Recombinant influenza viruses with stabilized ha for replication in eggs

Abstract

To disclose modified influenza virus neuraminidases that improve viral replication, thus improving the yield of vaccine viruses.SOLUTION: Expression of such modified neuraminidases by influenza virus stabilizes co-expressed hemagglutinins, ensuring that the hemagglutinins do not undergo mutation or decrease the need for HA binding to cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Application No. 62 / 892,241, filed August 27, 2019, the disclosure of which is incorporated herein by reference.

[0002] Statement of Government Funds This invention was made with Government support under HHSN272201400008C awarded by the National Institutes of Health (NUH). The Government has certain rights in this invention. [Background technology]

[0003] background Influenza is a major respiratory disease in some mammals, including horses, and is responsible for substantial morbidity and economic losses each year. In addition, influenza virus infection can cause severe systemic disease and mortality in some avian species. The segmented nature of the influenza virus genome allows for reassortment of segments during viral replication in cells infected with two or more influenza viruses. Segment reassortment combined with genetic mutation and drift can give rise to numerous diverse strains of influenza virus over time. Novel strains show antigenic variation in their hemagglutinin (HA) and / or neuraminidase (NA) proteins, and the genetic code for the HA protein in particular has a high degree of variability. The main current practice for preventing flu is vaccination. Most commonly, inactivated virus vaccines are used. Since the influenza HA protein is the primary target antigen of the host's protective immune response against the virus and is highly variable, the isolation of influenza viruses and the determination and characterization of the HA antigen of the viruses associated with the most recent pandemics are important for vaccine production. Based on prevalence and prediction, vaccines are designed to stimulate a protective immune response against the predominant and predicted influenza virus strains.

[0004] There are four general types of influenza viruses, A, B, C and D, 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 glycoprotein HA and NA proteins. All known HA and NA subtypes (H1 through H18 and N1 through N11) have been isolated from aquatic birds, which are thought to act as natural reservoirs of influenza.

[0005] Most influenza vaccines are produced in embryonated chicken eggs. However, WHO-recommended influenza vaccine strains often do not replicate efficiently in embryonated chicken eggs and require serial passage in eggs to allow adaptation of the viruses. During adaptation and amplification in eggs, the hemagglutinin (HA) protein of influenza viruses frequently acquires egg adaptation mutations. These egg adaptation mutations in HA frequently alter the antigenicity of the virus, resulting in vaccine viruses that are no longer optimally matched to circulating virus strains. Summary of the Invention

[0006] overview As described herein, influenza viruses were passaged seven times in eggs (in triplicate) to test the mutations that occurred in six non-immunogenic viral segments during adaptation. Surprisingly, the virus did not acquire HA mutations, but instead had mutations in the NA, PB2, NP, and M1 proteins. The NA mutations were identical in all three experiments, and included one deletion and four amino acid mutations. Only the NA mutations were tested, and it was found that they contributed to the effect of allowing efficient growth in eggs without HA mutations, e.g., alone or in various combinations.

[0007] Thus, the present disclosure relates to influenza mutations that prevent influenza viruses from acquiring antigenic-destroying mutations in the hemagglutinin (HA) segment during growth in eggs. Mutations in the neuraminidase (NA) protein of human influenza viruses were found to "stabilize" the HA during egg passage, e.g., when mutations were present in the NA, the HA protein did not acquire egg-adapted mutations. These NA mutations can also increase vaccine virus yields.

[0008] The present disclosure provides isolated recombinant influenza viruses, eg, reassortant influenza viruses, with selected amino acid residues or deletions at specific positions in the NA.

[0009] In one embodiment, the NA is selected not to code for a threonine at residue 32. In one embodiment, the NA is selected not to code for an aspartic acid (D) at position 147. In one embodiment, the NA is selected not to code for an asparagine (N) at residue 329. In one embodiment, the NA is selected not to code for a threonine (T) at residue 148 or residue 329. In one embodiment, the NA is selected not to code for a lysine (K) at residue 148 or residue 344. In one embodiment, the NA is selected not to code for a glycine (G) at residue 346. In one embodiment, the NA is selected not to code for a histidine (H) at residue 347. In one embodiment, the NA is selected not to code for an arginine (R) or asparagine at residue 347. In one embodiment, the NA is selected not to code for a threonine at residue 369. In one embodiment, the NA is selected not to code for an NA having a threonine or lysine at position 148. In one embodiment, the NA is selected not to code for NA with aspartic acid at position 151. In one embodiment, the NA is selected not to code for NA with asparagine at position 245. In one embodiment, the NA is selected not to code for NA with glycine at position 346. In one embodiment, the NA is selected to have one or more deletions at residues 46 to 50. The numbering for NA is based on N2. In one embodiment, the present disclosure provides isolated recombinant reassortant influenza viruses having any of the six "internal" viral segments from vaccine influenza viruses, such as PR8UW, NA viral segments with one or more specified residues or deletions of specified residues at specific positions, or any combination thereof, and HA viral segments, such as H1-H18, from circulating influenza viruses. Pharmaceutical compositions, such as compositions, vaccines, containing recombinant influenza viruses are also provided.

[0010] Thus, for vaccine viruses that are grown or passaged intracellularly, e.g. in eggs, the numbering is based on N2, and substitution, e.g. by mutation, of residues at positions 32, 147, 329, 347 in the NA, or one or more deletions of residues 46 to 50, or any combination thereof, or selection of the NA viral segment for the NA such that it does not encode a threonine at residue 32, does not encode an aspartic acid at position 147, does not encode an asparagine at residue 329, does not encode a histidine at residue 347, does not encode a threonine at residue 369, or has one or more deletions of residues 46 to 50, or any combination thereof, may result in stabilization of the HA and / or higher virus titers. In one embodiment, for vaccine viruses that are grown or passaged intracellularly, e.g., in eggs, the numbering is based on N2, and substitution, e.g., by mutation, of residues at positions 148, 151, 245, 346 in the NA, or any combination thereof, or selection of the NA viral segment for the NA such that it does not encode a threonine or lysine at residue 148, does not encode an aspartic acid at position 151, does not encode an asparagine at residue 245, does not encode a glycine at residue 346, or any combination thereof, may result in stabilization of the HA and / or higher viral titers.

