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of the isolated influenza virus of the present disclosure, the modified HA protein of the present disclosure, or the vaccine composition of the present disclosure, thereby eliciting an immune response in the subject.

[0027] In addition, the present disclosure provides a method for preventing and / or treating an influenza-related disease, disorder or condition in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of the isolated influenza virus of the present disclosure, the modified HA protein of the present disclosure or the vaccine composition of the present disclosure, thereby preventing and / or treating an influenza-related disease, disorder or condition.

[0028] The present disclosure also provides a method for identifying or screening modifications in HA protein that promote or improve the growth of influenza virus in cells, the method comprising the following steps:

[0029] (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 a trimer interface region thereof are modified; and

[0030] (b) Testing the ability of the modified influenza viruses to grow in cells.

[0031] Relatedly, the present disclosure provides a method for identifying modifications in HA protein that promote or improve influenza virus growth in cells, the method comprising the steps of:

[0032] (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 a trimer interface region thereof are modified; and

[0033] (b) performing one or more passages of the modified influenza virus expressing the modified HA protein in cells.

[0034] In addition, the present disclosure provides a method for improving the growth of influenza virus in cells, the method comprising the step of modifying the influenza virus to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in its trimer interface region are modified.

[0035] The present disclosure also provides a method for improving the stability of influenza virus in cells, the method comprising the step of modifying an influenza virus strain to express 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.

[0036] In addition, the present disclosure provides a method for improving the production of influenza virus strains, the method comprising the step of modifying influenza virus to express a modified HA protein comprising an amino acid sequence in which one or more amino acid residues in its trimer interface region are modified. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present disclosure. The present disclosure can be better understood by reference to one or more of these drawings and in conjunction with the detailed description of the specific embodiments proposed herein. Those skilled in the art will appreciate that many changes and / or modifications may be made to the above-mentioned embodiments without departing from the broad general scope of the present disclosure. Therefore, embodiments of the present invention are considered to be illustrative and non-restrictive in all respects.

[0038] Figure 1 . Schematic diagram of candidate vaccine virus (CVV) transfection and rescue.

[0039] Figure 2 . The observed mutations and variants were mapped to the structure of A / swine / Missouri / 2124514 / 2006_HA.

[0040] Figure 3Views of the outer surface and trimer interface of HA indicate that mutations in the HA of the A / chicken / Ohio / 494832 / 2007 rescue strain are restricted to the trimer interface region of the molecule.

[0041] Figure 4 Close-up structural view of the A405T mutated residue and its proximity to the K423 residue on the adjacent HA monomer.

[0042] Key to sequence listing

[0043] SEQ ID NO:1A Nucleotide sequence of the PB2 viral segment of chicken / Ohio / 494832 / 2007

[0044] SEQ ID NO:2A Nucleotide sequence of the PB1 viral segment of chicken / Ohio / 494832 / 2007

[0045] SEQ ID NO:3A Nucleotide sequence of the PA viral segment of chicken / Ohio / 494832 / 2007

[0046] SEQ ID NO:4A Nucleotide sequence of the HA viral segment of chicken / Ohio / 494832 / 2007

[0047] SEQ ID NO:5A Nucleotide sequence of the NP virus segment of chicken / Ohio / 494832 / 2007

[0048] SEQ ID NO:6A Nucleotide sequence of the NA viral segment of chicken / Ohio / 494832 / 2007

[0049] SEQ ID NO: 7A / chicken / Ohio / 494832 / 2007 nucleotide sequence of the M virus segment

[0050] SEQ ID NO: 8A Nucleotide sequence of the NS virus segment of chicken / Ohio / 494832 / 2007

[0051] SEQ ID NO:9A Amino acid sequence of PB2 protein of chicken / Ohio / 494832 / 2007

[0052] SEQ ID NO:10A Amino acid sequence of PB1 protein of chicken / Ohio / 494832 / 2007

[0053] SEQ ID NO:11A Amino acid sequence of PA protein of chicken / Ohio / 494832 / 2007

[0054] SEQ ID NO:12A Amino acid sequence of HA protein of chicken / Ohio / 494832 / 2007

[0055] SEQ ID NO:13A Amino acid sequence of NP protein of chicken / Ohio / 494832 / 2007

[0056] SEQ ID NO:14A Amino acid sequence of NA protein of chicken / Ohio / 494832 / 2007

[0057] SEQ ID NO:15A Amino acid sequence of the M1 protein of chicken / Ohio / 494832 / 2007

[0058] SEQ ID NO:16A Amino acid sequence of the M2 protein of chicken / Ohio / 494832 / 2007

[0059] SEQ ID NO: 17A Nucleotide sequence of the PB2 viral segment of / swine / Missouri / 2124514 / 2006

[0060] SEQ ID NO: 18 Nucleotide sequence of the PB1 viral segment of A / swine / Missouri / 2124514 / 2006

[0061] SEQ ID NO: 19A Nucleotide sequence of the PA viral segment of / swine / Missouri / 2124514 / 2006

[0062] SEQ ID NO: 20A Nucleotide sequence of the HA viral segment of / swine / Missouri / 2124514 / 2006

[0063] SEQ ID NO: 21A Nucleotide sequence of the NP virus segment of / swine / Missouri / 2124514 / 2006

[0064] SEQ ID NO: 22A Nucleotide sequence of the NA viral segment of / swine / Missouri / 2124514 / 2006

[0065] SEQ ID NO:23A Nucleotide sequence of the M viral segment of / swine / Missouri / 2124514 / 2006

[0066] SEQ ID NO:24A Nucleotide sequence of the NS virus segment of / swine / Missouri / 2124514 / 2006

[0067] Amino acid sequence of PB2 protein of SEQ ID NO:25A / Pig / Missouri / 2124514 / 2006

[0068] Amino acid sequence of PB1 protein of SEQ ID NO:26A / Pig / Missouri / 2124514 / 2006

[0069] Amino acid sequence of PA protein of SEQ ID NO:27A / Pig / Missouri / 2124514 / 2006

[0070] SEQ ID NO:28A Amino acid sequence of HA protein of / swine / Missouri / 2124514 / 2006

[0071] Amino acid sequence of NP protein of SEQ ID NO:29A / Pig / Missouri / 2124514 / 2006

[0072] Amino acid sequence of NA protein of SEQ ID NO:30A / Pig / Missouri / 2124514 / 2006

[0073] SEQ ID NO:31A Amino acid sequence of M1 protein of / swine / Missouri / 2124514 / 2006

[0074] SEQ ID NO:32A Amino acid sequence of the M2 protein of / swine / Missouri / 2124514 / 2006

[0075] Amino acid sequence of NS1 protein of SEQ ID NO:33A / Pig / Missouri / 2124514 / 2006

[0076] Amino acid sequence of NEP protein of SEQ ID NO:34A / Pig / Missouri / 2124514 / 2006

[0077] SEQ ID NO:35A Nucleotide sequence of the HA viral segment of the HS Sys 14 rescue isolate of HS / Chicken / Ohio / 494832 / 2007

[0078] SEQ ID NO:36A Amino acid sequence of the HA protein of the HS Sys 14 rescue isolate of chicken / Ohio / 494832 / 2007

[0079] SEQ ID NO:37A Nucleotide sequence of the HA viral segment of the HS Sys 15 rescue isolate of chicken / Ohio / 494832 / 2007

[0080] SEQ ID NO:38 Amino acid sequence of HA protein of HS Sys 15 rescue isolate of A / chicken / Ohio / 494832 / 2007

[0081] SEQ ID NO:39A Nucleotide sequence of the HA viral segment of the GDE 80.4A rescued isolate of chicken / Ohio / 494832 / 2007

[0082] SEQ ID NO:40A Amino acid sequence of the HA protein of the GDE 80.4A rescued isolate of chicken / Ohio / 494832 / 2007

[0083] SEQ ID NO:41A Nucleotide sequence of the HA viral segment of the GDE 80.4B rescue isolate of chicken / Ohio / 494832 / 2007

[0084] SEQ ID NO:42A Amino acid sequence of the HA protein of the GDE 80.4B rescue isolate of chicken / Ohio / 494832 / 2007

[0085] SEQ ID NO: 43A Nucleotide sequence of the HA viral segment of the GDE-80.7B rescue isolate of chicken / Ohio / 494832 / 2007

[0086] SEQ ID NO:44A Amino acid sequence of the HA protein of the GDE-80.7B rescue isolate of chicken / Ohio / 494832 / 2007

[0087] SEQ ID NO:45A Nucleotide sequence of the HA viral segment of the passaged isolate of / swine / Missouri / 2124514 / 2006

[0088] SEQ ID NO:46A Amino acid sequence of the HA protein of the subculture isolate of chicken / Ohio / 494832 / 2007

[0089] SEQ ID NO:47A Nucleotide sequence of the HA viral segment of the RG4 P2 rescue isolate of / swine / Missouri / 2124514 / 2006

[0090] SEQ ID NO:48A Amino acid sequence of the HA protein of the RG4 P2 rescue isolate of pig / Missouri / 2124514 / 2006

[0091] SEQ ID NO:49A Nucleotide sequence of the HA viral segment of the RG5 P2 rescue isolate of / swine / Missouri / 2124514 / 2006

[0092] SEQ ID NO:50A Amino acid sequence of the HA protein of the RG5 P2 rescue isolate of pig / Missouri / 2124514 / 2006

[0093] SEQ ID NO:51 Nucleotide sequence of the HA viral segment of the GDE 80.4A 3rd generation replication A isolate of A / chicken / Ohio / 494832 / 2007

[0094] SEQ ID NO:52 Amino acid sequence of HA protein of GDE 80.4A 3rd generation replication A isolate of A / chicken / Ohio / 494832 / 2007

[0095] SEQ ID NO:53A Nucleotide sequence of the HA viral segment of the GDE 80.4A 3rd generation replication B isolate of GDE 80.4A 3rd generation replication B isolate of GDE 80.4A 3rd generation replication B isolate of GDE 80.4A 3rd generation replication B isolate of GDE 80.4A

[0096] SEQ ID NO:54A Amino acid sequence of the HA protein of the GDE 80.4A 3rd generation replication B isolate of chicken / Ohio / 494832 / 2007

[0097] SEQ ID NO:55 Nucleotide sequence of the HA viral segment of the GDE 80.4B 3rd generation replication A isolate of A / chicken / Ohio / 494832 / 2007

[0098] SEQ ID NO:56 Amino acid sequence of HA protein of GDE 80.4B 3rd generation replication A isolate of A / chicken / Ohio / 494832 / 2007

[0099] SEQ ID NO:57A Nucleotide sequence of the HA viral segment of the GDE 80.4B 3rd generation replication B isolate of GDE 80.4B 3rd generation replication B isolate of GDE 80.4B

[0100] SEQ ID NO:58 Amino acid sequence of HA protein of GDE 80.4B 3rd generation replication B isolate of A / chicken / Ohio / 494832 / 2007

[0101] SEQ ID NO:59 Nucleotide sequence of the HA viral segment of the GDE-80.7B 3rd generation replication A isolate of A / chicken / Ohio / 494832 / 2007

[0102] SEQ ID NO:60 Amino acid sequence of HA protein of GDE-80.7B 3rd generation replication A isolate of A / chicken / Ohio / 494832 / 2007

[0103] SEQ ID NO:61 Nucleotide sequence of the HA viral segment of HS_Sys_14 3rd generation replication A isolate of A / chicken / Ohio / 494832 / 2007

[0104] SEQ ID NO:62 Amino acid sequence of the HA viral segment of HS_Sys_14 3rd generation replication A isolate of A / chicken / Ohio / 494832 / 2007

[0105] SEQ ID NO:63A Nucleotide sequence of the HA viral segment of HS_Sys_14 3rd generation replication B isolate of HS_Sys_14 3rd generation replication B isolate of HS_Sys_14 3rd generation replication B isolate of HS_Sys_14

[0106] SEQ ID NO:64A Amino acid sequence of the HA viral segment of HS_Sys_14 3rd generation replication B isolate of HS_Sys_14 3rd generation replication B isolate of HS_Sys_14

[0107] SEQ ID NO:65 Nucleotide sequence of the HA viral segment of HS_Sys_15 3rd generation replication A isolate of A / chicken / Ohio / 494832 / 2007

[0108] SEQ ID NO:66A Amino acid sequence of the HA viral segment of HS_Sys_15 3rd generation replication A isolate of HS_Sys_15 3rd generation replication A isolate of HS_Sys_15

[0109] SEQ ID NO:67A Nucleotide sequence of the HA viral segment of HS_Sys_15 3rd generation replication B isolate of HS_Sys_15 3rd generation replication B isolate of HS_Sys_15 3rd generation replication B isolate of HS_Sys_15

[0110] SEQ ID NO:68A Amino acid sequence of the HA viral segment of HS_Sys_15 3rd generation replication B isolate of HS_Sys_15 3rd generation replication B isolate of HS_Sys_15

[0111] Amino acid sequence of NS1 protein of SEQ ID NO:69A / chicken / Ohio / 494832 / 2007

[0112] SEQ ID NO:70A / chicken / Ohio / 494832 / 2007 NEP protein amino acid sequence

[0113] Amino acid sequence of PB1-F2 protein of SEQ ID NO:71A / chicken / Ohio / 494832 / 2007

[0114] Amino acid sequence of PB1-F2 protein of SEQ ID NO:72A / Pig / Missouri / 2124514 / 2006

[0115] SEQ ID NO:73 Nucleotide sequence of 5' non-coding region

[0116] SEQ ID NO:74 Nucleotide sequence of 3' non-coding region DETAILED DESCRIPTION

[0117] General techniques and definitions

[0118] Unless explicitly defined otherwise, all technical and scientific terms used herein should be deemed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0119] The present disclosure can be performed 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 and Maniatis, Molecular Cloning: A Laborat ory Manual, Cold Spring Harbor Laboratories, New York, 4th edition (2012), Volumes I, II and III in their entirety; DNA Cloning: A Practical Approach, Volumes I and II (DN Glover, 2nd edition, 1995), IRL Press, Oxford, in its entirety; Oligonucleotide Synthesis: A Practical Approach (MJ Gait, ed., 1984) IRL Press, Oxford, in its entirety, in particular Gait's article therein, pp. 1-22; Atkinson et al., pp. 35-81; Sproat et al., pp. 83-115; and Wu et al., pp. 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (BD Hames and SJ Higgins, eds., 1985) IRL Press, Oxford d, 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 Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), entire series.

[0120] It will be appreciated by those skilled in the art that the present disclosure is susceptible to variations and modifications other than those specifically described. It should be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, compositions and compounds mentioned or indicated in this specification, either individually or collectively, and any and all combinations of any two or more of the steps or features.

[0121] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for purposes of illustration only. As described herein, functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0122] Each feature of any particular aspect or embodiment or embodiment of the disclosure may be applied to any other aspect or embodiment or embodiment of the disclosure with appropriate modifications.

[0123] Throughout this specification, references to individual steps, compositions of matter, groups of steps, or groups of compositions of matter shall be considered to cover both one and more of those steps, compositions of matter, groups of steps, or groups of compositions of matter (i.e., one or more).

[0124] Thus, as used herein, the singular forms "a", "an", and "the" include plural forms of these words unless the context clearly dictates otherwise. For example, reference to "a bacterium" includes a plurality of such bacteria and reference to "an allergen" is a reference to one or more allergens.

[0125] The term "and / or", such as "X and / or Y", should be understood to mean "X and Y" or "X or Y", and should be considered to provide clear support for both meanings or either meaning.

[0126] Throughout this specification, the word "comprise" or variations such as "comprises or comprising" will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0127] In the context of an amino acid sequence, "consisting essentially of" means the amino acid sequence together with an additional one, two or three amino acids at the N-terminus or C-terminus.

[0128] The term "substantially" does not exclude "completely" (eg, a composition "substantially free" of Y may be completely free of Y).

[0129] The term "about" in relation to a value x is optional and means, for example, any value within 1%, 5%, or 10% of the recited value. In some instances, the term "about" encompasses the exact value recited.

[0130] All computer programs, algorithms, patents and scientific literature mentioned herein are incorporated by reference.

[0131] For the present disclosure, the database accession numbers or unique identifiers of the genes, proteins or viral strains provided herein, as well as one or more gene and / or protein sequences associated therewith, are incorporated herein by reference.

[0132] Modified HA protein

[0133] The inventors unexpectedly demonstrated that modifying specific amino acid residues in the interface region of the H2 protein trimer can promote or improve the growth of influenza viruses expressing modified hemagglutinin (HA) proteins in cell culture. These modified proteins can also increase virus yields, which may be beneficial for vaccine production.