[0011] In one embodiment, the disclosure provides an isolated recombinant influenza virus comprising PA, PB1, PB2, NP, NS, M, and HA viral segments, and an NA viral segment encoding an NA selected such that, numbering is based on N2, it does not encode a threonine at residue 32, it does not encode an aspartic acid at position 147, it does not encode an asparagine at residue 329, it does not encode a histidine at residue 347, it does not encode a threonine at residue 369, or has one or more deletions from residues 46 to 50, or any combination thereof, wherein the recombinant influenza virus has enhanced replication in avian eggs or has reduced HA mutations when propagated in avian eggs, compared to a corresponding influenza virus having an NA encoding a threonine at residue 32, it does not have a deletion at residues 46 or 50, it encodes an aspartic acid at position 147, it encodes an asparagine at residue 329, it encodes a histidine at residue 347, or any combination thereof. In one embodiment, the disclosure provides an isolated recombinant influenza virus comprising PA, PB1, PB2, NP, NS, M, and HA viral segments, and an NA viral segment encoding an NA selected to be, numbered based on N2, no threonine or lysine at residue 148, no aspartic acid at position 151, no asparagine at residue 245, no glycine at residue 346, no threonine at residue 369, or any combination thereof, wherein the recombinant influenza virus has enhanced replication in avian eggs or has reduced HA mutations when propagated in avian eggs, compared to a corresponding influenza virus having an NA encoding a threonine or lysine at residue 148, an aspartic acid at position 151, an asparagine at residue 245, a glycine at residue 346, or any combination thereof. In one embodiment, the isolated recombinant influenza virus is a reassortant.In one embodiment, the NA viral segment encodes an NA having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 1-3, 30-38, 48-50, or 54. In one embodiment, the NA viral segment encodes an NA having less than 100% amino acid sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3. In one embodiment, the NA viral segment encodes N2, N3, N7, or N9, and the positions of N3, N7, or N9 with the specified residue(s) correspond to the specified positions of N2. In one embodiment, the NA viral segment encodes N1, N4, N5, N6, N8, N10, or N11, and the positions of N1, N4, N5, N6, N8, N10, or N11 with the specified residue(s) correspond to the specified positions of N2. In one embodiment, the residue at position 32 is A, I, G, or L. In one embodiment, the deletion is a deletion of residues 46 to 50. In one embodiment, the residue at position 147 is N or Q. In one embodiment, the residue at position 148 is I or K. In one embodiment, the residue at position 151 is E, Q, H, or K. In one embodiment, the residue at position 245 is S, T, A, I, G, or L. In one embodiment, the residue at position 329 is S, V, I, L, A, G, D, or E. In one embodiment, the residue at position 344 is E, Q, N, H, or D. In one embodiment, the residue at position 346 is V, I, A, S, T, L, or L. In one embodiment, the residue at position 347 is G, Q, N, S, T, Y, C, or W. In one embodiment, the residue at position 369 is K, H, R, E, P, or D. In one embodiment, HA is H1, H3, H7, or H9. In one embodiment, the virus is an influenza A virus. In one embodiment, the PA, PB1, PB2, NP, M, and NS viral segments have at least 85% nucleic acid sequence identity to SEQ ID NO:24-29, or encode polypeptides having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to a polypeptide encoded by SEQ ID NO:24-29 or 39-44. In one embodiment, PB2 has an I, A, L, or G at residue 147.In one embodiment, the virus is an influenza B virus. In one embodiment, the selected NA viral segment does not have an aspartic acid at position 147, an asparagine at residue 329, and an arginine or histidine at residue 347. In one embodiment, the selected NA viral segment does not have a threonine or lysine at position 148, an aspartic acid at position 151, and an asparagine at position 245. In one embodiment, the selected NA viral segment has at least two of: N or Q at position 147, D or E at residue 329, or Q or G at residue 347. In one embodiment, the selected NA viral segment has at least two of: I, L, G, or A at position 148, E or Q at position 151, or S, I, T, V, or G at position 245. In one embodiment, the selected NA viral segment has at least two of: I or L at position 148, E or Q at position 151, or S, I, T, V, or G at position 245. In one embodiment, the selected NA viral segment has N or Q at position 147, S, D or E at residue 329, and Q or G at residue 347. In one embodiment, the selected NA viral segment has N or Q at position 147, S, D or E at residue 329, and V, S, I, or L at residue 346. In one embodiment, the residue at position 369 is K, H, R, E, P, or D. In one embodiment, the selected NA viral segment has I, L, G, or A at position 148, E or Q at position 151, S, I, T, V, or G at position 245, and K, H, R, E, P, or D at position 369. In one embodiment, the selected NA viral segment has an I or L at position 148, an E or Q at position 151, an S, I, T, V or G at position 245, and a K, H, R, E, P, or D at position 369. In one embodiment, the residue at position 369 is K, H, R, E, or D.

[0012] Further provided is an isolated recombinant nucleic acid, e.g., a vector, such as a viral vector, comprising a nucleic acid sequence encoding an influenza virus NA, selected such that the numbering is based on N2 and does not encode a threonine at residue 32, has one or more deletions of residues 46-50, does not encode an aspartic acid at position 147, does not encode an asparagine at residue 329, does not encode a histidine at residue 347, or any combination thereof. In one embodiment, the isolated recombinant nucleic acid does not encode a threonine or lysine at residue 148, does not encode an aspartic acid at position 151, does not encode an asparagine at residue 245, does not encode a glycine at residue 346, does not encode a threonine at residue 369, or any combination thereof. In one embodiment, the NA has at least 95% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49. In one embodiment, NA has less than 100% amino acid sequence identity to SEQ ID NO:2 or SEQ ID NO:3. In one embodiment, NA is N2, N3, N7, or N9. In one embodiment, NA is N1, N4, N5, N6, N8, N10, or N11. In one embodiment, the residue at position 32 is A, I, G, or L. In one embodiment, the deletion is a deletion of residues 46 to 50. In one embodiment, the residue at position 147 is N or Q. In one embodiment, the residue at position 329 is D or E. In one embodiment, the residue at position 347 is Q, N, S, T, Y, C, or W. In one embodiment, the residue at position 148 is I, L, G, or A. In one embodiment, the residue at position 148 is I or L. In one embodiment, the residue at position 151 is E, N, or Q. In one embodiment, the residue at position 245 is S, T, I, L, A, N, or V. In one embodiment, the residue at position 369 is K, H, R, E, P, or D.

[0013] Also provided is a method of preparing influenza virus, comprising administering to a vector for vRNA production comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operably linked to influenza virus NS DNA linked to a transcription termination sequence, wherein the PB1, PB2, PA, NP, NS, and M in the vector for vRNA production are in an amount effective to produce infectious influenza virus. the DNA is derived from one or more influenza vaccine virus isolates, wherein the NA DNA in the vector for vRNA production encodes an NA selected to not encode a threonine at residue 32, not encode an aspartic acid at position 147, not encode an asparagine at residue 329, not encode a histidine at residue 347, not encode a threonine or lysine at residue 148, not encode an aspartic acid at position 151, not encode an asparagine at residue 245, not encode a glycine at residue 346, not encode a threonine at residue 369, or have a deletion at one or more of residues 46 to 50, or any combination thereof, wherein the numbering of the NA residues is based on N2; andthe vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA, the vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, the vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and the vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M2, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS1, or a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS2. In one embodiment, the NA has at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity, for example, to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49. In one embodiment, the NA has at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity, for example, to SEQ ID NO:51-59 or 69-70. In one embodiment, the NA has less than 100% amino acid sequence identity to SEQ ID NO:2 or SEQ ID NO:3. In one embodiment, the NA is N2, N3, N7, or N9. In one embodiment, the NA is N1, N4, N5, N6, N8, N10, or N11. In one embodiment, the residue at position 32 is A, I, G, or L. In one embodiment, the deletion isThe deletion is from residue 46 to 50. In one embodiment, the residue at position 147 is N or Q. In one embodiment, the residue at position 329 is S, D or E. In one embodiment, the residue at position 347 is Q, N, S, T, Y, C or W. In one embodiment, the residue at position 148 is I, L, G or A. In one embodiment, the residue at position 148 is I or L. In one embodiment, the residue at position 151 is E, N or Q. In one embodiment, the residue at position 245 is S, T, I, L, A, N or V. In one embodiment, the residue at position 329 is S, I, L, A, N or V. In one embodiment, the residue at position 344 is E, Q, N, H or D. In one embodiment, the residue at position 346 is V, S, T, I, L, A or V. In one embodiment, the residue at position 347 is G, S, T, I, L, A, or V. In one embodiment, the residue at position 369 is K, H, R, E, P, or D.