[0134] Therefore, in one form, the present disclosure provides a modified hemagglutinin (HA) protein comprising an amino acid sequence in which one or more amino acid residues in its trimer interface region are modified. Suitably, influenza viruses expressing modified HA proteins are able to grow in cells. In this regard, when unmodified or wild-type modified HA proteins are expressed, influenza viruses are suitably unable to grow in cells or have limited or reduced ability to grow in cells. Therefore, modified HA proteins suitably provide influenza viruses expressing the HA proteins with the ability to grow in cells or improved ability to grow in cells.

[0135] In a related form, the present disclosure provides a modified H2 subtype HA protein comprising an amino acid sequence in which one or more amino acid residues in its trimer interface region are modified. In some examples, the one or more amino acid residues are modified at a position selected from the group consisting of: 39, 219, 233, 320, 383, 388, 390, 391, 392, 394, 405, 416, 430, 450 of the full-length H2 amino acid sequence and any combination thereof. More specifically, the one or more amino acid residues may be modified at a position 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 (e.g., as shown in SEQ ID NO: 12) and any combination thereof.

[0136] In another related form, the present disclosure provides a modified H2 subtype HA protein comprising an amino acid sequence wherein one or more amino acid residues thereof in the trimer interface region are modified, and wherein an influenza virus expressing the modified HA protein is capable of growing in a cell.

[0137] Influenza hemagglutinin (HA) is a glycoprotein encoded by the HA gene segment of influenza virus. HA is usually expressed on the surface of the virus capsid in the form of homotrimers and is an indispensable part of viral infectivity. For this reason, HA allows the identification of upper respiratory tract cells or red blood cells by binding to polysaccharides containing monosaccharide sialic acid thereon. This causes influenza virus to be internalized into endosomes by cells, and then promotes the conformational rearrangement of HA trimers. Then, HA protein fuses with the endosomal membrane, thereby allowing the viral gene segment (in the form of ribonucleoprotein complex (RNP)) to be released into the cytoplasm of the host cell together with nucleoprotein and polymerase complex. RNP is transported to the host cell nucleus, and then the transcription and replication of the viral genome are carried out. When influenza virus sprouts from infected host cells, HA protein is then incorporated into the envelope of influenza virions together with other newly generated viral proteins and replicated genomes. The HA protein on the new virus particle is still attached to the sialic acid group of the glycoprotein on the outer surface of the cell, and neuraminidase (NA) can cleave these groups, thereby effectively releasing the newly formed virions.

[0138] The terms "hemagglutinin", "hemagglutinin" and "HA" refer to any hemagglutinin protein known to those skilled 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 specific examples, the modified HA proteins described herein belong to H2, H1, H5, H3, H7 or H9 influenza A subtypes, or more specifically to H2, H1 or H5 influenza A subtypes. In some examples, the modified HA proteins described herein belong to H2 influenza A subtypes. HA can also be derived from influenza viruses isolated from any host species. In various examples, the modified HA proteins are derived at least in part from avian influenza virus isolates or strains. In other examples, the modified HA proteins are derived at least in part from swine influenza virus isolates or strains.

[0139] According to certain examples, the modified HA protein provided herein is an influenza hemagglutinin protein, such as an influenza A hemagglutinin protein or an influenza B hemagglutinin protein. A typical hemagglutinin protein comprises a signal peptide, a stem domain or a stalk domain, a globular head domain, a cavity domain, a transmembrane domain, and a cytoplasmic domain. In some examples, the modified hemagglutinin protein provided herein comprises a single polypeptide chain, such as HA0, HA1, or HA2. In other examples, the modified HA protein comprises more than one quaternary associated polypeptide chain (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 comprises a signal peptide (i.e., the modified hemagglutinin protein is a full-length hemagglutinin; for example, as shown 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).

[0140] As used herein, the term "trimer interface region", as far as it relates to HA proteins, refers to an external or surface region of a HA monomer that can associate, interact, contact or bind to adjacent HA monomers when forming an HA trimer. In this regard, one or more modified amino acid residues may be adjacent to and / or facing the trimer interface region of another HA molecule or monomer, for example when in a dimer or trimer arrangement. In addition, one or more modified amino acid residues may modulate structural features of the trimer interface region, such as tertiary structure. Suitably, the trimer interface region comprises one or more amino acid residues that approach and participate in interactions with one or more amino acid residues on adjacent HA monomers when the trimer is formed. Such interactions may include, for example, hydrogen bonding, electrostatic interactions, salt bridges, and the like. In specific examples, one or more modified amino acid residues in the trimer interface region are within or less than about 15 angstroms from one or more residues in the adjacent HA monomer when the trimer is formed (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 therein). In other examples, one or more modified amino acid residues in the trimer interface region are within or less than about 11 angstroms from one or more residues in the adjacent HA monomer when the trimer is formed. In various examples, one or more modified amino acid residues in the trimer interface region are within or less than about 8.5 angstroms from one or more residues in the adjacent HA monomer when the trimer is formed.

[0141] It is further contemplated that the amino acid residues of the trimer interface region may be found in any part, subunit or domain of the HA protein or molecule, such as in the globular head domain and / or the stem domain. In a specific example, one or more modified amino acids are present in the lower region of the stem domain (e.g., one or more of residues V39, K383, I388, N390, K391, V392, S394 and F450 of the full-length H2 HA protein). Suitably, the trimer interface region in the lower region of the stem domain may comprise, consist of or consist essentially of amino acid residues at positions 377 to 397, 439 to 452, 26 to 46 and optionally 325 to 335 (e.g., those positions as shown in SEQ ID NO: 12) of the full-length HA protein of the H2 subtype. Alternatively, the trimer interface region in the lower region of the stem domain may comprise, consist of, or consist essentially of amino acid residues at positions 383 to 394, 439 to 452, 31 to 40, and optionally 325 to 335 of a full-length HA protein of the H2 subtype (e.g., those positions as shown in SEQ ID NO: 12). In a further alternative, the trimer interface region in the lower region of the stem domain may comprise, consist of, or consist essentially of amino acid residues at positions 26 to 46, 325 to 335, 377 to 397, and 439 to 452 (e.g., N26 to D46, L325 to P335, D377 to E397, and L439 to D452) of a full-length HA protein of the H2 subtype (e.g., as shown in SEQ ID NO: 12 or SEQ ID NO: 28). In specific examples, the trimer interface region in the lower region of the stalk domain comprises, consists of, or consists essentially of the amino acid residues at positions 26 to 46, 377 to 397, and 439 to 452 (e.g., N26 to D46, D377 to E397, and L439 to D452) of the full-length HA protein of the H2 subtype.

[0142] Suitably, the modified HA protein comprises a modification of 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 a full-length H2 amino acid sequence (e.g., as shown 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 position 39 of the full-length H2 amino acid sequence. For other examples, the modified HA protein comprises a glutamic acid modification at amino acid residue position 383 of the full-length H2 amino acid sequence. With reference to certain examples, the modified HA protein comprises a threonine modification at amino acid residue position 388 of the full-length H2 amino acid sequence. In a specific example, the modified HA protein comprises an isoleucine modification at amino acid residue position 390 of the full-length H2 amino acid sequence. For other examples, the modified HA protein comprises an arginine or asparagine modification at amino acid residue position 391 of the full-length H2 amino acid sequence. With reference to various examples, the modified HA protein comprises an alanine modification at amino acid residue position 392 of the full-length H2 amino acid sequence. In some examples, the modified HA protein comprises a tyrosine modification at amino acid residue position 394 of the full-length H2 amino acid sequence. For other examples, the modified HA protein comprises a serine modification at amino acid residue position 450 of the full-length H2 amino acid sequence.

[0143] More specifically, the modification of one or more amino acid residues of the modified HA protein is suitably 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 shown in SEQ ID NO: 12 or SEQ ID NO: 28). Even more specifically, in some examples, the modification of one or more amino acid residues of the modified HA protein is 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 shown in SEQ ID NO: 12 or SEQ ID NO: 28).

[0144] In this regard, the trimer interface region can be divided into multiple discrete portions of the amino acid sequence of the 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 the HA protein. In addition, one or more amino acid residues of the trimer interface region can be found in the stem domain of the HA protein, and one or more additional amino acid residues of the trimer interface region can be found in the globular head domain of the HA protein.

[0145] The term "modified protein", such as "modified HA protein", is to be understood as a protein containing one or more modifications compared to a parent, consensus or wild-type protein (e.g., a wild-type HA protein). The wild-type HA protein sequence can be determined experimentally or can be publicly available in many databases. The present disclosure provides a suitable example, namely SEQ ID NO: 12. Another example is provided herein, namely SEQ ID NO: 28. The term "modification" or "modified" in the context of the present disclosure is to be understood to include chemical modification of proteins and genetic manipulation of the DNA encoding the protein. Such modifications can be replacement of one or more amino acid side chains, one or more substitutions, one or more deletions and / or one or more insertions in the protein of interest. In addition, it is contemplated that these terms include screening and / or selecting existing influenza virus isolates for having or expressing 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; these positions are numbered according to the conventional numbering system for full-length HA proteins, such as the positions exemplified in SEQ ID NO: 12 or SEQ ID NO: 28).

[0146] Suitably, the modified HA protein described herein, for example, exhibits or possesses a changed or regulated stability (e.g., increased stability) in its trimer formation, as compared to the wild-type or unmodified HA protein of the corresponding influenza virus isolate. Therefore, the phrase "increasing the stability of the HA trimer" or similar terms may mean that after modification in accordance with the present disclosure, the modified HA protein can form a more stable homotrimeric arrangement when present in cell culture compared to the starting (unmodified) HA protein. Without being bound by any theory, the modification of the trimer interface region described herein can be used to improve the ability of the modified HA protein monomer to form and maintain a homotrimeric arrangement, such as when the influenza virus isolate expressing such modified HA protein grows in cell culture. Therefore, in a specific example, when the influenza virus isolate expressing the modified HA protein grows in cell culture, the modified HA protein can form a HA trimer. In this regard, the interface region of the modified HA protein can suitably interact with one or more additional HA protein monomers to form its stable trimer arrangement. In some examples, the modified HA protein is capable of forming HA trimers with increased stability when compared to a wild-type or unmodified HA protein (e.g., an HA protein that does not include corresponding modifications to one or more amino acid residues in the interface region of the modified HA protein).

[0147] Thus, in a specific example, an HA trimer containing a modified HA protein as described herein exhibits increased stability, as compared to a corresponding HA trimer containing a wild-type or unmodified HA protein. "Enhanced," "increased," or "up-regulated," as used herein to describe the stability of a modified HA protein or trimer thereof, means that the level of stability is increased when compared to a control or reference sample (e.g., an HA trimer containing a wild-type or unmodified HA protein that does not include one or more modifications described herein).

[0148] The stability of the modified HA protein or more specifically its homotrimer can be evaluated by any method in the art, and can be evaluated directly or indirectly, for example, by its rescue ability, growth ability and / or replication ability when grown in cell culture (e.g., mammalian cells, such as MDCK cells). Therefore, the phrase "increasing the stability of the HA trimer" and the like can mean that compared with viruses with unmodified or wild-type HA proteins, viruses with modified HA proteins according to the present disclosure have improved rescue ability, growth ability and / or replication ability when grown in cell culture (e.g., in mammalian cells such as MDCK cells). Therefore, according to the present disclosure, the term "stability" can also be used in the context of viruses with modified HA proteins. For example, compared with viruses with unmodified or wild-type HA proteins, viruses with modified HA proteins according to the present disclosure may have increased or improved stability because they may have improved rescue ability, growth ability and / or replication ability when grown in cell culture (e.g., in mammalian cells such as MDCK cells). The degree of improvement of any one or more of these abilities (e.g., rescue ability, growth ability and / or replication ability) may vary. For example, such as the degree of improvement in rescue ability, growth ability and / or replication ability can be higher than the level observed in the influenza virus strain expressing the control HA protein (for example, unmodified HA protein or wild-type HA protein) by more 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%. For example, compared with the virus with or expressing unmodified or wild-type HA protein, the degree of improvement may exceed about 50%. In another example, the rescue ability, growth ability and / or replication ability of the increase can be determined by realizing the rescue of the threshold level of the replication virus grown on MDCK cells, which allows the production of the amount of virus for preparing the vaccine on a commercial scale.

[0149] Further exemplary methods for assessing the stability of the modified HA protein or its homotrimer can include computational or 3D modeling, co-immunoprecipitation, pull-down analysis and far-western assay. In this regard, the modified HA protein can be contained in a live virus or attenuated virus or in a virus-like particle (VLP).

[0150] In any method for detecting the stability of a modified HA protein or a homotrimer thereof, the stability level of the modified HA protein can be relative or absolute. In some examples, the stability level of the modified HA protein is 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% higher than the level observed in a control HA protein (e.g., an unmodified HA protein or a wild-type HA protein, such as SEQ ID NO: 12 or SEQ ID NO: 28).

[0151] The modified HA proteins described herein may be considered isolated. For the purposes of this disclosure, "isolated" means a material that has been removed from its natural state or, in other words, has been artificially processed. An isolated material may be substantially or essentially free of components that normally accompany it in its natural state, or may be processed to be in an artificial state with components that normally accompany it in its natural state. An isolated material may be in natural, chemically synthesized, or recombinant form.

[0152] "Protein" means a polymer of amino acids. The amino acids may be natural or unnatural amino acids, D-amino acids or L-amino acids as are well known in the art.

[0153] The term "protein" includes and encompasses "peptides," which are generally used to describe proteins having no more than fifty (50) amino acids (e.g., no more than 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 amino acids and any ranges therein), and "polypeptides," which are generally used to describe proteins having more than fifty (50) amino acids.

[0154] In specific 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, still 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).

[0155] It is contemplated that the modified HA proteins described herein suitably do not include or constitute the amino acid sequence of a wild-type HA protein (e.g., a wild-type HA protein of the H2 subtype). In specific examples, the modified HA proteins described herein share at least 70% or 75%, more specifically at least 80% or 85%, or even more specifically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a reference, unmodified or wild-type amino acid sequence of a HA protein (e.g., an amino acid sequence described in SEQ ID NO: 12 or SEQ ID NO: 28).

[0156] The present disclosure contemplates variants of modified HA proteins described herein. As used herein, a protein, polypeptide or peptide "variant" shares a definable amino acid sequence relationship with a reference amino acid sequence. In a specific example, the reference amino acid sequence is an amino acid sequence of a wild-type HA protein or a modified HA protein, including a mature amino acid sequence (i.e., no signal sequence) and an amino acid sequence (i.e., full-length amino acid sequence) comprising a signal sequence. The reference amino acid sequence can be, for example, an amino acid sequence of any one of SEQ ID NO: 12 or 28. A "variant" protein, polypeptide or peptide can have a deletion of one or more amino acids of a reference amino acid sequence or be replaced by different amino acids. Such modified HA protein variants can include, for example, naturally occurring variants of HA proteins and amino acid residues and / or amino acid sequences of orthologs (e.g., from different influenza strains or different host animals), including their consensus sequences. In this regard, it is contemplated that one or more amino acid variations described herein (e.g., V39I, L219P, V233A, V320A, V320I, K383E, I388T, N390I, K391R, K391N, V392A, S394Y, A405T, R416G, D430N, and F450S of the full-length HA protein of the H2 subtype) may be introduced into any HA protein known in the art. It is also contemplated that some amino acids may be substituted or deleted (i.e., conservative substitutions) without changing the activity of the variant protein. Thus, one or more residues of the modified HA protein described herein (e.g., residues defined by SEQ ID NO: 12 or 28) may be conservatively modified (e.g., by amino acid substitution or deletion) so as to substantially retain the functionality and / or immunogenicity of the modified HA protein. In addition, the modified HA protein may include additional amino acid variations located outside the trimer interface region, such as located in one or more hypervariable regions (e.g., hypervariable head regions) known in the art. Such additional amino acid variations can include, for example, variations at positions V129 (eg, V129I) and / or E184 (eg, E184K) of the full-length H2 HA protein, as described herein.

[0157] It is envisioned that protein or peptide variants share at least 70% or 75%, more specifically at least 80% or 85%, or even more specifically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a reference amino acid sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 28).

[0158] Terms commonly used herein to describe sequence relationships between individual proteins and nucleic acids include "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". Since individual nucleic acids / proteins may each 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 differ 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. For optimal individual sequence alignment, the comparison window may include additions or deletions (i.e., gaps) of about 20% or less compared to the reference sequence. The optimal sequence alignment for comparing the comparison window can be carried out by computerized implementation algorithm (Geneworks program of Intelligenetics; GAP, BESTFIT, FASTA and TFASTA in Wisconsin genetics software package version 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and optimal alignment (that is, causing the highest homology percentage on the comparison window), wherein the optimal alignment is generated by any selected method. Also with reference to, for example, Altschul et al., 1991, the BLAST program family disclosed in Nucl.Acids Res.25 3389, which is incorporated herein by reference. A detailed discussion about sequence analysis can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, the 19.3rd unit (John Wiley&Sons Inc NY, 1995-1999) compiled by Ausubel et al.