[0014] In one embodiment, the HA is H1, H3, H5, H7, or H9. In one embodiment, the virus is an influenza A virus. In one embodiment, the PA, PB1, PB2, NP, M, and NS viral segments have at least 85%, 85%, 90%, 95%, or 99% nucleic acid sequence identity to SEQ ID NO: 24-29 or 39-44, or encode a polypeptide having at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to a polypeptide encoded by SEQ ID NO: 24-29 or 39-44. In one embodiment, PB2 has an I, A, L, or G at residue 147. In one embodiment, the HA is any of H2, H4, H5, H6, H8, or H10-H18. In one embodiment, the virus is an influenza B virus.

[0015] Further provided is a method of immunizing a bird or mammal with a composition having an effective amount of the virus described herein. In one embodiment, the composition comprises at least one other different influenza virus. In one embodiment, the mammal is a human. In one embodiment, the composition is administered intranasally or by injection.

[0016] Thus, the present invention provides a method for selecting influenza viruses with enhanced replication in cell culture, e.g., in embryonated eggs. The method includes providing cells suitable for influenza vaccine production; serially culturing one or more influenza virus isolates in eggs; and isolating serially cultured viruses with enhanced growth compared to the one or more isolates prior to serial culture. Also provided is a method for identifying NA that stabilizes HA and / or results in altered growth of recombinant influenza virus, e.g., in eggs. The method includes introducing one or more substitutions or deletions as described herein into an NA viral segment to produce a mutant NA viral segment; and optionally determining whether the mutant NA viral segment, when present in a replication-competent recombinant influenza virus, results in enhanced replication of the recombinant influenza virus in eggs compared to a corresponding replication-competent influenza virus without the one or more substitutions and / or deletions in NA and optionally inhibits HA mutations.

[0017] In one embodiment, the disclosure provides an isolated influenza A virus with a hallmark residue(s) and / or deletions, or combinations thereof, in the NA described herein. In one embodiment, the isolated influenza A virus with the hallmark residue(s) and / or deletions, or combinations thereof, has an NA amino acid sequence with at least 80%, such as 90%, 92%, 95%, 97% or 99%, including any integer between 80-99, contiguous amino acid sequence identity to a polypeptide encoded by one of SEQ ID NOs: 1, 2, 3, or 30-38. In one embodiment, the isolated influenza A virus of the invention with hallmark residue(s) and / or deletions, or combinations thereof, has an HA from any one of HA subtypes 1-18. In one embodiment, the hallmark residue is a conservative substitution, for example, with SEQ ID NO: 2 or SEQ ID NO: 3. Conservative amino acid substitution refers to the interchangeability of residues having similar side chains. For example, the amino acids having aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; the amino acids having aliphatic-hydroxyl side chains are serine and threonine; the amino acids having amide-containing side chains are asparagine and glutamine; the amino acids having aromatic side chains are phenylalanine, tyrosine, and tryptophan; the amino acids having basic side chains are lysine, arginine, and histidine; and the amino acids having sulfur-containing side chains are cysteine ​​and methionine. In one embodiment, the conservative amino acid substitution groups are: threonine-valine-leucine-isoleucine-alanine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; glutamic acid-aspartic acid; and asparagine-glutamine.

[0018] In one embodiment, mutations are introduced into the NA viral segment of an influenza virus isolate by recombinant DNA techniques, including, for example, site-directed mutagenesis or replacement of a portion of the NA coding sequence with a portion containing the hallmark residue(s) or deletion. In one embodiment, the NA viral segment with the hallmark residues and / or deletions described herein is combined with the HA segment and an internal viral segment of an influenza vaccine virus.

[0019] The present disclosure provides multiple influenza virus vectors of the invention, such as those useful for preparing reassortant viruses, including 6:1:1 reassortants, 6:2 reassortants, and 7:1 reassortants. A 6:1:1 reassortant is an influenza virus with 6 internal viral segments derived from a vaccine virus, an HA viral segment derived from a (second) viral isolate different from the vaccine virus, and an NA viral segment with the characteristic residue(s) and / or deletion(s) or combination thereof described herein, which HA segment and NA viral segment are derived from a viral source different from the vaccine virus; a 6:2 reassortant is an influenza virus with 6 internal viral segments derived from a vaccine virus, an NA viral segment with characteristic residue(s) and / or deletion(s) or combination thereof, which segments are derived from the same source as the HA segment, and an HA viral segment from a viral isolate different from the vaccine virus; and a 7:1 reassortant is, in one embodiment, an influenza virus with 6 internal viral segments and an HA segment derived from a vaccine virus, and an NA segment modified to include characteristic residue(s) and / or deletion(s) or combination thereof, which NA segment is derived from a viral source different from the vaccine virus.

[0020] In one embodiment of the invention, the plurality comprises a vector for vRNA production selected from a vector comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to influenza virus NS DNA linked to a transcription termination sequence. In one embodiment, DNA for vRNA production of PB1, PB2, PA, NP, M, and NS has sequences from influenza viruses that replicate to high titers in cultured mammalian cells, such as MDCK cells, Vero cells, or PER.C6® cells, or embryonated eggs, and / or sequences from vaccine viruses, such as those that do not cause significant disease in humans. DNA for vRNA production of NA can be derived from any NA, such as any of N1-N11, and DNA for vRNA production of HA can be derived from any HA, such as any of H1-H18. In one embodiment, DNA for vRNA production can be for influenza B or C viruses. For example, DNA for vRNA production includes influenza B virus PA, PB1, PB2, NP, NS, and M, or influenza B virus PA, PB1, PB2, NP, NS, M, and NA, where for NA, vRNA has NA with characteristic residues and / or deletions as described herein.The DNA for NA and HA vRNA production may be from different strains or isolates (6:1:1 reassortants), or from the same strain or isolate (6:2 reassortants), or NA or HA may be from the same strain or isolate as for the internal genes (7:1 reassortants). The plurality also includes vectors for mRNA production selected from a vector encoding influenza virus PA, a vector encoding influenza virus PB1, a vector encoding influenza virus PB2, a vector encoding influenza virus NP, and optionally one or more vectors encoding NP, NS, M, e.g., M1 and M2, HA, or NA. The vector encoding the viral proteins may further comprise a transcription termination sequence.

[0021] Viruses capable of providing internal genes for reassortants within the scope of the present invention can be cultured in high titers, e.g., at least about 10 5 PFU / mL, e.g., at least 10 6 PFU / mL, 10 7 PFU / mL or 10 8 Titer of PFU / mL; high titer in embryonated eggs, e.g., at least about 10 7 EID 50 / mL, e.g., at least 10 8 EID 50 / mL, 10 9 EID 50 / mL or 10 10 EID 50 / mL titer; high titer in MDCK cells, e.g., at least about 10 7 PFU / mL, e.g., at least 10 8 These include viruses that have a titer in PFU / mL or high titers in two or more of these host cell types.