[0159] The term "sequence identity" is used in the broadest sense herein, including the number of accurate nucleotide or amino acid matches, depending on the appropriate comparison using a standard algorithm, depending on the degree of identity of the sequence in a comparison window. Therefore, "sequence identity percentage" is calculated by: comparing the sequences of two optimal comparisons in a comparison window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or amino acid residue occurs in two sequences to obtain the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window (i.e., window size), and the result is multiplied by 100 to obtain the sequence identity percentage. For example, "sequence identity" can be understood to mean "matching percentage" calculated by DNASIS computer program (windows version 2.5; Available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).

[0160] It is envisioned that the trimer interface region may be modified at any amino acid residue therein as 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 in question. For example, one class of substitutions is conservative amino acid substitutions. Such substitutions are those that replace a given amino acid in the HA protein with another amino acid having similar properties. Generally considered to be conservative substitutions are the substitution of one with another between the aliphatic amino acids Ala, Val, Leu, and Ile; the interchange of hydroxyl residues Ser and Thr; the exchange of acidic residues Asp and Glu; the substitution between amide residues Asn and Gln; the exchange of basic residues Lys and Arg; and the substitution between aromatic residues Phe and Tyr. Guidance on which amino acid changes may be phenotypically unchanged can be found in, for example, Bowie et al., Science 247: 1306-1310 (1990).

[0161] In certain examples, the modified HA protein includes 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 a respective peptide, polypeptide or protein.

[0162] According to a specific example, the modified HA protein includes mutations or substitutions 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. "Substituted" and "replacement" used herein refer to the replacement of amino acid residues in a parent or reference sequence (e.g., SEQ ID NO: 12 or SEQ ID NO: 28). In some examples, the substitution involves replacing naturally occurring or conservative residues. The modified HA protein herein encompasses replacing one or more amino acid residues in the trimer interface region described herein with any of the remaining nineteen amino acids. In a specific example, the modified HA protein includes replacing an amino acid of one size with an amino acid of a different size (e.g., replacing an amino acid with an amino acid of a relatively large or relatively small size), replacing a hydrophilic amino acid with an amino acid of a different hydrophilicity, replacing an amino acid of a polarity with an amino acid of a different polarity, and / or replacing an acidic amino acid with an amino acid of a different acidity. The modification in the modified HA protein may be or include replacing an amino acid at a specific position identified in any embodiment herein with a respective replacement amino acid identified in any example herein.

[0163] As described above, the modified HA protein may belong to the H2 subtype. In this context, and according to a specific example, one or more amino acid residue positions of the trimer interface region modified (e.g., 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), wherein the amino acid numbering is based on the full-length H2 amino acid sequence (e.g., the amino acid sequence described 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) is the first residue). In other examples, the one or more amino acid residues in the modified trimer interface region are selected from the group consisting of: V39, K383, I388, N390, K391, V392, S394, F450, and any combination thereof, wherein the amino acid numbering is based on the full-length H2 amino acid sequence. According to some examples, the modified HA protein includes one or more substitutions or mutations in 39I, 219P, 233A, 320A, 320I, 383E, 388T, 390I, 391R, 391N, 392A, 394Y, 405T, 416G, 430N, and 450S according to the amino acid numbering of the full-length H2 HA protein (e.g., SEQ ID NO: 12 or SEQ ID NO: 28). In a specific example, the modified HA protein includes one or more substitutions or mutations in V39I, L219P, V233A, V320A, V320I, K383E, I388T, N390I, K391R, K391N, V392A, S394Y, A405T, R416G, D430N, and F450S according to the amino acid numbering of the full-length H2 HA protein (e.g., SEQ ID NO: 12 or SEQ ID NO: 28). For certain examples, the modified HA protein includes one or more substitutions or mutations in 39I, 383E, 388T, 390I, 391R, 391N, 392A, 394Y, and 450S according to the amino acid numbering of the full-length H2 HA protein (e.g., SEQ ID NO: 12 or SEQ ID NO: 28). In various examples, the modified HA protein includes one or more substitutions or mutations of V39I, K383E, I388T, N390I, K391R, K391N, V392A, S394Y, and F450S according to the amino acid numbering of the full-length H2 HA protein (e.g., SEQ ID NO: 12 or SEQ ID NO: 28).

[0164] 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.

[0165] Suitably, 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 suitably comprises a modification, such as a substitution, of 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 alanine with threonine at a position corresponding to position 405 (i.e., A405T) of the full-length H2 HA protein. More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence described in SEQ ID NO:38, or a fragment, variant, or derivative thereof. An influenza virus isolate (e.g., a reassortant influenza virus) expressing such a modified HA protein may be particularly suitable or suitable for large-scale manufacturing.

[0166] Suitably, 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 suitably comprises a modification, such as a substitution, of valine at a position corresponding to position 39 of the full-length H2 HA protein. In certain examples, the modified HA protein comprises a substitution of valine with isoleucine at a position corresponding to position 39 (i.e., V39I) of the full-length H2 HA protein. 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.

[0167] Suitably, 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 suitably comprises one or more of the following modifications (e.g., substitutions): 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 certain examples, the modified HA protein comprises the following substitutions:

[0168] (a) substitution of tyrosine for serine at a position corresponding to position 394 of the full-length H2 HA protein (i.e., S394Y); and / or

[0169] (b) Substitution of arginine with alanine at the position corresponding to position 416 (ie, R416G).

[0170] In some examples, the modified HA protein comprises modification of one or more amino acid residues at the following positions of the full-length H2 HA protein: (a) S394; (b) R416; or (c) S394 and R416. In other examples, the modified HA protein comprises the following modifications of the full-length H2 HA protein: (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 described in SEQ ID NO: 36, or a fragment, variant, or derivative thereof. Likewise, influenza virus isolates (e.g., reassortant influenza viruses) expressing such modified HA proteins may be particularly suitable or amenable to large-scale manufacturing thereof.

[0171] Suitably, 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 suitably comprises one or more of the following modifications (e.g., substitutions): 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 certain examples, the modified HA protein comprises the following substitutions:

[0172] (a) replacing valine with alanine at the position corresponding to position 233 of the full-length H2 HA protein (ie, V233A); and / or

[0173] (b) Substitution of valine with alanine at the position corresponding to position 320 (ie, V320A).

[0174] In some examples, the modified HA protein comprises modifications of one or more amino acid residues at the following positions of the full-length H2 HA protein: (a) V233; (b) V320; or (c) V233 and V320. In other examples, the modified HA protein comprises the following modifications of the 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.

[0175] Suitably, 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 suitably comprises a modification, such as a substitution, of lysine 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 lysine with glutamic acid at a position corresponding to position 383 (i.e., K383E) of the full-length H2 HA protein. More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence described in SEQ ID NO: 48, or a fragment, variant, or derivative thereof.

[0176] Suitably, the modified HA protein comprises a modification, e.g., a substitution, at a position corresponding to position 320 of the full-length H2 HA protein, at a position corresponding to position 390, and / or at a position corresponding to position 391. More specifically, the modified HA protein suitably comprises one or more of the following modifications (e.g., substitutions): a valine at a position corresponding to position 320 of the full-length H2 HA protein; an asparagine at a position corresponding to position 390; and a lysine at a position corresponding to position 391. In certain examples, the modified HA protein comprises the following substitutions:

[0177] (a) substitution of isoleucine for valine at the position corresponding to position 320 of the full-length H2 HA protein (i.e., V320I);

[0178] (b) substituting isoleucine for asparagine at the position corresponding to position 390 (ie, N390I); and / or

[0179] (c) Substitution of lysine with arginine at the position corresponding to position 391 (ie, K391R).

[0180] In some examples, the modified HA protein comprises modifications of one or more amino acid residues at the following positions of the 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 the following modifications of the full-length H2 HA protein: (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 depicted in SEQ ID NO: 46, or a fragment, variant, or derivative thereof.

[0181] Suitably, 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 suitably 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 leucine with proline at a position corresponding to position 219 (i.e., L219P) of the full-length H2 HA protein. More specifically, the modified HA protein may comprise, consist of, or consist essentially of the amino acid sequence described in SEQ ID NO: 50, or a fragment, variant, or derivative thereof.

[0182] Suitably, the modified HA protein comprises a modification, e.g., 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 suitably comprises one or more of the following modifications (e.g., substitutions): 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 certain examples, the modified HA protein comprises the following substitutions:

[0183] (a) substitution of valine with alanine at the position corresponding to position 233 of the full-length H2 HA protein (i.e., V233A);

[0184] (b) substituting serine for phenylalanine at the position corresponding to position 450 (ie, F450S); and / or

[0185] (c) Substitution of lysine with asparagine at the position corresponding to position 391 (ie, K391N).

[0186] In some examples, the modified HA protein comprises modifications of one or more amino acid residues at the following positions of the 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 the following modifications of the full-length H2 HA protein: (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 depicted in SEQ ID NO: 54, or a fragment, variant, or derivative thereof.

[0187] Suitably, the modified HA protein comprises a modification, e.g., 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 suitably comprises one or more of the following modifications (e.g., substitutions): 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 certain examples, the modified HA protein comprises the following substitutions:

[0188] (a) substituting serine for phenylalanine at a position corresponding to position 450 of the full-length H2 HA protein (ie, F450S); and / or

[0189] (b) Substitution of lysine with asparagine at the position corresponding to position 391 (ie, K391N).

[0190] In some examples, the modified HA protein comprises modification of one or more amino acid residues at the following positions of the full-length H2 HA protein: (a) F450; (b) K391; or (c) F450 and K391. In other examples, the modified HA protein comprises the following modifications of the full-length H2 HA protein: (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.

[0191] Suitably, the modified HA protein comprises a modification, e.g., 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 suitably comprises one or more of the following modifications (e.g., substitutions): 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 certain examples, the modified HA protein comprises the following substitutions:

[0192] (a) replacing valine with isoleucine at the position corresponding to position 39 of the full-length H2 HA protein (ie, V39I); and / or

[0193] (b) Substitution of aspartic acid with asparagine at the position corresponding to position 430 (ie, D430N).

[0194] In some examples, the modified HA protein comprises modification of one or more amino acid residues at the following positions of the full-length H2 HA protein: (a) V39; (b) D430; or (c) V39 and D430. In other examples, the modified HA protein comprises the following modifications of the full-length H2 HA protein: (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.

[0195] Suitably, the modified HA protein comprises a modification, e.g., a substitution, at a position corresponding to position 39 of the full-length H2 HA protein, at a position corresponding to position 388, and / or at a position corresponding to position 392. More specifically, the modified HA protein suitably comprises one or more of the following modifications (e.g., substitutions): 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. In certain examples, the modified HA protein comprises the following substitutions:

[0196] (a) substitution of isoleucine for valine at the position corresponding to position 39 of the full-length H2 HA protein (i.e., V39I);

[0197] (b) substituting threonine for isoleucine at the position corresponding to position 388 (ie, I388T); and / or

[0198] (c) Substitution of valine with alanine at the position corresponding to position 392 (ie, V392A).

[0199] In some examples, the modified HA protein comprises modifications of one or more amino acid residues at the following positions of the 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 the following modifications of the full-length H2 HA protein: (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 depicted in SEQ ID NO: 58, or a fragment, variant, or derivative thereof.

[0200] Suitably, the modified HA protein comprises a modification, e.g., a substitution, at a position corresponding to position 39 of the full-length H2 HA protein, at a position corresponding to position 391, and / or at a position corresponding to position 430. More specifically, the modified HA protein suitably comprises one or more of the following modifications (e.g., substitutions): a valine at a position corresponding to position 39 of the full-length H2 HA protein; a lysine at a position corresponding to position 391; and an aspartic acid at a position corresponding to position 430. In certain examples, the modified HA protein comprises the following substitutions:

[0201] (a) substitution of isoleucine for valine at the position corresponding to position 39 of the full-length H2 HA protein (i.e., V39I);

[0202] (b) substituting asparagine for lysine at the position corresponding to position 391 (ie, K391N); and / or

[0203] (c) Substitution of aspartic acid with asparagine at the position corresponding to position 430 (ie, D430N).

[0204] In some examples, the modified HA protein comprises modifications of one or more amino acid residues at the following positions of the 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 the following modifications of the full-length H2 HA protein: (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 depicted in SEQ ID NO: 60, or a fragment, variant, or derivative thereof.

[0205] It is further contemplated that, as compared to individual wild-type HA proteins, modified HA proteins with one or more additional amino acid modifications or substitutions at other positions. Therefore, as compared to individual wild-type HA proteins, modified HA proteins may have at least about 2, 3, 4, 5, 6, 7 or more different residues at other positions. It will be appreciated by those skilled in the art that the number of additional positions that may have amino acid substitutions will depend on the wild-type HA protein or encoding nucleic acid used to generate the variant. For this reason, the modified HA protein provided herein may be derived from any known HA sequence in 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 / ). In addition, databases of influenza HA wild-type or MDCK cell and egg passage sequences can be obtained from the EpiFlu database of the Global Shared Influenza Database Initiative (GIS AID).

[0206] Modified HA proteins can be produced by any method known in the art, including but not limited to chemical synthesis, recombinant DNA technology (including site-specific mutagenesis), or replacement of a portion of the HA coding sequence with a portion including characteristic residues, and proteolytic cleavage to produce peptide fragments.

[0207] Chemical synthesis includes solid phase synthesis and liquid phase synthesis. Such methods are well known in the art, but can refer to SYNTHETIC VACCINES, Nicholson compiles (Blackwell Scientific Publications) Chapter 9 and CURRENT PROTOCOLS IN PROTEIN SCIENCE, Coligan et al. compile (John Wiley & Sons, Inc. NY USA 1995-2008) The example of chemical synthesis technology provided in Chapter 15. In this regard, also refer to International Publication WO 99 / 02550 and International Publication WO 97 / 45444.

[0208] Those skilled in the art can conveniently prepare recombinant proteins using standard protocols, such as described in, for example, Sambrook et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), specifically Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al., ed. (John Wiley & Sons, Inc. NY USA 1995-2008), specifically Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE, Coligan et al., ed. (John Wiley & Sons, Inc. NY USA 1995-2008), specifically Chapters 1, 5, and 6. Typically, recombinant protein preparation includes expressing a nucleic acid encoding the protein in a suitable host cell. Modified HA proteins can be obtained, for example, by mutating one or more genes (i.e., viral gene segments) encoding the HA protein of interest by site-directed or random mutagenesis. Such mutations may include point mutations, deletion mutations, and insertion mutations. For example, one or more point mutations (eg, replacing one or more amino acids with one or more different amino acids) may be used to construct the modified HA proteins described herein.

[0209] According to a specific example, the modified HA protein has been modified or mutated by one or more passages (e.g., 1, 2, 3, 4, 5, 6, 7, etc. passages), e.g., by serial passages, of an influenza virus isolate expressing an unmodified or wild-type HA protein in cells and / or eggs (e.g., those provided herein). In some examples, the modified HA protein has been modified or mutated by one or more passages of an influenza virus isolate expressing an unmodified or wild-type HA protein in mammalian cells (e.g., MDCK cells).

[0210] Suitably, the modified HA proteins described herein are immunogenic. Thus, the modified HA proteins may be suitable for use as immunogens in vaccines to treat or prevent 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 produces an immune response.

[0211] In specific examples, the modifications provided herein do not or do not significantly change or modulate (i.e., increase or decrease) the immunogenicity / antigenicity of the modified HA protein (e.g., relative to or compared to its wild-type or unmodified version). To this end, it is noteworthy that the trimer interface region is internalized when the HA trimer is formed and is generally inaccessible to the host's immune system during influenza virus infection. The immunogenicity or antigenicity of the modified HA protein can be assessed by any method known in the art, such as by using standard immunoassays and / or determining predicted or actual T cell reactivity to determine the presence or amount of neutralizing antibodies or antibodies that recognize the modified HA protein (e.g., in a trimer arrangement).

[0212] It is further contemplated that the modified HA protein may include one or more further modifications known in the art. For example, the modified HA protein may be further modified to remove determinants that cause the virus to be highly pathogenic (e.g., high base regions around the HA1 / HA2 cleavage sites). In some examples, the modified HA protein is further modified to remove the polybasic cleavage site therein. This site allows HA to mature independently of trypsin and generally defines "highly pathogenic" avian influenza and "lowly pathogenic" avian influenza.

[0213] The modified HA protein can also be manipulated to form a chimeric HA protein (i.e., containing amino acid sequences from more than one influenza virus strain). For example and appropriately, in addition to one or more modified amino acid residues in the trimer interface region, the chimeric HA may contain the cytoplasmic portion or the cytoplasmic and transmembrane portion of the HA from one influenza strain, and at least the extracellular antigenic portion of the HA from a different influenza strain. This approach has previously been described as a technique for producing influenza viruses containing antigenic portions of HA proteins in the absence of a low yield of unmodified HA segments.

[0214] 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 virus strain. In such examples, although the modified HA protein sequence is a non-chimeric sequence, it may comprise other modifications as described herein.

[0215] Coding nucleic acid

[0216] The present disclosure also provides isolated nucleic acids encoding the modified HA proteins described herein.

[0217] As used herein, the term "nucleic acid" designates 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 may also be DNA-RNA hybrids. Nucleic acids comprise a nucleotide sequence that typically includes nucleotides comprising A, G, C, T or U bases. However, the nucleotide sequence may include other bases, such as modified purines (e.g., inosine, methylinosine and methyladenosine) and modified pyrimidines (e.g., thiouridine and methylcytosine).

[0218] It is contemplated that the encoding nucleic acids described herein may encode the modified HA proteins of the present disclosure directly (e.g., via viral segments or viral mRNA) or indirectly (e.g., via viral segments or complementary DNA sequences encoding viral segments). In specific examples, the isolated nucleic acids are or comprise HA viral segments (i.e., influenza RNA segments) encoding the modified HA proteins provided herein. Suitably, the HA viral segments comprise, consist of, or consist essentially of the nucleotide sequences described 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 fragments, derivatives, or variants thereof. In some examples, the isolated nucleic acids are or comprise nucleotide sequences complementary to the HA viral segments encoding the modified HA proteins provided herein. In alternative examples, the isolated nucleic acids are or comprise DNA or cDNA sequences encoding HA viral segments (i.e., viral RNA), which encode the modified HA proteins provided herein. In various examples, the isolated nucleic acid is or comprises a viral mRNA sequence encoding a modified HA protein provided herein.

[0219] As used herein, a "polynucleotide" is generally a nucleic acid having eighty (80) or more consecutive nucleotides, while an "oligonucleotide" generally has less than eighty (80) consecutive nucleotides. A "primer" is generally a single-stranded oligonucleotide, preferably having 15 to 50 consecutive nucleotides, which is capable of annealing to a complementary nucleic acid "template" and being cleaved by a DNA polymerase (e.g., Taq polymerase, RNA-dependent DNA polymerase, or Sequenase TM ) in a template-dependent manner. A "probe" may be a single-stranded or double-stranded oligonucleotide or polynucleotide that is appropriately labeled for use in detecting complementary sequences in Northern or Southern blotting.

[0220] Fragments, variants and derivatives of isolated nucleic acids are also contemplated herein. Variants may include nucleotide sequences having at least 70%, at least 75%, preferably at least 80%, at least 85%, more preferably at least 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity with any nucleotide sequence encoding a variant or modified HA protein disclosed herein (e.g., SEQ ID NO: 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65 or 67). Nucleic acid derivatives may include chemically modified nucleic acids, modified internucleotide linkages, nucleic acid analogs, artificial nucleic acids and combinations thereof known in the art.

[0221] Fragments of the isolated nucleic acid may 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 a modified HA protein (e.g., encode at least a portion of a trimer interface region of a HA protein). In general, a segment may contain 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, 510, 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 encoding a portion of a modified HA protein described herein (e.g., SEQ ID NO: 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65 or 67).

[0222] In specific examples, the isolated nucleic acids described herein can be modified to include or alternatively to exclude the 5' non-coding region (e.g., AGC[A / G]AAAGCAG G (SEQ ID NO:73), wherein [A / G] indicates a nucleotide variation of A or G at this position) and / or the 3' non-coding region (e.g., CCTTGTTTCTACT (SEQ ID NO:74)) of influenza virus, as known in the art.

[0223] The present disclosure also provides nucleic acids that have been modified, for example, by exploiting codon sequence redundancy. In more specific examples, codon usage can be modified to optimize expression of a nucleic acid in a specific organism or cell type.

[0224] 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).

[0225] Nucleic acids of the present disclosure can be produced, isolated, detected by using nucleic acid sequence amplification techniques and / or subject to recombinant DNA techniques.

[0226] 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-β replicase amplification, recombinase polymerase amplification (RPA) and helicase-dependent amplification.

[0227] Genetic constructs

[0228] The present disclosure also provides a genetic construct comprising the aforementioned isolated nucleic acid. The genetic construct can be a vector.

[0229] In a specific example, the genetic construct comprises an isolated nucleic acid operably linked or linked to one or more other genetic components. The genetic construct may be suitable for therapeutic delivery of isolated nucleic acids (e.g., DNA or RNA vaccines) or for recombinant production of the modified HA protein of the present disclosure in a host cell. In addition, the genetic construct can be used to produce or generate influenza viruses (e.g., variants or modified strains of influenza virus isolates or reassortant influenza virus isolates) expressing modified HA proteins.

[0230] In a broad sense, the genetic construct can be in the form of a plasmid, phage, clay, yeast or bacterial artificial chromosome, or comprise the genetic components of a plasmid, phage, clay, yeast or bacterial artificial chromosome, as is well known in the art. The genetic construct may be suitable for maintaining and breeding isolated nucleic acids in bacteria or other host cells, suitable for operating and / or expressing nucleic acids of the present disclosure or encoded proteins by recombinant DNA techniques. The vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of DNA and RNA in the same chain, a polylysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, etc. Such vectors may or may not replicate autonomously.

[0231] For the purpose of host cell expression, the genetic construct is an expression construct. Suitably, the expression construct comprises a nucleic acid of the present disclosure operably linked to one or more additional sequences in an expression vector. A "vector" can be an extrachromosomal vector, such as a plasmid, that replicates autonomously, or a vector that is integrated into the host genome. The expression construct can alternatively be a linear expression construct. Such linear expression constructs will generally not contain any amplification and / or selection sequences. However, linear constructs comprising such amplification and / or selection sequences are also within the scope of the present disclosure. For example, a linear expression construct can include a separate linear expression construct for each viral segment. It is also possible to include more than one viral segment, such as two, three, four, five or six viral segments on the same linear expression construct.

[0232] The expression construct suitable for the disclosed method can be a unidirectional or bidirectional expression construct. Since influenza virus requires protein to be infectious, it is generally preferred to use a bidirectional expression construct because this can reduce the total number of expression constructs required for the host cell. The bidirectional expression construct contains at least two promoters, which drive expression in different directions (i.e., 5' to 3' and 3' to 5') from the same construct. The two promoters can be operably linked to different chains of the same double-stranded DNA. Suitably, one of the promoters is a pol I promoter, and at least one other promoter is a pol II promoter. Therefore, the disclosed method can utilize at least one bidirectional expression construct, wherein at least one gene or cDNA is located between an upstream pol II promoter and a downstream non-endogenous pol I promoter. The gene or cDNA is transcribed from the pol II promoter to produce a capped positive viral mRNA, which can be translated into protein, while transcription from a non-endogenous pol I promoter produces a negative-sense viral RNA (vRNA).

[0233] "Operably linked" means that the additional nucleotide sequence is positioned relative to the nucleic acid of the present disclosure, preferably to initiate, regulate or otherwise control transcription.

[0234] The regulatory nucleotide sequence is generally suitable for the host cell used for expression. As described herein, various types of suitable expression vectors and suitable regulatory sequences suitable for various host cells are known in the art. Expression vectors can be designed to express the modified HA protein 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, which is incorporated herein by reference in its entirety), yeast cells, plant cells, algae or mammalian cells).

[0235] Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosome binding site, a polyadenylation sequence, a transcription start and stop sequence, a translation start and stop sequence, and an enhancer or activator sequence. The present disclosure contemplates constitutive, repressible or inducible promoters known in the art.

[0236] In some examples, the genetic construct includes one or more untranslated 5' and / or 3' regions that are operably linked or attached to the HA viral segment encoding the modified HA protein. To this end, the UTR may be derived from the same influenza virus isolate as the source of the modified HA protein or a different influenza virus isolate.

[0237] The expression construct may also include additional nucleotide sequences encoding a fusion partner (usually provided by the expression vector) so that the recombinant protein is expressed as a fusion protein.

[0238] The expression construct may also include additional nucleotide sequences encoding a selectable marker, such as amp R 、neo R or R , but not limited to this.

[0239] Suitably, the genetic constructs provided herein are suitable or applicable to the production or generation of reassortant influenza viruses expressing modified HA proteins by reverse genetics methods or hybrid reverse genetics-classical reassortment methods. Thus, any method of introducing expression constructs known in reverse genetics techniques can be used to introduce one or more genetic constructs provided herein into a host cell.

[0240] 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 by using electroporation, DEAE-dextran, calcium phosphate precipitation, liposomes, microinjection or microparticle bombardment. In some instances, the genetic constructs can be in the form of naked nucleic acids. Naked nucleic acids may have been purified from influenza viruses. 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 replicons based on alphaviruses.

[0241] Genetic constructs provided herein may include RNA transcription terminator sequences.Terminator sequences may be endogenous terminator sequences or non-host cell endogenous terminator sequences.Suitable terminator sequences are apparent to those skilled in the art and include, but are not limited to, RNA polymerase I transcription terminator sequences, RNA polymerase II transcription terminator sequences and ribozymes.In addition, expression constructs may contain one or more polyadenylation signals for mRNA, particularly at the gene ends that are controlled to express by pol II promoters.

[0242] In another form, the present disclosure also provides a plurality of genetic constructs, including a genetic construct containing a nucleic acid encoding a modified HA protein as described herein and one or more additional genetic constructs that can be used to prepare reassortant viruses, including 6:1:1 reassortants, 6:2 reassortants, and 7:1 reassortants. The additional genetic constructs may contain or encode one or more of the NA, PA, PB1, PB2, NP, NS, and M viral segments (i.e., encoding one or more of the NA, PA, PB1, PB1-F2, PB2, NP, NS1, NEP, M1, and M2 viral proteins).

[0243] Host cells

[0244] The disclosure also provides host cells transformed with the isolated nucleic acids and / or genetic constructs described herein.

[0245] In a related form, the present disclosure relates to a method for 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).

[0246] The host cell may be any host cell known in the art. A well-known method of growing influenza virus is to use specific pathogen-free (SPF) embryonated hen eggs, where the virus is inoculated into the egg contents (i.e., allantoic fluid), grown and purified therefrom. Influenza virus can also be grown in animal cell culture, and this culture method is preferred for reasons of replication accuracy, speed, and patient allergies.

[0247] With reference to the cells described herein, the methods of the present invention will generally use cell lines, but primary cells may also be used as an alternative. Such cells or cell lines may 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 NSO cells, 45.6TG1.7 cells, AF-2 clone 9B5 cells, AF-2 clone 9B5 cells, J558L cells, MOPC 315 cells, MPC-11 cells, NCI-H929 cells, NP cells, NSO / 1 cells, P3 NS1 Ag4 cells, P3 / NS1 / 1-Ag4-1 cells, P3U1 cells, P3X63Ag8 cells, P3X63Ag8.653 cells, P3X63Ag8U.1 cells, RPMI 8226 cells, Sp20-Ag14 cells, U266B1 cells, X63AG8.653 cells, Y3.Ag.1.2.3 cells and YO cells. Non-limiting examples of insect cells include SJ9, SJ21, Trichoplusia ni, Spodoptera fugiperda, and Bombyx mori. Exemplary plant cell systems for expressing modified HA proteins are provided in U.S. Pat. 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.

[0248] In a specific example, the host cell is a mammal. Suitable mammalian cells include, but are not limited to, hamsters, cattle, primates (including humans and monkeys) and dog cells. Various cell types can be used, such as kidney cells, fibroblasts, retinal cells and lung cells known in the art. An example of a suitable hamster cell is a cell line named BHK21 or HKCC. Suitable monkey cells include African green monkey cells, such as kidney cells in 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 dog cells include canine kidney cells, such as cells in the CLDK and MDCK cell lines (WO97 / 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.

[0249] It is contemplated that the cells or cell lines described herein may be suitable for expressing modified HA proteins, for example, for producing subunit vaccines containing such proteins. According to other examples, the cells or cell lines described herein are suitable for influenza virus growth. Such cell lines may include: MDCK cells from Madin Darby canine kidneys; Vero cells from African green monkeys (Cercopit hecus aethiops) kidneys; or PE R.C6 cells from human embryonic retinoblasts (Pau et al. (2001) Vaccine 19: 2716-21). These cell lines are widely available, for example, from the American Type Cell Culture (ATCC) Collection, Coriell Cell Bank, and the European Collection of Cell Cultures (ECACC). Alternative cell lines may include avian cell lines (see, for example, WO2003 / 076601; WO2005 / 042728; WO2003 / 043415), including cell lines derived from ducks (e.g., duck retinal cells) or hens (e.g., chicken embryonic fibroblasts (CEF)). Examples include avian embryonic stem cells, including the EBx cell line, EB45, EB14, EB14-074, and EB66 derived from chicken embryonic stem cells.

[0250] Suitably, the cell or cell line is an MDCK cell derived from Madin Darby canine kidney. Original MDCK cells can be obtained from ATCC in the form of CCL-34. Derivatives of MDCK cells can also be used. For example, MDCK cell lines can adapt to suspension culture growth (e.g., "MDCK 33016", deposited as DSMACC 2219). Similarly, WO2001 / 064846 discloses a MDCK derivative cell line ("B-702", deposited as FERM BP-7449) grown in suspension in serum-free culture. 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 the cell lines provided herein, can be used in the methods of the present disclosure.

[0251] For the growth or propagation of viruses on cell lines (e.g., MDCK cells), influenza viruses can be grown on cells in suspension or adherent culture. In addition, the cells described herein can be cultured in various serum-free media or substantially serum-free media known to those skilled in the art (e.g., Iscove's medium, ultra CHO medium (Bio Whittaker), EX-CELL (JRH Biosciences)). In addition, cells for replication can alternatively be cultured in serum-containing media (e.g., MEM or DMEM media 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 process of the methods described herein can be containers known to those skilled in the art, such as spinner bottles, roller bottles, or fermenters.

[0252] In a specific example, the cells are suitably grown in serum-free medium and / or protein-free medium, for example, for cell proliferation and / or to support influenza virus replication. If the culture medium does not contain additives from human or animal-derived serum or is substantially free of additives (e.g., less than 0.5%, 0.25%, or 0.1% by weight), in the context of the present disclosure, the culture medium is referred to as serum-free medium. Protein-free refers to a culture medium that does not contain proteins, growth factors, other protein additives, and non-serum proteins when cells are manipulated therein, but may optionally include proteins necessary for viral growth, such as trypsin or other proteases. The cells grown in such cultures themselves naturally contain proteins.

[0253] Cell lines that support influenza virus replication are suitable for culturing at a temperature below 37°C during viral replication (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). When the virus is grown in the cell line, the culture medium and the virus inoculum used to start the culture are suitably free of (e.g., will be detected and give a negative result for contamination) contaminating viruses, such as herpes simplex virus, respiratory syncytial virus, parainfluenza virus 3, SARS coronavirus, adenovirus, rhinovirus, reovirus, polyoma virus, gemoma virus, circovirus, and / or parvovirus.

[0254] Influenza virus

[0255] In one form, the present disclosure provides an isolated influenza virus comprising an HA viral segment encoding a modified HA protein, such as the HA protein described above, comprising an amino acid sequence in which one or more amino acid residues in the trimer interface region are modified.

[0256] Suitably, the HA viral segment has been modified, for example, by one or more passages in a cell and / or a recombination method, to encode a modification of one or more amino acid residues in the trimer interface region. More specifically, the HA viral segment has been appropriately modified by a recombinant method prior to integration into an isolated influenza virus to encode one or more modified amino acid residues of a modified HA protein. In a specific example, the HA viral segment comprises a nucleotide sequence selected from the group consisting of SEQ ID NQ: 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, consisting of them or consisting essentially of them. In various examples, the HA viral segment encodes an amino acid sequence as described in any one of 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.

[0257] Influenza virus is an enveloped RNA virus belonging to the family Orthomyxoviridae (Palese and Shaw (2007) Orthomyxoviridae: The Viruses and Their Replication, 5th edition, Fields' Virology, edited by BN Fields, DMKnipe and PM Howley, Wolters Kluwer Health / Lippincott Williams & Wilkins, Philadelphia, USA, pp. 1647-1689). Influenza A and B viruses are major pathogens in humans, causing respiratory diseases ranging in severity from subclinical infection to primary viral pneumonia, and can even lead to death. The clinical effects of infection vary depending on the virulence of the influenza virus strain and the exposure, medical history, age and immune status of the host. The natural host of influenza virus is mainly avian, but influenza virus, particularly influenza A virus (including those influenza A viruses of avian origin), can also infect humans and other animal hosts (bats, dogs, pigs, horses, marine mammals and mustelids) and cause disease.