[0022] Other reassortants with internal genes from other PR8 isolates or vaccine viruses may be utilized in the recombinant reassortant viruses.

[0023] In one embodiment, the DNA for the internal genes of PB1, PB2, PA, NP, M, and NS encodes a protein with substantially the same activity as the corresponding polypeptide encoded by one of SEQ ID NOs: 24-29 or 39-44. As used herein, "substantially the same activity" includes about 0.1%, 1%, 10%, 30%, 50%, 90%, e.g., up to 100% or more activity, or about 80%, 90% or more detectable protein levels, respectively, of the activity or protein level of the corresponding full-length polypeptide. In one embodiment, the nucleic acid is a sequence encoding a polypeptide that is substantially the same, e.g., at least 80%, e.g., 90%, 92%, 95%, 97% or 99% contiguous amino acid sequence identity, including any integer between 80 and 99, to a polypeptide encoded by one of SEQ ID NOs: 24-29 or 39-44. In one embodiment, the isolated and / or purified nucleic acid molecule comprises a nucleotide sequence that is substantially the same, e.g., at least 50%, e.g., 60%, 70%, 80% or 90% or more contiguous nucleic acid sequence identity, including any integer between 50 and 100, to one of SEQ ID NOs: 24-29, and in one embodiment also encodes a polypeptide having at least 80%, e.g., 90%, 92%, 95%, 97% or 99% contiguous amino acid sequence identity, including any integer between 80 and 99, to a polypeptide encoded by one of SEQ ID NOs: 24-29 or 39 to 44. In one embodiment, the influenza virus polypeptide has one or more, e.g., 2, 5, 10, 15, 20 or more conservative amino acid substitutions, e.g., up to 10% or 20% conservative substitutions of residues, to a polypeptide encoded by one of SEQ ID NOs: 24-29 or 39 to 44. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.For example, the amino acids having aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; the amino acids having aliphatic-hydroxyl side chains are serine and threonine; the amino acids having amide-containing side chains are asparagine and glutamine; the amino acids having aromatic side chains are phenylalanine, tyrosine, and tryptophan; the amino acids having basic side chains are lysine, arginine, and histidine; and the amino acids having sulfur-containing side chains are cysteine ​​and methionine. In one embodiment, the conservative amino acid substitution groups are: valine-leucine-isoleucine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; glutamic acid-aspartic acid; and asparagine-glutamine. In one embodiment, the influenza virus polypeptide has one or more, e.g., two, three, or four, non-conservative amino acid substitutions relative to a polypeptide encoded by one of SEQ ID NOs:24-29.

[0024] In one embodiment, the nucleic acid is a sequence encoding a polypeptide encoded by one of SEQ ID NOs: 1-3 or 48-49, or one of SEQ ID NOs: 51-59, or a NA polypeptide that is substantially identical, e.g., at least 80%, e.g., 90%, 92%, 95%, 97% or 99% contiguous amino acid sequence identity, including any integer between 80 and 99, to one of the accession numbers ACP41107.1 (N1) (SEQ ID NO: 36), AIK26357.1 (N7) (SEQ ID NO: 37), ALH21372.1 (N9) (SEQ ID NO: 45), or BAK86313.1 (N2) (SEQ ID NO: 50), the sequences of which are incorporated herein by reference. In one embodiment, the isolated and / or purified nucleic acid molecule encodes a polypeptide having at least 80%, such as 90%, 92%, 95%, 97% or 99% contiguous amino acid sequence identity to one of SEQ ID NOs: 1, 3, 30-35, or 48-49, one of Accession Nos. ACP41107.1 (N1), AIK26357.1 (N7), ALH21372.1 (N9), or BAK86313.1 (N2), or to a NA encoded by one of SEQ ID NOs: 51-59, the sequences of which are incorporated herein by reference. In one embodiment, the influenza virus polypeptide has one or more, e.g., 2, 5, 10, 15, 20 or more conservative amino acid substitutions, e.g., up to 10% or 20% conservative substitutions of residues, to the NA encoded by SEQ ID NO: 1, 3, 30-35, 48-49, or one of Accession Nos. ACP41107.1 (N1), AIK26357.1 (N7), ALH21372.1 (N9), or BAK86313.1 (N2), or one of SEQ ID NOs: 51-59, the sequences of which are incorporated herein by reference. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.For example, the amino acids having aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; the amino acids having aliphatic-hydroxyl side chains are serine and threonine; the amino acids having amide-containing side chains are asparagine and glutamine; the amino acids having aromatic side chains are phenylalanine, tyrosine, and tryptophan; the amino acids having basic side chains are lysine, arginine, and histidine; and the amino acids having sulfur-containing side chains are cysteine ​​and methionine. In one embodiment, the conservative amino acid substitution groups are: valine-leucine-isoleucine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; glutamic acid-aspartic acid; and asparagine-glutamine. In one embodiment, the influenza virus polypeptide has one or more, e.g., two, three or four, non-conservative amino acid substitutions relative to a polypeptide having an NA encoded by one of SEQ ID NOs: 1, 3, 30-35, 48-49, or one of Accession Nos. ACP41107.1 (N1), AIK26357.1 (N7), ALH21372.1 (N9), or BAK86313.1 (N2), or one of SEQ ID NOs: 51-59, the sequences of which are incorporated herein by reference.

[0025] Thus, the invention includes the use of isolated and purified vectors or plasmids expressing or encoding influenza viral proteins or expressing or encoding influenza vRNA, both native and recombinant vRNA. The vectors contain influenza cDNA, such as influenza A (e.g., any influenza A gene, including any of the 18 HA subtypes or 11 NA subtypes), B or C DNA (see Fields Virology (Fields et al. (eds.), Lippincott, Williams and Wickens (2013), which is specifically incorporated herein by reference). Any suitable promoter or transcription termination sequence may be utilized to express a protein or peptide, such as a viral protein or peptide, a protein or peptide of a non-viral pathogen, or a therapeutic protein or peptide.

[0026] The composition or vectors of the invention may also include a heterologous gene or open reading frame of interest, e.g., a foreign gene encoding an immunogenic peptide or protein useful as a vaccine or in gene replacement, e.g., an epitope useful in cancer therapy or vaccine, or a peptide or polypeptide useful in gene therapy. When preparing a virus, the vector or plasmid containing the gene or cDNA of interest may replace the vector or plasmid for the influenza virus gene, or may be added to the vector or plasmid for all influenza virus genes. Thus, another embodiment of the invention includes a composition or vector as described above, where one of the vectors is replaced by or further comprises a desired nucleic acid sequence, e.g., a 5' influenza virus sequence, optionally including a 5' influenza virus coding sequence or a portion thereof, linked to a desired cDNA, optionally including a 3' influenza virus coding sequence or a portion thereof, linked to a 3' influenza virus coding sequence or a portion thereof. In one embodiment, the desired nucleic acid sequence, e.g., a cDNA, is in the antisense (antigenome) orientation. Introduction of such a vector, in combination with other vectors described above, into a host cell permissive for influenza virus replication results in recombinant virus that contains vRNA corresponding to the heterologous sequence of that vector.

[0027] The promoter in the vector for vRNA production is an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T7 promoter, or a T3 promoter, and optionally the vector includes a transcription termination sequence, such as an RNA polymerase I transcription termination sequence, an RNA polymerase II transcription termination sequence, an RNA polymerase III transcription termination sequence, or a ribozyme. Ribozymes within the scope of the present invention include, but are not limited to, Tetrahymena ribozyme, RNase P, hammerhead ribozyme, hairpin ribozyme, hepatitis ribozyme, and synthetic ribozymes. In one embodiment, the RNA polymerase I promoter is a human RNA polymerase I promoter.

[0028] The promoter or transcription termination sequence in a vRNA or viral protein expression vector may be the same or different from the promoter or any other vector. In one embodiment, the vector or plasmid expressing influenza vRNA contains a promoter suitable for expression in at least one particular host cell, including, for example, an avian or mammalian host cell, such as a canine, feline, equine, bovine, ovine, or primate cell, or in more than one host cell, including a human cell.

[0029] In one embodiment, at least one vector for vRNA comprises an RNA polymerase II promoter linked to another ribozyme sequence linked to a viral coding sequence linked to a ribozyme sequence, optionally linked to an RNA polymerase II transcription termination sequence. In one embodiment, at least two, for example 3, 4, 5, 6, 7 or 8 vectors for vRNA production comprise an RNA polymerase II promoter, a first ribozyme sequence, which is 5' to a sequence corresponding to a viral sequence including a viral coding sequence, which is 5' to a second ribozyme sequence, which is 5' to a transcription termination sequence. Each RNA polymerase II promoter in each vRNA vector may be the same or different from the RNA polymerase II promoter in any other vRNA vector. Similarly, each ribozyme sequence in each vRNA vector may be the same or different from the ribozyme sequence in any other vRNA vector. In one embodiment, the ribozyme sequences in a single vector are not identical.

[0030] In one embodiment, at least one vector comprises a sequence encoding PB1, PB2, PA, NP, M, or NS having substantially the same activity as a corresponding polypeptide encoded by one of SEQ ID NOs: 24-29 or 39 to 44, or a sequence corresponding to a portion thereof, such as a sequence encoding a polypeptide with at least 80%, such as 85%, 90%, 92%, 95%, 98%, 99% or 100% amino acid identity to a polypeptide encoded by one of SEQ ID NOs: 24-29, including any integer between 88 and 100. Optionally, two vectors may be utilized instead of a vector comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, such as a vector comprising a promoter operably linked to an influenza virus M1 cDNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to an influenza virus M2 cDNA linked to a transcription termination sequence.

[0031] The vectors of the present invention may be physically linked, or each vector may be present on a separate plasmid or other, e.g., linear, nucleic acid delivery vehicle.In one embodiment, each vRNA producing vector is present on a separate plasmid.In one embodiment, each mRNA producing vector is present on a separate plasmid.

[0032] The present invention also provides a method for preparing influenza virus. The method includes contacting a cell with a plurality of vectors of the present invention, e.g., sequentially or simultaneously, in an amount effective to produce infectious influenza virus. The present invention also includes isolating the virus from a cell contacted with a plurality of vectors. Thus, the present invention further provides isolated viruses, as well as host cells contacted with a plurality of vectors or viruses of the present invention. In another embodiment, the present invention includes contacting a cell with one or more vectors, either vRNA or protein producing vectors, before the other vector, either vRNA or protein producing vector. In one embodiment, the promoter for the vRNA vector utilized in the method is an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T3 promoter, or a T7 promoter. In one embodiment, the RNA polymerase I promoter is a human RNA polymerase I promoter. In one embodiment, each vRNA vector utilized in the method is on a separate plasmid. In one embodiment, the vRNA vector utilized in the method is on one plasmid, or on two or three different plasmids. In one embodiment, each mRNA vector utilized in the method is on a separate plasmid. In one embodiment, the mRNA vectors for PA, PB1, PB2 and NP utilized in the method are on one plasmid or on two or three different plasmids.

[0033] The methods of making viruses described herein do not require helper virus infection, which is useful in viral mutagenesis studies and in the production of vaccines (e.g., for AIDS, influenza, hepatitis B, hepatitis C, rhinovirus, filovirus, malaria, herpes, foot and mouth disease, etc.) and gene therapy vectors (e.g., for cancer, AIDS, adenosine deaminase, muscular dystrophy, ornithine transcarbamylase deficiency, central nervous system tumors, etc.) Thus, viruses for use in medical treatments (e.g., for vaccines or gene therapy) are provided.

[0034] The invention also provides isolated viral polypeptides, as well as methods of preparing and using the recombinant viruses of the invention. The methods include administering to a host organism, e.g., a mammal, an effective amount of an influenza virus, e.g., an inactivated virus preparation, of the invention, optionally in combination with an adjuvant and / or carrier, in an amount effective to prevent or ameliorate infection of an animal, e.g., a mammal, with the virus or an antigenically closely related virus. In one embodiment, the virus is administered intramuscularly, while in another embodiment, the virus is administered intranasally. In some dosing protocols, all doses may be administered intramuscularly or intranasally, while in others a combination of intramuscular and intranasal administration is utilized. The vaccine may further include other isolates of influenza virus, including recombinant influenza viruses, other pathogen(s), additional biological material, or microbiological components, e.g., to form a multivalent vaccine. In one embodiment, intranasal vaccination with, e.g., inactivated influenza virus, and a mucosal adjuvant, can induce nasopharyngeal virus-specific IgA and neutralizing antibodies, as well as serum IgG.

[0035] The influenza viruses of the present invention may be utilized in conjunction with other antiviral drugs, such as amantadine, rimantadine, and / or neuraminidase inhibitors, and may be administered, for example, in combination with these antiviral drugs, separately, e.g., before, during and / or after administration.

[0036] Thus, the modified neuraminidase comprises at least one, or at least two, or at least three modifications, wherein the modifications comprise one or more amino acids within positions 29-35, one or more amino acids within positions 44-52, one or more amino acids within positions 144-154, one or more amino acids within positions 240-250, one or more amino acids within positions 326-333, one or more amino acids within positions 344-350, one or more amino acids within positions 365-375, or a combination thereof, where the numbering is with respect to N2. In one embodiment, the NA comprises a deletion of at least one proline, asparagine, glutamine, valine, or a combination of proline, one or more asparagine(s), glutamine, and valine in positions 44-52; a substitution of threonine in positions 29-35; a substitution of threonine or aspartic acid in positions 145-155; a substitution of asparagine in positions 240 to 250 or 326-333; a substitution of histidine in positions 345-350; or a combination thereof. [Brief description of the drawings]

[0037] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1. Nucleotide sequences of viral segments of A / Yokohama / 2017 / 2003 (sequence numbers 4-11) and the amino acid sequence of NA of A / Yokohama / 2017 / 2003 (sequence number 3).