[0258] Influenza viruses referred to herein encompass any virus type, subtype or strain, including but not limited to naturally occurring strains, variants or mutants, mutagenized viruses, reassortant viruses and / or genetically modified viruses (e.g., modified by reverse genetics or recombinant DNA technology).

[0259] Influenza virus of the present disclosure can be influenza A virus or influenza B virus. According to some examples, influenza virus is influenza A virus. According to alternative examples, influenza virus is influenza B virus. Influenza A virus or influenza B virus can be any strain. For example, influenza A virus provided herein can include HA subtypes selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16. In a specific example, influenza A virus is H2 subtype (i.e., containing modified H2 subtype HA protein). In addition, such viruses may contain influenza A virus NA subtype N1, N2, N3, N4, N5, N6, N7, N8 or N9. In some examples, NA protein belongs to N1, N2, N3 or N5 subtype. More specifically, NA protein can belong to N1, N2 or N3 subtype. In some instances, the influenza A virus can 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 instances, the influenza A virus is an H2N1 strain. In an alternative instance, the influenza A virus is an H2N2 strain. In other instances, the influenza A virus is an H2N3 strain.

[0260] In view of the above, the influenza virus referred to herein is appropriately a pandemic influenza strain. In this regard, the modified HA protein provided herein may be derived at least in part from a pandemic influenza strain. The term "pandemic influenza strain" used herein refers to an influenza strain associated with or susceptible to an influenza disease outbreak. In general, the characteristics of an influenza strain that is likely to cause a pandemic outbreak are: (a) compared to the hemagglutinin in the currently circulating human strain, it contains a new hemagglutinin, i.e., a hemagglutinin that has not been apparent in the human population for more than a decade (e.g., H2), or a hemagglutinin that has not been seen in the human population at all before (e.g., H5, H6 or H9, which are usually only found in bird populations), so that the human population will be immune to the hemagglutinin of the strain; (b) it can be horizontally transmitted in the human population; and (c) it is pathogenic to humans. Therefore, the modified HA protein may be suitable for producing reassortant viruses for use in vaccines to prevent potential pandemic strains that may or have been transmitted to humans from non-human animal populations. Thus, in some instances, the term pandemic influenza virus strain refers to an influenza A virus strain. Suitable pandemic strains include, but are not limited to, H5N1, H9N2, H7N7, H2N2, H2N3, H7N1, and H1N1. Other suitable human pandemic strains include H7N3, H10N7, and H5N2. In one example, the pandemic strain may be an influenza A H1 subtype other than the (H1N1)pdm09 strain.

[0261] The present disclosure contemplates isolated recombinant influenza viruses.A "recombinant" virus is a virus that has been manipulated in vitro, such as using recombinant DNA techniques, to alter the viral genome.

[0262] Suitably, the influenza virus is a reassortant virus, such as a recombinant reassortant virus. The term "reassortant virus" refers to a virus containing genetic material composed of genetic material of at least two donor viruses (e.g., one or more gene segments from a first parent influenza virus strain (donor, backbone or seed strain), and one or more gene segments from a second parent influenza virus strain (vaccine strain)). When the reassortant virus is used to prepare a vaccine composition, its genetic material generally contains at least the HA gene from a seasonal or pandemic virus, while other genes (i.e., backbone genes) are from one or more other donors or seed viruses, which are selected based on the ability to grow easily on a production matrix (e.g., the allantoic cavity of an embryonated hen's egg or a permissive cell line) used to make an influenza vaccine and / or low or no pathogenicity to humans. Examples of donor or seed viruses 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 hybrid versions thereof.

[0263] Influenza donor strains are strains that usually provide backbone segments in reassortment influenza viruses, even if they may sometimes provide NA segments of the virus. Vaccine strains are influenza strains that provide HA and / or NA segments. In general, the HA and NA segments in the reassortment influenza virus will all come from vaccine strains. Vaccine strains are usually circulating strains, such as seasonal or pandemic influenza strains. Suitably, vaccine strains are different or heterologous from donor strains. The genome segments present in the reassortment virus can be described with a gene constellation ratio, which represents the number of segments provided by each parent influenza virus strain. For example, when the reassortment virus contains genome segments from two parent influenza virus strains (such as donor strains and vaccine strains), its gene constellation ratio may be 1:7, 2:6, 3:5, 4:4, 5:3, 6:2 or 7:1.

[0264] Typically, most of the gene segments of the reassortant virus are from the donor strain, because it is desirable to utilize the characteristics of the donor strain (e.g., to improve replication and / or yield in cell culture) by reassorting the donor strain segments with the vaccine strain segments. In specific examples, the reassortant influenza viruses produced by the methods provided herein have a gene constellation ratio of 5:3, 6:2, or 7:1, where the first number of the ratio represents the number of segments from the donor strain, and the second number of the ratio represents the number of segments from the vaccine strain.

[0265] According to a specific example, the donor strain is a strain that has been approved by regulatory authorities for use in vaccine manufacturing. Using a donor strain with regulatory approval is advantageous because the reassortant virus generated by the methods provided herein can be used to prepare a vaccine that may be more easily marketed than if the donor strain had not been approved by pre-existing regulatory authorities.

[0266] In certain examples, the gene constellation ratio of the reassortant influenza virus is 6: 2. For these examples, the reassortant influenza virus comprises 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 as provided herein.

[0267] In other examples, the gene constellation ratio of the reassortant influenza virus is 7: 1. In such examples, the reassortant influenza virus may include six backbone segments from the donor strain, the HA segment from the vaccine strain, and the NA segment from the donor strain. In other words, the reassortant influenza virus includes the 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 the HA segment from the donor strain, and the NA segment from the vaccine strain. In other words, the reassortant influenza virus includes the NA segment from the vaccine strain, and the remaining seven segments are from the donor strain.

[0268] In further examples, the gene constellation ratio of the reassortant influenza virus is 5:3. 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 a backbone segment (i.e., a segment selected from the group consisting of PB1, PB2, PA, NP, M and NS). In a specific 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.

[0269] According to a specific embodiment, the isolated reassortant influenza virus comprises:

[0270] (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);

[0271] (b) an HA viral segment (including chimeric versions thereof) derived from a second influenza virus isolate (e.g., a vaccine virus), wherein the HA viral segment has been modified to encode a modification of one or more amino acid residues in its trimer interface region; and

[0272] (c) an NA viral segment optionally derived from a first influenza virus isolate, a second influenza virus isolate, or a third influenza virus isolate, including chimeric versions thereof.

[0273] To this end, the NA viral segment encoding the modified HA protein and the HA viral segment can be from the same influenza virus isolate. Alternatively, the NA viral segment can be from the same influenza virus isolate (e.g., donor virus) as the backbone viral segment. In addition, the NA viral segment can be from an influenza virus isolate different from the influenza virus isolate from which the backbone viral segment and the HA viral segment are derived.

[0274] Suitably, the isolated influenza viruses provided herein can grow or replicate in cells, more specifically in mammalian cells such as MDCK cells. For this reason, the isolated influenza viruses may be able to form virions with stabilized or more stable HA trimers when cultured in cells (e.g., when compared to isolated influenza viruses with unmodified HA viral segments encoding unmodified HA proteins). In a specific example, when compared to isolated influenza viruses with unmodified HA viral segments encoding unmodified HA proteins, the isolated influenza viruses provided herein can enhance or improve growth or replication in cells (e.g., MDCK cells).

[0275] Therefore, in another form, the present disclosure provides a method for improving the growth of influenza virus in a cell, the method comprising the step of modifying the influenza virus to express a modified HA protein (e.g., a modified HA protein described herein), 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.

[0276] It is envisioned that the methods of the invention may include the further step of passage 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. passages). As demonstrated by the passage experiments in Example 1, this may help to induce further mutations (i.e., in addition to the mutations initially included 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 specifically H2 subtype influenza viruses.

[0277] Thus, for vaccine viruses to be grown or passaged in cells (e.g., MDCK cells), for example, by encoding a polypeptide having a specific amino acid residue modification or substitution in HA (e.g., V39I, L219P, V233A, V320A, V320I, K383E, I388T, N390I, K391R, K391N, V392A, S394Y, A405T, R416G, D430N, F450S, or any combination thereof, wherein the numbering is based on the full-length H2) Mutation / substitution or selection of the HA viral segment that modifies the HA protein can modify 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 or any combination thereof of the HA protein of the H2 subtype), which can result in increased stability or stabilization of HA trimer formation and / or higher viral titers at the end of infection.

[0278] In a particular example, the present disclosure provides an isolated influenza virus comprising PA, PB1, PB2, NP, NS, M and NA viral segments (including heterologous or chimeric versions thereof) and a HA viral segment (including heterologous or chimeric versions thereof), wherein the HA viral segment encodes an HA selected to encode one or more modified amino acid residues of H2 (e.g., V39, L219, V233, V320, K383, I388, N390, K391, V392, S394, A405, R416, D430, F450, or any combination thereof), wherein the numbering is based on full-length H2, wherein the recombinant influenza virus may be capable of growing in cell culture, have enhanced growth / replication in cell culture and / or be capable of forming HA trimers with enhanced stability, for example, during vaccine production. More specifically, the isolated influenza virus can comprise an HA viral segment encoding an HA protein comprising one or more modified amino acid residues in the lower region of its stem domain (e.g., one or more of V39, K383, I388, N390, K391, V392, S394, and F450 of the full-length H2 HA protein, or any combination thereof is modified).

[0279] As used herein, a "heterologous" influenza virus gene or viral segment is from an influenza virus source or isolate that is different from most other influenza virus genes or gene segments in a recombinant influenza virus (including reassortant influenza viruses). Thus, a heterologous NA viral segment is suitably derived from an influenza virus source or isolate that is different from the influenza virus source or isolate from which one or more of the PA, PB1, PB2, NP, NS, and M viral segments are derived. For such examples, a heterologous NA viral segment may be derived from the same or a different influenza virus source or isolate from which an HA viral segment (e.g., a segment encoding a modified HA protein described herein) is derived.

[0280] Method for preparing influenza virus

[0281] Also provided herein is a method for preparing or producing influenza virus in a cell. Such methods suitably include a step of contacting a 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. The present disclosure also contemplates that classical reassortment methods (or portions thereof) may be used to prepare or produce influenza virus in a cell. For example, such methods may include contacting a cell with an influenza virus isolate expressing a modified HA protein (e.g., an influenza virus isolate comprising an HA viral segment 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.

[0282] In view of the above, the method of the present invention can be used to prepare or produce modified or variant strains of influenza virus, such as those provided herein, which include modified HA virus segments encoding modified HA proteins 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 examples, the influenza virus isolate is suitably a pandemic influenza virus isolate, such as those pandemic influenza virus isolates described above. In addition, such modified or variant strains of influenza virus should not be considered as reassortant influenza viruses.

[0283] Alternatively, the methods of the invention can be used to prepare or produce reassortant influenza viruses, such as those provided herein, which include an HA viral segment encoding a modified HA protein described herein.

[0284] Suitably, the inventive method can be or at least partially include a reverse genetics method for generating reassortant viruses. In reverse genetics, the genetic information required for generating the desired influenza virus is transferred to the cell, and then the cell can generate influenza virus. Reverse genetics initially requires that viral ribonucleoprotein (RNP) be assembled in vitro and transfected into cells infected by helper viruses (Luytjes et al. (1989) Cell 59 (6): 1107-1113; Enami et al. (1990) PNAS 87 (10): 3802-3805). Subsequent technology involves transfection of RNA polymerase I plasmids encoding all viral RNA (vRNA) together with protein expression constructs of polymerase and NP genes (Fodor et al. (1999) J Virol. 73 (11): 9679-9682). Recently, reverse genetics methods involve the use of a modified RNA polymerase I system that allows the expression of negative sense vRNA and positive mRNA from the same template (Hoffmann et al. (2000) PNAS 97 (11): 6108-6113). In this type of method, each desired gene is cloned into a pHW2000 plasmid consisting of a viral cDNA inserted between an RNA polymerase I promoter and a termination sequence, flanked by a CMV promoter and polyadenylation signal. After eight plasmids are transfected into cells, synthesis of vRNA and mRNA occurs, thereby producing viruses. Further improvements have led to the development of systems that use linear DNA expression constructs instead of plasmids (WO2009 / 000891) and the use of single expression constructs (WO2011 / 012999).

[0285] Therefore, the method of the present invention may further include the step of 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 PA protein, PB1 protein, PB1-F2 protein, PB2 protein, NP protein, NS1 protein, NEP protein, M1 protein, M2 protein and NA protein. In a specific example, the method of the present invention includes one or more of the following steps:

[0286] (a) contacting a cell with one or more expression constructs, such as those described above, comprising one or more nucleic acid molecules comprising and / or encoding one or more of a PA viral segment, a PB1 viral segment, a PB2 viral segment, a NP viral segment, an M viral segment, and a NS viral segment derived from a first influenza virus (e.g., a donor influenza virus);

[0287] (b) contacting the cell with one or more expression constructs, such as those described above, comprising one or more nucleic acid molecules comprising or encoding an HA viral segment and optionally a NA viral segment derived from a second influenza virus (e.g., a vaccinal influenza virus), wherein the HA viral segment encodes a modified HA protein;

[0288] (c) culturing the cells to produce one or more reassortant viruses; and

[0289] (d) selecting a reassortant virus comprising the HA viral segment and optionally the NA viral segment from the second influenza virus.

[0290] In other examples, the inventive method may involve a hybrid method of classical reassortment and reverse genetics. For example, a method in which a host cell is infected by a first influenza strain (e.g., a donor strain) and transfected by one or more expression constructs (e.g., before, after, or simultaneously), the expression constructs encode at least one viral segment from a second influenza strain (e.g., HA and optional NA viral segments from a vaccine strain). An example of such a method is outlined in WO2021099419, which is incorporated herein by reference.

[0291] Therefore, in some embodiments, the method of the present invention includes one or more of the following steps:

[0292] (a) contacting a cell with a donor influenza virus strain comprising a first HA viral segment and a first NA viral segment;

[0293] (b) contacting the cell with one or more expression constructs, such as those described 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 vaccinal influenza virus strain, wherein the second HA viral segment encodes a modified HA protein;

[0294] (c) culturing the cells to produce one or more reassortant viruses; and

[0295] (d) selecting a reassortant virus comprising a second HA viral segment and, optionally, a second NA viral segment.

[0296] The term "vaccine influenza virus strain" as used herein refers to an influenza virus strain suitable for use in an immunogenic composition or an immunogenic virus (e.g., a reassortant virus). Vaccine influenza virus strains may include, but are not necessarily limited to, pathogenic strains, non-pathogenic strains or relatively non-pathogenic strains, inactivated strains, and / or attenuated strains. In a specific example, the vaccine influenza virus strain is a pandemic influenza virus strain.

[0297] The vector or expression construct used in the method of the present invention may be those known in the art, including those described above. Therefore, the present disclosure contemplates the use of isolated and purified vectors or plasmids that express or encode influenza virus proteins, or express or encode influenza vRNA (both natural and recombinant vRNA). The vector may comprise influenza cDNA (see, e.g., Fields Virology (Fields et al. (eds.), Lippincott, Williams, and Wickens (2013), which is incorporated herein by reference). Any suitable promoter or transcription termination sequence may be used to express proteins or peptides, e.g., viral proteins, such as the modified HA proteins described herein. For example, one or more expression constructs may be suitable for: (a) vRNA production, and comprising a promoter operably linked to an influenza virus DNA molecule linked to a transcription termination sequence; and / or (b) mRNA production, and comprising a promoter operably linked to a DNA segment encoding an influenza virus segment.

[0298] As described above, the present disclosure also contemplates additional selection steps to enhance the production of reassortant viruses comprising modified HA viral segments (i.e., HA viral segments of vaccine strains that have been modified to encode modified HA proteins). The selection step may include any method that enhances the selection of reassortant viruses comprising HA viral segments encoding modified HA proteins derived from vaccine strains. Suitably, the selection step is performed after host cell culture to produce reassortant influenza viruses that can express modified HA proteins.

[0299] Suitably, the methods provided herein include a step of separating the reassortant virus from the host cells prior to the selection step. For example, the cell culture supernatant containing the reassortant virus is separated from the host cells, and the supernatant containing the reassortant virus is subjected to the selection step.