[0038] [Diagram 2] Figure 2. Amino acid sequence of NA of A / Saitama / 103 / 2014 (sequence number 2).

[0039] [Diagram 3] Figure 3. Nucleotide sequence of the NA viral segment (SEQ ID NO: 12) and the amino acid sequence of the NA of the A / Yokohama / 2017 / 2003 mutant (SEQ ID NO: 1) and the nucleotide sequences of other viral segments of that mutant (SEQ ID NOs: 12-21).

[0040] [Figure 4] Figure 4. Graph showing titers in eggs of various reassortants with PB2, M, NA and NP segments of mutant and wild type A / Yokohama / 2017 / 2003. Viral inoculation: 2x103 pfu / egg into allantoic fluid, incubated at 37°C for 72 h.

[0041] [Diagram 5] Figure 5. Location of NA mutations on the 3D structure of N2 NA.

[0042] [Figure 6] Figure 6. Graph showing titers in eggs for recombinant viruses with specific mutations found in the mutant of A / Yokohama / 2017 / 2003 ("Y2017-M3L4"). Virus inoculation: 2 x 103 pfu / egg into allantoic fluid, incubated at 37°C for 72 h.

[0043] [Figure 7] Figure 7. Graph showing titers in eggs of reassortants with two different backbones (PA, PB1, PB2, NP, NS and M) and two different HA and NA combinations (e.g., PB2-I504V, PB1-M40L / G180W, PA-R401K, NP-I116L, NS1-A30P / R118K; and Y2017-M3L4 NA containing the following mutations: NA-T32A, D147N, N329D, H347Q and deletion 46-50aa). Viral inoculation: 2x103 pfu / egg into allantoic fluid, incubated at 37°C for 72h.

[0044] [Figure 8] Figure 8. Amino acid sequence comparison of Yokohama / 2017 / 2003 NA wild type (sequence number 3) and Y2017-M3L4 (sequence number 1).

[0045] [Figure 9] Figure 9. Illustrative NA sequences of N3, N4, N6, N7, N8, and N9 (SEQ ID NOs:30-35).

[0046] [Figure 10] FIG. 10. Illustrative sequences of internal viral segments of the master vaccine strain (SEQ ID NOs: 39-44).

[0047] [Figure 11] Figure 11. Illustrative NA sequences (SEQ ID NOs:51-54).

[0048] [Figure 12] FIG. 12. Titers in eggs for various NA mutants.

[0049] [Figure 13] Figure 13. Titers of HK4801HA, Y2017-M3L4NA and HY-PR8 (PB2 C4U, I504V; PB1 C4U, M40L / G180W; PA C4U, R401K; NP I116L; NS A30P / R118K) over time and analysis of HA mutations in infected eggs.

[0050] [Figure 14] FIG. 14 shows data for viruses passaged in eggs that carried specific NA mutations but no substitutions in the HA.

[0051] [Figure 15] FIG. 15 is a schematic diagram of the locations of specific NA residues.

[0052] [Figure 16] FIG. 16 is a schematic diagram of the locations of specific NA residues.

[0053] [Figure 17] FIG. 17 shows virus titers for an egg-passaged isolate (HK4801NA (T148K, D151E, H347G, and T369K)) that resulted in efficient replication in the allantoic cavity against viruses carrying either HK4801HA or Singapore0019 HA (HY-PR8 backbone).

[0054] [Figure 18] FIG. 18 shows egg titers for different combinations of selected residues at positions 153, 329, 347, and 369 in NA.

[0055] [Figure 19] FIG. 19 summarizes virus titers over time (HK4801HA, Y2017-M3L4NA and HY-PR8 (PB2 C4U, I504V; PB1 C4U, M40L / G180W; PA C4U, R401K; NP I116L; NS A30P / R118K) and HA status.

[0056] [Figure 20] FIG. 20 summarizes the viral titers and HA status for viruses with different NA.

[0057] [Figure 21] FIG. 21 provides inoculated and harvested virus titers at allantoic passage (HA-K189E / N158K / A212T mutant virus).

[0058] [Figure 22] FIG. 22 shows detection of HA status after multiple passages.

[0059] [Figure 23] FIG. 23 shows egg titers for viruses with different NA.

[0060] [Figure 24] Figure 24 is a close-up of the NA active center. Most egg-adapted mutations are located in / around the NA active site.

[0061] [Diagram 25] Figure 25. Location of amino acid substitutions in egg-adapted influenza A / Hong Kong / 4801 / 2014 (H3N2) and A / Alaska / 232 / 2015 (H3N2) neuraminidase proteins (SEQ ID NOs: 51-52).

[0062] [Figure 26] Figure 26. Introduction of the NA mutation (see Figure 25) into the NA of the H3N2 virus (SEQ ID NO:53) from the 2017 / 18 season enhanced growth of the HY-PR8 backbone virus without an HA mutation.

[0063] [Figure 27] FIG. 27. Mutations observed in the NA mutant viruses (HY-PR8 backbone) of FIG. 26 upon egg passaging.

[0064] [Figure 28] Figure 28. Introduction of NA mutations into the NA of H3N2 viruses derived from the 2017 / 18 season enhanced the growth of HY-PR8 backbone viruses without HA mutations.

[0065] [Figure 29] FIG. 29. Mutations observed in the NA mutant viruses (HY-PR8 backbone) of FIG. 28 upon egg passaging.

[0066] [Diagram 30] Figure 30. HY-PR8 backbone viruses harboring A / Yokohama / 48 / 2018HA and A / Yokohama / 48 / 2018NA (T148K, D151E, N245S, H347G, and T369K) acquired the same NA-K148I mutation, and no HA mutation (SEQ ID NO: 54) was detected.

[0067] [Diagram 31] Figure 31. HY-PR8 backbone viruses harboring A / Yokohama / 48 / 2018HA and A / Yokohama / 48 / 2018NA (T148I, D151E, N245S, H347G, and T369K) acquired only the HA-435L mutation in the stem region.

[0068] [Diagram 32]Figure 32. HA-H435L is located in the stem region of the HA trimer, and previous studies have reported that HA-H435L does not affect antigenicity (Kuwahara et al., Jpn. J. Infect. Dis., 2018).

[0069] [Diagram 33] Figure 33. Effect of introduction of NA-T148I, D151E, N245S, H347G, and T369K into the NA of H3N2 viruses from the 2017 / 18 season.

[0070] [Diagram 34] Figure 34. Effect of introduction of NA-T148I, D151E, N245S, H347G, and T369K into the NA (SEQ ID NOs: 55-56) of H3N2 viruses from the 2018 / 19 season.

[0071] [Diagram 35] Figure 35. Yokohama48NA (T148I, D151E, N245S, H347G, and T369K) enhanced the growth of viruses having the HA (sequence numbers 57-58) of H3N2 viruses from the 2017-18 and 2018-19 seasons.

[0072] [Diagram 36] FIG. 36. Yokohama48NA (T148I, D151E, N245S, H347G, and T369K) has reduced sialidase activity.

[0073] [Figure 37] Figure 37. Growth of Kansas / 14 / 2017 (SEQ ID NO:59) was enhanced by introduction of NA mutations T148I, D151E, N245S, H347G, and T369K or by carrying Yokohama48NA (T148I, D151E, N245S, H347G, and T369K).