[0300] In some instances, the selection step includes negative selection for the reassortment virus comprising the HA protein from the donor strain. Negative selection can include, for example, contacting the host cell, the reassortment virus separated therefrom and / or the cell culture supernatant with one or more specific binding or antibodies produced for the HA protein from the donor strain. Negative selection can also include exposing the host cell to an inhibitor (e.g., short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) or small interfering DNA (siDNA)), which preferentially or specifically reduces the transcription and / or translation of the HA gene or protein of the donor strain relative to the HA gene or protein of the vaccine strain. In addition to the above, the selection step can also include negative selection for the reassortment virus comprising the NA protein from the donor strain, for example, by utilizing one or more specific binding or antibodies produced for the NA protein from the donor strain.

[0301] In other examples, the selection step is or includes a positive selection step. The positive selection step may include contacting the host cell, the reassortment virus separated therefrom and / or the cell culture supernatant with one or more antibodies specific to the modified HA protein derived from the vaccine virus isolate or strain. In this way, reassortment viruses containing modified HA viral segments can be positively selected from the host cell or cell culture supernatant. Suitably, one or more antibodies for positive selection are labeled (e.g., with magnetic beads). The labeling facilitates subsequent separation of reassortment viruses containing HA genes encoding modified HA proteins, such as by affinity chromatography.

[0302] The methods described herein may include one or more positive selection steps and / or negative selection steps. For example, the reassortant virus can be passaged multiple times in the presence of one or more antibodies described above to perform positive selection and / or negative selection. Multiple selection steps can be performed to enhance the selection of reassortant influenza viruses comprising HA viral segments encoding modified HA proteins.

[0303] The method of the present invention can suitably produce a pool of reassortant viruses from which a specific class of reassortant viruses can be isolated. Reassortant viruses having, for example, high growth characteristics and comprising a modified HA protein and optionally a NA protein of a seasonal or pandemic influenza strain can be isolated for use in vaccine production. Therefore, the above method may also include the step of isolating a reassortant influenza virus comprising an HA viral segment encoding a modified HA protein.

[0304] To this end, the inventive method may include harvesting or isolating complete or whole virions from a cell culture medium. Alternatively or additionally, the inventive method may include harvesting or isolating split virions from a culture medium. In such examples, the harvesting or separation step suitably includes contacting the influenza virus with a splitting agent (e.g., a detergent). The inventive method may alternatively or additionally include harvesting or isolating one or more specific influenza virus proteins, such as modified HA proteins, from a culture medium or from a harvested influenza virus, such as by affinity chromatography.

[0305] During the harvest or separation of influenza virus and / or influenza virus protein, cells can be separated from the culture medium by standard methods such as centrifugation, separation, filtration or ultrafiltration. Then the influenza virus or the viral protein produced therefrom can be concentrated and / or purified according to methods well known to persons skilled in the art, such as gradient centrifugation (such as gradient ultracentrifugation (GUC)), filtration, precipitation, chromatography and any combination thereof. In a specific example, influenza virus and / or one or more proteins produced therefrom are separated or harvested from the culture medium by gradient ultracentrifugation. Suitably, influenza virus is inactivated during or after purification. For example, virus inactivation can be carried out by contacting influenza virus with an inactivator (such as, adding b-propiolactone or formaldehyde) at any point in the purge process.

[0306] Suitably, the method of the present invention may also include the step of selecting an influenza virus (e.g., a reassortant influenza virus) that can grow or rescue in a cell, more specifically in a mammalian cell such as an MDCK cell. In a specific example, the method of the present invention includes the step of determining whether an influenza virus (e.g., a reassortant virus) forms a virion that can form an HA trimer (or a stable HA trimer) when cultured in a cell. Therefore, in a broad form, the present disclosure provides a method for preparing an influenza virus that can form a stable HA trimer and / or grow in a cell.

[0307] Based on the above, the present disclosure also provides isolated influenza viruses prepared by the methods described herein.

[0308] In a related form, the present disclosure also provides modified HA proteins prepared by the methods described herein.

[0309] vaccine

[0310] The present disclosure contemplates that influenza viruses produced according to the methods described herein and / or modified HA proteins derived therefrom can be used in vaccine compositions.

[0311] Thus, in one form, the present disclosure provides a method of preparing a vaccine composition, the method comprising the steps of:

[0312] (a) providing an isolated influenza virus and / or a modified HA protein as provided herein; and

[0313] (b) combining the isolated influenza virus and / or modified HA protein with an adjuvant and / or treating the isolated influenza virus with an agent that inactivates or attenuates the virus.

[0314] In a related form, the present disclosure relates to a vaccine composition, wherein the vaccine composition is produced according to the method provided herein.

[0315] In another related form, the present disclosure relates to a vaccine composition, wherein the vaccine composition:

[0316] (a) comprising an isolated influenza virus as described herein and a pharmaceutically acceptable carrier, diluent or excipient; or

[0317] (b) comprises the modified HA protein described herein and a pharmaceutically acceptable carrier, diluent or excipient.

[0318] Flu vaccines are usually based on live attenuated viruses or inactivated viruses. Inactivated vaccines may be based on intact virions, "split" virions, or purified surface antigens. Viral antigens can also be presented in the form of viral particles. The method of the present invention can be used to manufacture any of these types of vaccines. When inactivated influenza virus is used, the vaccine can contain intact virions, split virions, or purified surface antigens (e.g., hemagglutinin and optional neuraminidase). Chemical methods for inactivating viruses include treatment with an effective amount of one or more of the following inactivators: detergent, formaldehyde, b-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), diethylamine, acetylethyleneimine, or a combination thereof. Non-chemical virus inactivation methods are also known in the art, such as UV light or gamma rays. Subunit vaccines comprising modified HA proteins described herein are also contemplated.

[0319] Virions can be harvested from virus-containing fluids (e.g., cell culture supernatants) by a variety of methods. For example, the purification process may involve zonal centrifugation or affinity chromatography using a linear sucrose gradient solution (optionally including a detergent for disrupting the virions). The antigen can then be purified by diafiltration after optional dilution.

[0320] The vaccine composition may contain a pharmaceutically acceptable carrier, diluent or excipient. "Pharmaceutically acceptable carrier, diluent or excipient" means a solid or liquid filler, diluent or encapsulated material that can be safely used for systemic administration. Depending on the specific route of administration, various carriers, diluents and excipients well known in the art can be used. These carriers, diluents and excipients can be selected from the group including: sugar, starch, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oil, synthetic oil, polyol, alginic acid, phosphate buffer solution, emulsifier, isotonic saline and salts (e.g., mineral acid salts including hydrochloride, bromide and sulfate), organic acids (e.g., acetate, propionate and malonate), water and pyrogen-free water.

[0321] A useful reference text describing acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ USA, 1991), which is incorporated herein by reference.

[0322] Suitably, certain immunological or immunogenic agents may be used in combination with the immunogenic proteins described herein in order to elicit an immune response. The term "immunogenic agent" includes within its scope carriers, delivery agents, immunostimulants and / or adjuvants that are 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 the composition. Non-limiting examples of suitable immunostimulants and adjuvants include squalane and squalene (or other oils of plant or animal origin), including squalene oil-in-water emulsions (e.g., MF59, AS03, and AF03); block copolymers; TLR agonists, such as pathogen-derived compounds, including lipopeptides, glycolipids, nucleotides, small molecule inhibitors, and bacterial-derived components, such as flagellin; detergents, such as -80; A. Mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium derived adjuvants, such as Corynebacterium parvum; Propionibacterium derived adjuvants, such as Propionibacterium acne; Mycobacterium bovis (Bacille Calmette and Guerin or BCG); Bordetella pertussis pertussis) antigens; tetanus toxoid; diphtheria toxoid; surfactants such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecyl ammonium bromide, N,N-dioctadecyl-N′,N′bis(2-hydroxyethyl-propylenediamine), methoxyhexadecylglycerol and pluronic polyols; polyamines such as pyranose, dextran sulfate, polyIC carbopol; peptides such as muramyl dipeptide and derivatives, dimethylglycine, phasin; oily 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 factor; 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; and

[0323] Adjuvants; mycobacterial cell wall extracts; synthetic glycopeptides such as muramyl dipeptide or other derivatives; Avridine; lipid A derivatives; dextran sulfate; DEAE-dextran alone or in combination with aluminum phosphate; carboxypolymethylenes such as Carbopol'EMA; acrylic copolymer emulsions such as Neocryl A640 (e.g., U.S. Pat. No. 5,047,238); water-in-oil emulsifiers such as Montanide ISA 720; poliovirus, vaccinia or animal poxvirus proteins; or mixtures thereof.

[0324] Immunogenic agents can include carriers, such as thyroglobulin; albumins, such as human serum albumin; toxins, toxoids, or any mutated cross-reactive substances (CRMs) from tetanus, diphtheria, pertussis, Pseudomonas, Escherichia 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, etc. Alternatively, a fragment or epitope of a carrier protein or other immunogenic protein can be used. For example, a T cell epitope of a bacterial toxin, toxoid, or CRM can be used. In this regard, reference can be made to U.S. Patent No. 5,785,973, which is incorporated herein by reference. It is envisioned that a carrier protein or other immunogenic protein can be directly or indirectly connected (e.g., via a linker known in the art) to the modified HA protein described herein.

[0325] It has been found that water-in-oil emulsions are particularly suitable for use as adjuvants for influenza virus vaccines. Various such emulsions are known, which typically contain at least one oil and at least one surfactant, wherein the oil and surfactant are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion are typically less than 5 microns in diameter and may even have a submicron diameter, and these small sizes can be achieved by microfluidics to provide stable emulsions. Droplets with an average size of less than 220 nm are preferred because they can be sterilized by filtration.

[0326] In each example, the oil-in-water emulsion is uniform. The feature of uniform emulsion is that most of the droplets (particles) dispersed therein are in the specified size range (for example, diameter). Suitable specified size range can be for example between about 50-220nm (for example about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220nm or any scope therein), between about 50-180nm, between about 80-180nm, between about 100-175nm, between about 120-185nm, between about 130-190nm, between about 135-175nm or between about 150-175nm. In some instances, the droplet (particle) that is less than 10% quantity in the uniform emulsion exceeds the specified diameter range. In certain embodiments, the average particle size of the oil droplets in the oil-in-water emulsion formulation is between about 135-175 nm, for example, about 155 nm ± 20 nm, as measured by dynamic light scattering, and such formulations contain no more than 1 x 10 7As used herein, "large particles" means particles with a diameter of ≥1.2 μm, typically between about 1.2-400 μm. In a specific example, the uniform emulsion has less than 10%, less than 5%, or less than 3% of the droplets exceeding the preferred size range. In some examples, the average droplet size of the particles in the oil-in-water emulsion formulation is between about 125-185 nm, such as 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 because less than 5% of the number of droplets in the formulation exceeds the range of about 125-185 nm.

[0327] The vaccine compositions described herein can be used with oils, such as those from animal (e.g., fish) or plant sources. The sources of vegetable oils include nuts, seeds, and grains. The most common nut oils are peanut oil, soybean oil, coconut oil, and olive oil. Jojoba oil obtained from jojoba beans can also be used. Seed oils include safflower oil, cottonseed oil, sunflower oil, sesame seed oil, etc. In the cereal group, corn oil is the most readily available, but oils from other cereals, such as wheat, oats, rye, rice, teff, triticale, etc., can also be used. Although 6-10 carbon fatty acid esters of glycerol and 1,2-propylene glycol do not naturally exist in seed oils, they can be prepared by hydrolyzing, separating, and esterifying appropriate materials starting from nuts and seed oils. Fats and oils in mammalian milk are metabolizable and can therefore be used in the vaccine compositions described herein. The procedures for separation, purification, saponification, and other methods 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 (eg, spermaceti) exemplify several fish oils that may be used herein.

[0328] Many branched chain oils are biochemically synthesized in 5-carbon isoprene units, commonly referred to as terpenoids. Shark liver oil contains a branched unsaturated terpenoid called 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 of the present invention. Fish oils, including squalene and squalane, are readily available from commercial sources or can be obtained by methods known in the art. Other suitable oils are tocopherols. Mixtures of oils are also contemplated.

[0329] Surfactants can be classified according to their "HLB" (hydrophile / lipophile balance). Suitably, the HLB of the surfactants described herein is at least 10, more specifically at least 15, and even more specifically at least 16. The vaccine composition may include one or more surfactants, including but not limited to: polyoxyethylene sorbitan ester surfactants (commonly known as Tweens), particularly polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO) and / or butylene oxide (BO), such as DOWFAX; TM The present invention discloses a kind of surfactants which are sold under the trade names of OCEPAL®, such as linear EO / PO block copolymers; octylphenol polyethers, the number of which repeating ethoxy(oxy-1,2-ethanediyl) groups can vary, with octylphenol polyether-9 (Triton X-100, or tert-octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids, such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as SPAN), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Nonionic surfactants are preferred. Exemplary surfactants for inclusion in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin, and Triton X-100.

[0330] Mixtures of surfactants may also be used (e.g., Tween 80 / Span 85 mixtures). Combinations of polyoxyethylene sorbitan esters (e.g., polyoxyethylene sorbitan monooleate (Tween 80)) and octylphenol polyethers (e.g., tert-octylphenoxypolyethoxyethanol (Triton X-100)) are also suitable. Another contemplated combination includes laureth-9 plus polyoxyethylene sorbitan esters and / or octylphenol polyethers.

[0331] Exemplary amounts (weight %) of surfactants are: polyoxyethylene sorbitan esters (e.g., Tween 80) 0.01% to 1%, particularly about 0.1%; octyl or nonylphenoxypolyoxyethanol (e.g., Triton X-100, or other detergents in the Triton series) 0.001% to 0.1%, particularly 0.005% to 0.02%; polyoxyethylene ethers (e.g., laureth-9) 0.1% to 20%, more particularly 0.1% to 10%, even more particularly 0.1% to 1% or about 0.5%.

[0332] In a specific example, the oil-in-water emulsion is a squalene-in-water emulsion, and more specifically a submicron squalene-in-water emulsion. According to some examples, the vaccine composition comprises MF59.

[0333] Any suitable procedure is contemplated for use in producing vaccine compositions. Exemplary procedures include those described in, for example, New Generation Vaccines (1997, Levine et al., Marcel Dekker, Inc. New York, Basel, Hong Kong), which is incorporated herein by reference.

[0334] Any safe route of administration may be used, including, but not limited to, oral, rectal, parenteral, sublingual, buccal, intravenous, intraarticular, intramuscular, intradermal, subcutaneous, inhalation, intranasal, intraocular, intraperitoneal, intracerebroventricular, topical, mucosal, and transdermal administration.

[0335] Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, lozenges, capsules, nasal sprays, suppositories, aerosols, transdermal patches, etc. These dosage forms may also include injection or implantation of controlled release devices designed specifically for this purpose, or other forms of implants modified to additionally function in this manner. Controlled release may be achieved by coating with hydrophobic polymers including acrylic resins, waxes, higher fatty alcohols, polylactic acid and polyglycolic acid, and certain cellulose derivatives (e.g., hydroxypropyl methylcellulose). In addition, controlled release may also be achieved by using other polymer matrices, liposomes, and / or microspheres.

[0336] The composition can be present in the form of discrete units, such as capsules, pouches, functional foods / feeds or tablets, each containing a predetermined amount of one or more therapeutic agents of the present disclosure, can be present in the form of powders or particles, or in the form of solutions or suspensions in aqueous liquids, non-aqueous liquids, oil-in-water emulsions or water-in-oil liquid emulsions. Such compositions can be prepared by any pharmaceutical method, but all methods include the step of combining one or more of the above-mentioned agents with carriers that may constitute one or more essential components. Typically, the composition is prepared by uniformly and intimately mixing the agent of the present disclosure with a liquid carrier or a subdivided solid carrier or both, and then, if necessary, the product is shaped into a desired presentation form.

[0337] The above-mentioned composition can be administered in a manner and effective amount compatible with the dosage form. In the context of the present disclosure, the dosage administered to the subject should be sufficient to affect the beneficial response of the subject (e.g., produce a protective immune response) within an appropriate time period. The amount of the administered agent may depend on the subject being treated, including its age, sex, weight and general health status, and these factors will depend on the judgment of the practitioner.

[0338] Also disclosed herein are containers comprising the immunogenic or vaccine compositions 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 tank, an ampoule, a cartridge, and a disposable pen. In one example, the container is a vial, an ampoule, or a syringe.

[0339] The container may be made of glass, metal (eg, steel, stainless steel, aluminum, etc.) and / or polymer (eg, thermoplastic, elastomer, thermoplastic elastomer). The container may be at least partially siliconized.

[0340] The vaccine composition disclosed herein may also include 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 a 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.

[0341] The buffer may be selected from a USP compatible buffer for parenteral use, particularly when the pharmaceutical formulation is for parenteral use. For example, the buffer may be selected from the group consisting of: monoacids such as acetic acid, benzoic acid, gluconic acid, glyceric acid, and lactic acid; diacids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, and tartaric acid; polyacids such as citric acid and phosphoric acid; and bases such as ammonia, diethanolamine, glycine, triethanolamine, and TRIS.

[0342] Methods of eliciting immune responses and treatments

[0343] The influenza virus proteins and vaccine compositions described herein may be suitable for administration to human or non-human animal subjects, such that the present disclosure provides methods for increasing immune responses and / or preventing and / or treating influenza-related diseases, disorders or conditions in subjects. The present disclosure also provides a composition for use as a medicament, and provides the use of the compositions of the present disclosure in the manufacture of a medicament for increasing immune responses and / or preventing and / or treating influenza-related diseases, disorders or conditions in subjects.

[0344] Thus, in one form, the present disclosure provides a method of eliciting an immune response in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of an isolated influenza virus, modified HA protein, or vaccine composition provided herein, thereby eliciting an immune response in the subject.

[0345] In a related form, the present disclosure provides a method for preventing and / or treating an influenza-related 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, modified HA protein or vaccine composition described herein, thereby preventing and / or treating an influenza-related disease, disorder or condition.

[0346] For the purposes of the aspects described herein, the terms "subject," "patient," and "individual" include, but are not limited to, mammals, including humans, performance animals (e.g., horses, camels, greyhounds), livestock (e.g., cattle, sheep, horses, pigs, chickens, ducks), and companion animals (e.g., cats and dogs). Suitably, the subject is a human.

[0347] "Triggering an immune response" means generating or stimulating the production or activity of one or more elements of the immune system (including cellular immune system, humoral immune system (i.e., antibodies) and / or 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 proinflammatory 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 neutrophil chemoattractant IL-8. In some instances, the immune response triggered by the vaccine composition described herein is protective.

[0348] As generally used herein, the terms "immunization," "vaccination," and "vaccine" refer to methods and / or compositions for eliciting a protective immune response against influenza virus, thereby at least partially preventing or minimizing subsequent infection with influenza virus or related serotypes, strains, or variants.

[0349] "Protective immunity" means a level of immunity sufficient in response to one or more antigens to result in rapid binding and / or elimination of the antigens and thereby at least partially ameliorate or prevent subsequent influenza virus infection in the subject.

[0350] A "protective immune response" means a level of immune response sufficient to prevent or reduce the severity, symptoms, aspects or features of an active infection and / or influenza virus infection in a subject.

[0351] The terms "treating", "treat" or "treatment" as used herein refer to therapeutic interventions that at least partially improve, eliminate or alleviate the symptoms or pathological signs of an influenza-related disease, disorder or illness (e.g., influenza infection) after it has begun to develop. Treatment need not be absolutely beneficial to the subject. Any method or criterion known to those skilled in the art can be used to determine the beneficial effect.

[0352] As used herein, "preventing, prevent or prevention" refers to a course of action initiated before infection or exposure to influenza virus or its molecular components and / or before the onset of symptoms or pathological signs of a disease, disorder or illness in order to prevent infection and / or alleviate symptoms or pathological signs. It should be understood that such prevention need not be absolutely beneficial to the subject. "Preventive" treatment is a treatment administered to a subject who does not show signs of a disease, disorder or illness or who only shows early signs, with the goal of reducing the risk of symptoms or pathological signs of a disease, disorder or illness.

[0353] Vaccines described herein can be used to treat children and adults. Flu vaccines are currently recommended for pediatric and adult immunizations starting at 6 months of age. Therefore, human subjects may be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old or at least 55 years old. Preferred subjects receiving vaccines are elderly people (e.g., ≥50 years old, ≥60 years old, preferably ≥65 years old), young people (e.g., ≤5 years old), hospitalized subjects, health care workers, armed forces and military personnel, pregnant women, chronic diseases, immunodeficient subjects, subjects who have taken antiviral compounds 7 days before receiving the vaccine, people who are allergic to eggs and people traveling abroad. However, these vaccines are not only suitable for these groups, but can be used more generally in groups. For pandemic strains, it is preferred that all age groups are administered.

[0354] Treatment can adopt single dose schedule or multiple dose schedule.Multiple doses can be used in the primary immunization schedule and / or booster immunization schedule.In the multiple dose schedule, each dosage can be given by the same or different approach (for example, parenteral for the first time and mucosal reinforcement, mucosal for the first time and parenteral reinforcement).For immune initial patients (for example, subjects who have never received influenza vaccine before) or for new HA subtypes, vaccination (for example, when a pandemic breaks out), it is particularly useful to apply more than one dose (usually two doses).Multiple doses will be usually applied at intervals of at least 1 week (for example, 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.).

[0355] Screening methods

[0356] The present disclosure also relates to methods involving testing or screening for modifications of HA proteins (eg, HA proteins of the H2 subtype) that can confer favorable growth characteristics to the influenza virus when the influenza virus is grown in cells (eg, MDCK cells).

[0357] Thus, in one form, the present disclosure provides a method for identifying a modification in an HA protein (e.g., an HA protein of the H2 subtype) that promotes or improves the growth of an influenza virus in a cell, the method comprising the steps of:

[0358] (a) passage one or more candidate influenza viruses expressing HA protein in cells one or more times;

[0359] (b) selecting those candidate influenza viruses that are able to grow or exhibit improved growth in the cells;

[0360] (c) screening the candidate influenza viruses selected in step (b) for one or more modifications of the HA protein (e.g., its trimer region or the lower region of its stalk domain).

[0361] Suitably, the candidate influenza virus is unable to grow or is capable of only limited growth in cells when expressing a wild-type or unmodified version of the HA protein.

[0362] In another form, the present disclosure provides a method for identifying or screening for modifications in HA proteins (e.g., HA proteins of the H2 subtype) that promote or improve the growth of influenza viruses in cells, the method comprising the steps of:

[0363] (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 a trimer interface region thereof are modified; and

[0364] (b) Testing the ability of the modified influenza viruses to grow in cells.

[0365] Suitably, the method of the present invention further comprises the step of selecting modifications to the HA protein or modified HA protein that can promote or improve the growth of influenza viruses in cells. Thus, one or more modifications in the modified HA protein can be identified by the method of the present invention, the modification being selected based on its ability to promote or improve the growth of influenza viruses in cell culture. Such modifications can then be introduced into the HA protein of wild-type, recombinant or reassortant viruses, for example, by the methods described herein, so as to promote or improve the growth of these influenza viruses in cell culture.

[0366] Suitably, the modified HA protein comprises an amino acid sequence in which one or more amino acid residues in the lower region of the stem domain of its trimer interface region are modified. As described herein, the lower region of the stem domain may comprise amino acid residues N26 to D46, L325 to P335, D377 to E397 and L439 to D452 of the full-length HA protein of the H2 subtype. In other examples, the lower region of the stem domain comprises amino acid residues N26 to D46, D377 to E397 and L439 to D452 of the full-length HA protein of the H2 subtype.

[0367] With reference to some examples, the modified one or more amino acid residues are at positions selected from the group consisting of: 39, 219, 233, 320, 383, 388, 390, 391, 392, 394, 405, 416, 430, 450 of the full-length H2 amino acid sequence and any combination thereof. More specifically, the modified one or more 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 of the full-length H2 amino acid sequence and any combination thereof.

[0368] In a related form, the present disclosure provides a method for identifying a modification in an HA protein (e.g., an HA protein of the H2 subtype) that promotes or improves the growth of an influenza virus in a cell, the method comprising the steps of:

[0369] (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 a trimer interface region thereof are modified; and

[0370] (b) performing one or more passages of the modified influenza virus expressing the modified HA protein in cells.

[0371] Suitably, the method of the invention further comprises the step of screening the modified influenza virus for one or more additional modifications of the modified HA protein after one or more passages in the cell. 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, pyrophosphate sequencing, and mass spectrometry.

[0372] According to a specific example of the method of the present invention, the modified HA protein is the protein described above.

[0373] In order that the preferred embodiments of the present disclosure may be fully understood and put into practical use, reference is made to the following non-limiting examples.

[0374] Example

[0375] Example 1.

[0376] The goal 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.

[0377] Methods and Results

[0378] However, the inventors have found that the vast majority of attempts to rescue synthetic viruses using the WT H2N3 allele from A / chicken / Ohio / 494832 / 2007 have failed to produce viruses. Those that did produce viruses had mutations or variants in the HA viral segment of the produced viruses. All variants were identified by visual analysis of Sanger sequencing results. If multiple peaks were observed at a given nucleotide base in all reads, and the minimum read depth was 2 reads, variants were identified. The present embodiment has only further studied variants that cause amino acid changes.

[0379] Identification of HA mutations / variants by one of 4 different approaches

[0380] 1. Passage of WT A / Chicken / Ohio / 494832 / 2007. Sequencing of this virus has revealed 4 identifiable variants (1x)

[0381] aA / Chicken / Ohio / 484832 / 2007

[0382] i.V129I variant – This variant is not at or near the trimer interface. It is located outside the head domain and is unlikely to contribute to trimer stability.

[0383] ii. V320I variant

[0384] iii.N390I variant

[0385] iv.K391R variant

[0386] 2. Rescue of A / swine / Missouri / 2124514 / 2006 (2x independent rescues).

[0387] a.RG4 A / Pig / Missouri / 2124514 / 2006

[0388] i.K383E variant

[0389] b.RG5 A / Pig / Missouri / 2124514 / 2006

[0390] i.L219P mutation

[0391] 3. A / chicken / ohio / 494832 / 2007 rescue

[0392] a.GDE 80.4AA / Chicken / Ohio / 484832 / 2007

[0393] i.V233A variant

[0394] ii. V320A variant

[0395] b.GDE 80.4BA / Chicken / Ohio / 484832 / 2007

[0396] i.V39I mutation

[0397] c.GDE 80.7BA / Chicken / Ohio / 484832 / 2007

[0398] i.F450S

[0399] d.HS_Sys_14A / Chicken / Ohio / 484832 / 2007

[0400] i.S394Y variant

[0401] ii.R416G variant

[0402] e.HS_Sys_15A / Chicken / Ohio / 484832 / 2007

[0403] i.A405T variant

[0404] Synthetic seed technology and procedures

[0405] The method described by Dormitzer et al. (Sci Transl Med. 2013 May 15; 5(185)) was used (see, for example, Dormitzer et al. Figure 1 ) The HA and NA sequences of A / chicken / Ohio / 494832 / 2007 were recombinantly assembled into an expression construct.

[0406] MDCK cells are then transfected with expression constructs for the backbone viral segments PA, PB1, PB2, NP, NS and M from a high growth parental strain (e.g. A / Puerto Rico / 8 / 1934 or a cell-adapted form thereof, e.g. PR8X) and HA and NA sequences from A / Chicken / Ohio / 494832 / 2007

[0407] The reassortant viruses are then rescued and characterized

[0408] · Figure 1 A schematic diagram of the transfection and rescue procedures is provided.

[0409] The first two rescue attempts failed to generate any rescue isolates of the H2N3 strain.

[0410] · Amplification of viral RNA in the supernatant of transfected cells confirmed the absence of viral RNA amplification.

[0411] Additional transfection and rescue experiments were performed, resulting in rescued virus isolates in 5 out of a total of 28 experiments.

[0412] Sequence analysis

[0413] After sequencing eight rescued or serially passaged H2N3 isolates, all isolates were shown to contain variant nucleotide bases in the HA viral segment encoding modified amino acid residues in the HA protein (see Figure 2 ).

[0414] • No other modifications were observed in any of the remaining 7 viral segments of these strains.

[0415] Mutation Mapping

[0416] The structure of the A / Pig / Missouri / 2124514 / 2006HA protein has been solved.

[0417] When the variants identified in these rescues were mapped to the structure, they all resided at the interface between adjacent monomers in the HA trimer structure, as shown in Figure 3 It can be observed in.

[0418] · Figure 4 We further showed that the A405T mutation in the HS Sys 15 isolate causes this residue to associate more tightly with the K423 residue on the adjacent HA monomer when arranged in a homotrimeric manner, which may function to stabilize the trimer structure.

[0419] Without being bound by any theory and based on the position of the variants, and the fact that the inventors were unable to rescue the WT virus in the absence of at least one variant position, we hypothesize that the wild-type H2 monomer is unable to form functional trimers when grown in cells, thereby preventing or limiting the propagation of viruses expressing this HA in cell culture.

[0420] Clonal isolation

[0421] • Clonal isolation experiments were performed to determine the stability of the A405T mutation in the HS Sys 15 isolate.

[0422] Several 10-fold limiting dilutions were performed:

[0423] 10x plates, serial dilutions from -4 to -9, 60 plates total

[0424] Titer check on day 3

[0425] Ideally, the fewer colonies there are in a plate, the greater the likelihood that they are clones.

[0426] The more rounds of clonal isolation, the greater the probability that the isolate is clonal.

[0427] • 28 clones were isolated from 60 plates.

[0428] 12 clones from -5 dilution plate

[0429] 16 clones from -4 dilution plate

[0430] All 28 clones were sequenced, with the vast majority (25 of 28) showing that each clone had a clean virus population with the A405T mutation, while the remaining 3 clones showed evidence of a mixture of A405T mutant viruses and WT viruses, of which the WT virus was just a minor variant.

[0431] • The data indicate that the A405T mutation in the HS Sys 15 isolate is stable.

[0432] Similar to the V129I variant, the E184K variant is not at or near the trimer interface. Again, it is located outside the head domain and is unlikely to contribute to the stability of the trimer.

[0433] Further passage

[0434] The inventors took five H2N3 isolates and performed an additional 3 passages (in duplicate) to determine the fidelity of these variants. Sanger sequencing of this passage material revealed additional variants, as shown in Table 1.

[0435] • In HS_Sys_15, the A405T variant became the dominant allele and no additional mutations occurred.

[0436] Both variants found in HS_Sys_14 were stable during passage and no additional mutations occurred.

[0437] • Many of the additional variants that emerged appeared more than once, thus, there appears to be some convergence towards HA viral segments that acquired specific mutations.

[0438] • Residues 39, 383, 388, 390, 391, 392, 394 and 450 all map to the lower region of the stem (ie, the lower stem cluster).

[0439] Table 1. List of variants identified in the rescued viruses after 3 passages.

[0440]

[0441] in conclusion

[0442] The present embodiment identifies many mutations that can support the rescue or growth of H2 influenza virus on MDCK cells. These mutations occur along the length of the molecule, and consistently appear in the trimer interface region between the HA monomers. The specific region in the lower stem seems to be a mutation hotspot supporting virus rescue, but mutations are also identified in other positions of the HA molecule. The data from passages show that some mutations are more stable than other mutations over time, but at least the present embodiment shows that all these mutations on the trimer interface at least contribute to the viability of the virus when it grows in cell culture. For this reason, the present embodiment shows that wild-type or unmodified H2 HA protein is close to being able to support the growth of H2 influenza virus in cells. In addition, the above data support such a viewpoint, that the mutation of the dynamic range of the entire trimer interface region can promote the rescue and growth of H2 influenza virus in cell culture, and the virus can be further converted and changed by passage to further support their stability or growth in cells.

[0443] Itemized list of implementation options

[0444] 1. A modified hemagglutinin (HA) protein, comprising an amino acid sequence in which one or more amino acid residues in its trimer interface region are modified, wherein an influenza virus expressing the modified HA protein is able to grow in a cell.

[0445] 2. The modified HA protein of claim 1, which is capable of forming HA trimers when expressed by influenza virus grown in cells.

[0446] 3. The modified HA protein of claim 1 or claim 2, wherein modification of the one or more amino acid residues in the trimer interface region increases the stability of the HA trimer formed by the modified HA protein relative to the HA trimer formed by the corresponding unmodified HA protein.

[0447] 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.

[0448] The modified HA protein of claim 4 , wherein the modified HA protein belongs to the H2, H1 or H5 subtype.

[0449] 6. The modified HA protein of claim 4 or claim 5, wherein the modified HA protein is of the H2 subtype.

[0450] 7. The modified HA protein of claim 6, wherein the modified one or more 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.