[0074] [Figure 38]Figure 38. Neutralization of viruses bearing Aichi / 2 / 68 HA and wild-type or mutant NA from 2017-18 season H3N2 viruses by human monoclonal IgG clone F045-092.

[0075] [Figure 39A] Figure 39A. Position of sialic acid relative to residues in NA. [Figure 39B] FIG. 39B. Enlarged view of FIG. 38A.

[0076] [Diagram 40] Figure 40. Illustrative NA sequences for modification (sequence numbers 51-59) and modified NA sequences (sequence numbers 69-70).

[0077] [Diagram 41] Figure 41. Illustrative HA sequences from stabilized strains (SEQ ID NOs:60-68).

[0078] [Diagram 42] Figure 42. Illustrative NA residues in 6M viruses observed in egg-grown A / HongKong / 4801 / 2014 and A / Alaska / 232 / 2015.

[0079] [Diagram 43] Figure 43. Viruses in which the 6M residue was introduced into the NA of A / Delaware / 33 / 2018 and A / Saint-Petersburg / RII-324S / 2019, as well as viruses with Yokohama / 147 / 2017NA(6M)NA, enhanced viral growth of HY-PR8-backbone viruses with wild-type HA of A / Delaware / 33 / 2018 or A / Saint-Petersburg / RII-324S / 2019. Harvested viruses with NA(6M) or Yokohama / 147 / 2017NA(6M) of each strain were sequenced but did not have additional mutations in the HA and NA.

[0080] [Diagram 44]Figure 44. Viruses in which 6M was introduced into the NA of A / Tokyo / UT-GR85 / 2019 and A / Kanagawa / IC1820 / 2019 did not enhance HY-PR8-backbone virus growth. However, viruses carrying Yokohama / 147 / 2017NA(6M) showed enhanced virus growth of HY-PR8-backbone viruses carrying wild-type HA of A / Tokyo / UT-GR85 / 2019 or A / Kanagawa / IC1820 / 2019 without HA mutations. The collected viruses carrying Yokohama / 147 / 2017NA(6M) were sequenced but did not have additional mutations in HA and NA.

[0081] [Diagram 45] FIG. 45. Mutations observed in the HA and NA proteins of HY-PR8 backbone virus carrying Yokohama147NA(6M) upon 10 passages in eggs.

[0082] [Diagram 46] Figure 46. Location of HA mutations (shown in Figure 43) that occurred during egg passaging on the 3D structure of the HA protein.

[0083] [Figure 47] Figure 47. Location of NA mutations (shown in Figure 43) that occurred during egg passaging on the 3D structure of the NA protein.

[0084] [Figure 48] Figure 48. Reduced molecular weight of Yokohama147NA(6M). VP40-induced VLPs carrying FLAG-tagged Yokohama147NA or Yokohama147NA(6M) were analyzed by immunoblotting with anti-FLAG and anti-VP40 antibodies.

[0085] [Figure 49] Figure 49. Loss of glycosylation sites in the NA protein due to mutations.

[0086] [Figure 50] Figure 50. Receptor-binding specificity of Yokohama147HA, Yokohama147NA, and Yokohama147NA(6M).

[0087] [Figure 51] Figure 51. Introduction of 6M into Yokohama147NA reduced sialidase activity.

[0088] [Figure 52] Figure 52. HY-PR8 backbone viruses harboring wild-type HA and mutant NA (T148I, D151E, N245S, T329S, K344E, H347G and T369K) (=6M+T148I+T329S+K344E) derived from A / Kansas / 14 / 2017NA did not acquire HA and NA mutations upon egg passage.

[0089] [Figure 53] Figure 53. Location of NA mutations on the 3D structure of the NA protein: T148I, D151E, N245S, T329S, K344E, G346V, H347G and T369K. 6M is shown in purple and T148I, T329S, K344E are shown in green. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] Detailed Description definition The term "isolated" as used herein refers to an in vitro preparation and / or separation of a nucleic acid molecule, such as a vector or plasmid, a peptide or polypeptide (protein), or a virus of the invention, and thus is not associated with in vivo materials or is substantially purified from in vitro materials. Isolated virus preparations are generally obtained by in vitro culture and propagation and / or through passage in eggs, and are substantially free of other infectious materials.

[0091] As used herein, "substantially purified" means that the subject species is the predominant species, e.g., on a molecular basis, that is more abundant than any other individual species in a composition, and preferably is at least about 80% of the species present, and optionally greater than 90%, e.g., 95%, 98%, 99% or more of the species present in the composition.

[0092] As used herein, "substantially free" means below the level of detection for a particular infectious agent using standard methods for that agent.

[0093] A "recombinant" virus is one that has been manipulated in vitro, e.g., using recombinant DNA techniques, to introduce changes into the viral genome. Reassortant viruses can be prepared by recombinant or non-recombinant techniques.

[0094] As used herein, the term "recombinant nucleic acid" or "recombinant DNA sequence or segment" refers to a nucleic acid, e.g., DNA, that has been derived or isolated from a source and subsequently chemically altered in vitro, such that its sequence corresponds to a naturally occurring sequence that is not naturally occurring or is not located as it is in the native genome. An example of DNA "derived" from a source is a DNA sequence that has been identified as a useful fragment, which is then chemically synthesized in essentially pure form. An example of such DNA that has been "isolated" from a source is a useful DNA sequence that has been cleaved or removed from the source by chemical means, e.g., by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the present disclosure, by genetic engineering techniques.

[0095] As used herein, a "heterologous" influenza virus gene or viral segment is derived from an influenza virus source that is different from the majority of the other influenza virus genes or viral segments in the recombinant influenza virus, eg, a reassortant.

[0096] The terms "isolated polypeptide," "isolated peptide," or "isolated protein" include a polypeptide, peptide, or protein, or some combination thereof, encoded by cDNA or recombinant RNA, including those of synthetic origin.

[0097] As used herein, the term "recombinant protein" or "recombinant polypeptide" refers to a protein molecule that is expressed from a recombinant DNA molecule. In contrast, the term "native protein" is used herein to refer to a protein that has been isolated from a natural (i.e., non-recombinant) source. Molecular biology techniques may be used to create recombinant forms of a protein that have the same characteristics as the native form of the protein.

[0098] Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm.

[0099] Computer implementations of these mathematical algorithms can be used to compare sequences to determine sequence identity. Alignments using these programs can be performed using default parameters. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves the initial identification of high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that either match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the similar word score threshold. These initial similar word hits act as seeds to initiate searches to find longer HSPs that contain them. These word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by a quantity X from its maximum achieved value, and the cumulative score becomes zero or below due to the accumulation of one or more negatively scoring residue alignments, or reaching the end of either sequence.

[0100] In addition to calculating the percent sequence identity, the BLAST algorithm can also perform statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered to be similar to a reference nucleic acid sequence if the minimum sum probability in the comparison of the test nucleic acid sequence with the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0101] The BLASTN program (for nucleotide sequences) can use as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program can use as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See http: / / www.ncbi.n1m.nih.gov. Alignments can also be performed manually, with verification.