[0451] 8. The modified HA protein of claim 7, wherein the one or more amino acid residues that are modified comprise:

[0452] (a) V39;

[0453] (b) L219;

[0454] (c) V233;

[0455] (d) V320;

[0456] (e) K383;

[0457] (f) I388;

[0458] (g) N390;

[0459] (h) K391;

[0460] (i) V392;

[0461] (j) S394;

[0462] (k) A405;

[0463] (l) R416;

[0464] (m) D430;

[0465] (n) F450;

[0466] (o) S394 and R416;

[0467] (p) V233 and V320;

[0468] (q) V320, N390 and K391;

[0469] (r) V320 and N390;

[0470] (s) V320 and K391;

[0471] (t) N390 and K391;

[0472] (u) F450 and K391;

[0473] (v) V233, F450 and K391;

[0474] (w) V233 and F450;

[0475] (x) V233 and K391;

[0476] (y) V39 and D430;

[0477] (z) V39, I388 and V392;

[0478] (aa) V39 and I388;

[0479] (ab) V39 and V392;

[0480] (ac)I388 and V392;

[0481] (ad) V39, K391, and D430;

[0482] (ae) V39 and K391; or

[0483] (af)K391 and D430.

[0484] 9. The modified HA protein of claim 7 or claim 8, wherein the modification of the one or more amino acid residues is 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.

[0485] 10. The modified HA protein of claim 9, wherein the modification of the one or more amino acid residues comprises:

[0486] (a) V39I;

[0487] (b) L219P;

[0488] (c) V233A;

[0489] (d) V320A;

[0490] (e) V320I;

[0491] (f) K383E;

[0492] (g) I388T;

[0493] (h)N390I;

[0494] (i) K391R;

[0495] (j) K391N;

[0496] (k) V392A;

[0497] (l) S394Y;

[0498] (m)A405T;

[0499] (n) R416G;

[0500] (o)D430N;

[0501] (p) F450S;

[0502] (q) S394Y and R416G;

[0503] (r) V233A and V320A;

[0504] (s) V320I, N390I and K391R;

[0505] (t) V320I and N390I;

[0506] (u) V320I and K391R;

[0507] (v) N390I and K391R;

[0508] (w) F450S and K391N;

[0509] (x) V233A, F450S and K391N;

[0510] (y) V233A and F450S;

[0511] (z) V233A and K391N;

[0512] (aa) V39I and D430N;

[0513] (ab) V39I, I388T, and V392A;

[0514] (ac) V39I and I388T;

[0515] (ad) V39I and V392A;

[0516] (ae)I388T and V392A;

[0517] (af) V39I, K391N, and D430N;

[0518] (ag) V39I and K391N; or

[0519] (ah)K391N and D430N.

[0520] 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.

[0521] 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 at positions 377 to 397, 439 to 452, 26 to 46, and optionally 325 to 335 of the H2 subtype full-length HA protein.

[0522] 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 at positions 383 to 394, 439 to 452, 31 to 40, and optionally 325 to 335 of the H2 subtype full-length HA protein.

[0523] 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 the full-length HA protein of the H2 subtype.

[0524] 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 the full-length HA protein of the H2 subtype.

[0525] 16. The modified HA protein of any one of claims 11 to 15, wherein the one or more amino acid residues that are modified 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.

[0526] 17. The modified HA protein of any one of claims 11 to 16, wherein the modification of the one or more amino acid residues is 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.

[0527] 18. The modified HA protein of any of the preceding claims, comprising, consisting of, or consisting essentially of an amino acid sequence as described in any one of 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.

[0528] 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.

[0529] 20. A modified H2 subtype HA protein, 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 of the full-length H2 amino acid sequence and any combination thereof.

[0530] 21. The modified HA protein of claim 20, wherein the influenza virus expressing the modified HA protein is capable of growing in a cell.

[0531] 22. The modified HA protein of claim 21, wherein the cell is an MDCK cell.

[0532] 23. A modified H2 subtype HA protein, comprising an amino acid sequence in which one or more amino acid residues in a trimer interface region are modified, and wherein an influenza virus expressing the modified HA protein is capable of growing in a cell.

[0533] 24. An isolated nucleic acid comprising a nucleotide sequence encoding the modified HA protein of any one of claims 1 to 23 or a nucleotide sequence complementary thereto.

[0534] 25. An isolated nucleic acid as described in claim 24, wherein the isolated nucleic acid comprises, consists of or essentially consists 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; or a nucleotide sequence complementary thereto.

[0535] 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 linked to one or more regulatory sequences.

[0536] 27. A host cell transformed with the isolated nucleic acid of claim 24 or claim 25 or the genetic construct of claim 26.

[0537] 28. A method for producing the modified HA protein of any one of claims 1 to 23, the method comprising the steps of: (i) culturing the previously transformed host cell of claim 27; and (ii) isolating the modified HA protein from the host cell cultured in step (i).

[0538] 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 a trimer interface region thereof are modified.

[0539] 30. The isolated influenza virus of claim 29, wherein the modified HA protein is the modified HA protein of any one of claims 1 to 23.

[0540] 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 NQ: 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.

[0541] 32. The isolated influenza virus of any one of claims 29 to 31, which is a recombinant influenza virus.

[0542] 33. The isolated influenza virus of any one of claims 29 to 32, which is a reassortant influenza virus.

[0543] 34. The isolated influenza virus of any one of claims 29 to 33, which is capable of growing in a cell.

[0544] 35. The isolated influenza virus of claim 34, which is capable of growing in MDCK cells.

[0545] 36. The isolated influenza virus of any one of claims 29 to 35, which is capable of forming HA trimers comprising the modified HA protein when grown in a cell.

[0546] 37. The isolated influenza virus of any one of claims 29 to 36, further comprising:

[0547] (a) one or more of the PA, PB1, PB2, NP, NS, and M viral segments from a donor influenza virus; and

[0548] (b) Heterologous or chimeric NA viral segments.

[0549] 38. A method for preparing influenza virus in a cell, the method 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.

[0550] 39. The method of claim 38, wherein the modified HA protein is the modified HA protein of any one of claims 1 to 23, the nucleic acid is the isolated nucleic acid of claim 24 or claim 25, and / or the genetic construct is the genetic construct of claim 26.

[0551] 40. The method of claim 38 or claim 39, further comprising the step of 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 PA protein, PB1 protein, PB1-F2 protein, PB2 protein, NP protein, NS1 protein, NEP protein, M1 protein, M2 protein, and NA protein.

[0552] 41. The method of claim 40, wherein the NA protein is of the N1, N2 or N3 subtype.

[0553] 42. The method of any one of claims 38 to 41, wherein a modified pandemic influenza virus is prepared, the modified pandemic influenza virus comprising the nucleic acid encoding the modified HA protein.

[0554] 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.

[0555] 44. An isolated influenza virus prepared by the method of any one of claims 38 to 43.

[0556] 45. A modified HA protein prepared by the method of any one of claims 28 or 38 to 43.

[0557] 46. ​​A method for preparing a vaccine composition, the method comprising the steps of:

[0558] (a) providing an isolated influenza virus according to any one of claims 29 to 37 or 44 and / or a modified HA protein according to any one of claims 1 to 23 or 45; and

[0559] (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.

[0560] 47. The method of claim 46, wherein the adjuvant comprises an immunostimulatory DNA sequence, a bacterially derived component, an aluminum salt (alum), or a squalene oil-in-water emulsion system.

[0561] 48. A vaccine composition, wherein the vaccine composition is produced according to the method of claim 46 or claim 47.

[0562] 49. A vaccine composition, wherein the vaccine composition comprises:

[0563] (a) the isolated influenza virus of any one of claims 29 to 37 or 44 and a pharmaceutically acceptable carrier, diluent or excipient; or

[0564] (b) The modified HA protein of any one of claims 1 to 23 or 45 and a pharmaceutically acceptable carrier, diluent or excipient.

[0565] 50. A method for eliciting an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the isolated influenza virus of any one of claims 29 to 37 or 44, the modified HA protein of any one of claims 1 to 23 or 45, or the vaccine composition of claim 48 or claim 49, thereby eliciting the immune response in the subject.

[0566] 51. A method for preventing and / or treating an influenza-related disease, disorder or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of an isolated influenza virus as described in any one of claims 29 to 37 or 44, a modified HA protein as described in any one of claims 1 to 23 or 45, or a vaccine composition as described in claim 48 or claim 49, thereby preventing and / or treating the influenza-related disease, disorder or condition.

[0567] 52. A method for identifying or screening for modifications in HA protein that promote or improve the growth of influenza virus in a cell, the method comprising the steps of:

[0568] (a) modifying the influenza virus to express 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 thereof are modified; and

[0569] (b) testing the ability of the modified influenza virus to grow in cells.

[0570] 53. The method of claim 52, further comprising the step of selecting the modified HA protein that promotes or improves growth of the influenza virus in a cell.

[0571] 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 stem region of the trimer interface region thereof are modified.

[0572] 55. A method for identifying modifications in HA protein that promote or improve the growth of influenza virus in a cell, the method comprising the steps of:

[0573] (a) modifying the influenza virus to express 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 thereof are modified; and

[0574] (b) performing one or more passages of the modified influenza virus expressing the modified HA protein in a cell.

[0575] 56. The method of claim 55, further comprising the step of screening the modified influenza virus for one or more additional modifications of the modified HA protein after one or more passages in cells.

[0576] 57. A method for improving the growth of influenza virus in a cell, the method comprising the step of modifying the influenza virus to express a modified HA protein, the modified HA protein comprising an amino acid sequence in which one or more amino acid residues in its trimer interface region are modified.

[0577] 58. The method of claim 57, further comprising the step of performing one or more passages of the influenza virus expressing the modified HA protein in cells.

[0578] 59. The method of any one of claims 55 to 58, wherein the modified HA protein is a modified HA protein of any one of claims 1 to 23.

[0579] 60. A method for improving the stability of an influenza virus strain in a cell, the method comprising the step of modifying the influenza virus strain to express a modified HA protein, the modified HA protein comprising an amino acid sequence in which one or more amino acid residues in its trimer interface region are modified.

[0580] 61. The method of claim 60, further comprising the step of performing one or more passages of the influenza virus expressing the modified HA protein in cells.

[0581] 62. The method of any one of claims 60-61, wherein the modified HA protein is a modified HA protein of any one of claims 1 to 23.

[0582] 63. A method for improving the production of an influenza virus strain, the method comprising the step of modifying the influenza virus strain to express a modified HA protein, the modified HA protein comprising an amino acid sequence in which one or more amino acid residues in its trimer interface region are modified.

[0583] 64. The method of claim 63, wherein the influenza virus strain is incapable of growing in culture when expressing a wild-type or unmodified HA protein.

[0584] 65. The method of claim 63 or 64, wherein the influenza virus strain is produced in a cell.

[0585] 66. The method of any one of claims 60-62 or 65, wherein the cell is selected from the group consisting of mammals, avians, yeast, and plants.

[0586] 67. The method of claim 66, wherein the cell is a mammalian cell.

[0587] 68. The method of claim 67, wherein the cells are canine kidney cells.

[0588] 69. The method of claim 68, wherein the canine kidney cells are Madin Darby canine kidney (MDCK) cells.

[0589] 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) expressing an HA protein that is not present in currently circulating human strains, or an HA protein that has not been previously detected in the human population, such that the human population will be immune-naive to the HA protein of the influenza virus strain; (b) being able to spread horizontally in the human population; and (c) being pathogenic to humans.

[0590] Sequence Listing

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Claims

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

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

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

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

7. The modified HA protein of claim 6, wherein the modified one or more 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 HA protein of claim 7, wherein the one or more amino acid residues that are modified comprise: (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.

9. The modified HA protein of claim 7, wherein the modification of the one or more amino acid residues is 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.

10. The modified HA protein of claim 9, wherein the modification of the one or more amino acid residues comprises: (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.

11. 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. 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 at positions 377 to 397, 439 to 452, 26 to 46, and optionally 325 to 335 of the H2 subtype full-length HA protein.

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 at positions 383 to 394, 439 to 452, 31 to 40, and optionally 325 to 335 of the H2 subtype full-length HA protein.

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 the full-length HA protein of the H2 subtype.

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 the full-length HA protein of the H2 subtype.

16. The modified HA protein of claim 11, wherein the one or more amino acid residues that are modified 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. The modified HA protein of claim 11, wherein the modification of the one or more amino acid residues is 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.

18. The modified HA protein of claim 1, comprising, consisting of, or consisting essentially of an amino acid sequence as described in any one of 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.

19. The modified HA protein of claim 1, 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.

20. A modified H2 subtype HA protein, 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 of the full-length H2 amino acid sequence and any combination thereof.

21. The modified HA protein of claim 20, wherein the influenza virus expressing the modified HA protein is capable of growing in a cell.

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

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

24. An isolated nucleic acid comprising a nucleotide sequence encoding a modified HA protein or a nucleotide sequence complementary thereto, 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, wherein an influenza virus expressing the modified HA protein is capable of growing in a cell.

25. An isolated nucleic acid as described in claim 24, comprising, consisting of or essentially consisting of a nucleotide sequence selected from the group consisting of SEQ ID NQ: 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.

26. A genetic construct comprising: (i) an isolated nucleic acid comprising a nucleotide sequence 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, wherein an influenza virus expressing the modified HA protein is capable of growing in a cell; or (ii) a nucleotide sequence complementary thereto; operably linked or linked to one or more regulatory sequences.

27. A host cell transformed with: an isolated 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 its trimer interface region are modified, wherein an influenza virus expressing the modified HA protein is able to grow in the cell, or a nucleotide sequence complementary thereto; or a genetic construct comprising: (i) the isolated nucleic acid; or (ii) a nucleotide sequence complementary thereto; operably linked or linked to one or more regulatory sequences.

28. A method for producing a modified HA protein, the method comprising the steps of: (i) culturing a host cell, wherein the host cell is transformed with: an isolated 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, wherein an influenza virus expressing the modified HA protein is able to grow in the cell, or a nucleotide sequence complementary thereto; or a genetic construct, wherein the genetic construct comprises: (i) the isolated nucleic acid; or (ii) a nucleotide sequence complementary thereto; operably linked or linked to one or more regulatory sequences; and (ii) isolating the modified HA protein from the host cell cultured in step (i).

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 a trimer interface region thereof are modified.

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

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

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

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

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

36. The isolated influenza virus of claim 29, which is capable of forming HA trimers comprising the modified HA protein when grown in a cell.

37. The isolated influenza virus of claim 29, further comprising: (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 viral segments.

38. A method for preparing influenza virus in a cell, the method 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. The method of claim 38, wherein the modified HA protein is of the H2 subtype of claim 1.

40. The method of claim 38, further comprising the step of 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 PA protein, PB1 protein, PB1-F2 protein, PB2 protein, NP protein, NS1 protein, NEP protein, M1 protein, M2 protein, and NA protein.

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

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

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

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

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

46. ​​A method for preparing a vaccine composition, the method comprising the steps of: (a) providing an isolated influenza virus and / or a modified HA protein, wherein the isolated influenza virus comprises an HA virus segment encoding the modified HA protein, wherein the modified HA protein comprises an amino acid sequence in which one or more amino acid residues in the trimer interface region thereof are modified; 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.

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

48. A vaccine composition, wherein the vaccine composition is produced according to the method of claim 46.

49. A vaccine composition, wherein the vaccine composition comprises: (a) 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 thereof 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 thereof are modified, and a pharmaceutically acceptable carrier, diluent or excipient.

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

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

52. A method for identifying or screening for modifications in HA protein that promote or improve the growth of influenza virus in a cell, the method comprising the steps of: (a) modifying the influenza virus to express 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 thereof are modified; and (b) testing the ability of the modified influenza virus to grow in cells.

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

54. 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 stem region of the trimer interface region thereof are modified.

55. A method for identifying modifications in HA protein that promote or improve the growth of influenza virus in a cell, the method comprising the steps of: (a) modifying the influenza virus to express 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 thereof are modified; and (b) performing one or more passages of the modified influenza virus expressing the modified HA protein in a cell.

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

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

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

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

60. 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 the step of modifying the influenza virus strain to express a modified HA protein, the modified HA protein comprising an amino acid sequence in which one or more amino acid residues in its trimer interface region are modified.

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

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

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

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

66. The method of claim 64, wherein the influenza virus strain is produced in a cell.

67. The method of claim 61, wherein the cell is selected from the group consisting of mammals, birds, yeast, and plants.

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

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

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

71. The method of claim 61, wherein the influenza virus strain is characterized by at least one of the following: (a) expressing an HA protein that is not present in currently circulating human strains, or an HA protein that has not been previously detected in the human population, such that the human population will be immune-naive to the HA protein of the influenza virus strain; (b) being able to spread horizontally in the human population; and (c) being pathogenic to humans.

72. The method of claim 64, wherein the cell is selected from the group consisting of mammals, birds, yeast, and plants.

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

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

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

76. The method of claim 64, wherein the influenza virus strain is characterized by at least one of the following: (a) expressing an HA protein that is not present in currently circulating human strains, or an HA protein that has not been previously detected in the human population, such that the human population will be immune-naive to the HA protein of the influenza virus strain; (b) being able to spread horizontally in the human population; and (c) being pathogenic to humans.

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