[0102] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0103] Influenza virus structure and proliferation Influenza A viruses carry a genome of eight single-stranded negative-sense viral RNAs (vRNAs) that code for at least ten proteins. The influenza virus life cycle begins with the binding of hemagglutinin (HA) to sialic acid-containing receptors on the surface of the host cell, followed by receptor-mediated endocytosis. The low pH in late endosomes causes a conformational shift in HA, which exposes the N-terminus of the HA2 subunit (the so-called fusion peptide). This fusion peptide initiates fusion of the virus with the endosomal membrane, and the matrix protein (M1) and RNP complex are released into the cytoplasm. The RNP consists of the nucleoprotein (NP), which encapsulates the vRNA, and the viral polymerase complex formed by the PA, PB1, and PB2 proteins. The RNP is transported to the nucleus, where transcription and replication occur. The RNA polymerase complex catalyzes three distinct reactions: synthesis of mRNA with a 5' cap and 3' polyA structure, synthesis of full-length complementary RNA (cRNA), and synthesis of genomic vRNA using cRNA as a template. The newly synthesized vRNA, NP, and polymerase proteins are then assembled into RNPs, exported from the nucleus, and transported to the cell membrane, where budding of progeny virus particles occurs. The neuraminidase (NA) protein plays a key role in the later stages of infection by removing sialic acid from sialyloligosaccharides, thus releasing newly assembled virions from the cell surface, and preventing self-assembly of virus particles. Viral assembly involves protein-protein and protein-vRNA interactions, but the nature of these interactions is largely unknown.

[0104] Influenza B and C viruses are structurally and functionally similar to influenza A viruses, but there are some differences. For example, influenza B viruses do not have the M2 protein with ion channel activity, but have BM2, and have viral segments with both NA and NB sequences. Influenza C viruses have only seven viral segments.

[0105] Cells that can be used to produce viruses Any cell that supports efficient replication of influenza virus, for example any avian or mammalian cell, such as avian eggs, human, such as 293T or PER.C6® cells, or canine, bovine, equine, feline, porcine, ovine, rodent, such as mink, such as MvLu1 cells, or hamster, such as CHO cells, or non-human primate, such as Vero cells, including mutant cells, can be used to isolate and / or grow influenza virus. The isolated virus can be used to prepare reassortant virus. In one embodiment, the host cell for vaccine production is a continuous mammalian or avian cell line or cell line. A complete characterization of the cells used may be performed to include appropriate testing for purity of the final product. Data that can be used to characterize a cell include: (a) information regarding its origin, derivation, and passage history; (b) information regarding its growth and morphological characteristics; (c) results of tests for adventitious substances; (d) identifying characteristics, such as biochemical, immunological, and cytogenetic patterns, that allow the cell to be clearly recognized among other cell lines; and (e) results of tests for tumorigenicity. In one embodiment, the passage level, or population doublings, of the host cells used is as low as possible.

[0106] In one embodiment, the cells are WHO certified or certifiable continuous cell lines. The requirements for certification of such cell lines include characterization for at least one of phylogeny, growth characteristics, immunological markers, viral susceptibility, tumorigenicity, and storage conditions, and by testing in animals, eggs, and cell cultures. Such characterization is used to ensure that the cells are free of detectable adventitious material. In some countries, karyology may also be required. In addition, tumorigenicity may be tested in cells at the same passage level as those used for vaccine production. These viruses may be purified prior to vaccine production by a process shown to produce consistent results (see, e.g., World Health Organization, 1982).

[0107] Viruses produced by host cells may be highly purified prior to formulation as a vaccine or gene therapy. Generally, purification procedures result in the extensive removal of cellular DNA and other cellular components, as well as adventitious material. Procedures that extensively degrade or denature DNA may also be used.

[0108] Influenza vaccine The vaccine comprises an isolated recombinant influenza virus of the invention, and optionally one or more other isolated viruses, including other isolated influenza viruses, one or more immunogenic proteins or glycoproteins of one or more isolated influenza viruses or one or more other pathogens, such as one or more bacterial, non-influenza virus, yeast or fungal derived immunogenic proteins, or isolated nucleic acids encoding one or more viral proteins (e.g., DNA vaccines) including one or more immunogenic proteins of an isolated influenza virus of the invention. In one embodiment, the influenza virus of the invention may be a vaccine vector for influenza virus or other pathogens.

[0109] Whole virion vaccines may be concentrated by ultrafiltration and then purified by zonal centrifugation or chromatography. Viruses other than those of the invention, such as those contained in multivalent vaccines, may be inactivated before or after purification, for example using formalin or β-propiolactone.

[0110] Subunit vaccines contain purified glycoproteins. Such vaccines may be prepared as follows: virus suspensions fragmented by treatment with detergents are used and surface antigens are purified, for example by ultracentrifugation. Subunit vaccines therefore contain mainly HA proteins, and also NA. The detergents used may be cationic detergents, such as hexadecyltrimethylammonium bromide (Bachmeyer, 1975), anionic detergents, such as ammonium deoxycholate (Laver & Webster, 1976); or non-ionic detergents, such as those marketed under the name TRITON X100. Hemagglutinin may also be isolated after treatment of the virions with a protease, such as bromelain, and subsequently purified. Subunit vaccines may be combined with the attenuated viruses of the invention in multivalent vaccines.

[0111] Split vaccines comprise virions that have been subjected to treatment with a substance that dissolves lipids. Split vaccines can be prepared as follows: an aqueous suspension of purified virus, inactivated or not, obtained as described above, is treated with a lipid solvent, such as ethyl ether or chloroform, combined with a detergent, under stirring. Dissolution of the viral envelope lipids results in fragmentation of the viral particles. The aqueous phase, which contains the split vaccine, composed mainly of hemagglutinin and neuraminidase, freed from their original lipid environment, as well as the core and its degradation products, is recuperated. The remaining infectious particles are then inactivated, if they have not already been inactivated. Split vaccines may be combined with the attenuated viruses of the invention in multivalent vaccines.

[0112] Inactivated vaccinesInactivated influenza virus vaccines are provided by inactivating replicated viruses using known methods, such as, but not limited to, formalin or β-propiolactone treatment. Inactivated vaccine types that may be used in the present invention can include whole-virus (WV) vaccines or subvirion (SV) (split) vaccines. WV vaccines contain intact, inactivated viruses, whereas SV vaccines contain purified viruses that have been disrupted by detergents that solubilize the lipid-containing viral envelope, after which the remaining viruses are chemically inactivated.

[0113] In addition, vaccines that can be used include those that contain separated HA and NA surface proteins, referred to as surface antigen vaccines or subunit vaccines.

[0114] Live attenuated virus vaccines Attenuated live influenza virus vaccines, such as those comprising the recombinant viruses of the invention, can be used for the prevention or treatment of influenza virus infection. Attenuation is achieved in one step by transfer of the attenuated genes from the attenuated donor virus to the cloned isolate or reassorted virus according to known methods. Since resistance to influenza A viruses is primarily mediated by the development of an immune response against the HA and / or NA glycoproteins, the genes encoding these surface antigens are derived from the reassorted virus or clinical isolate. The attenuated genes are derived from the attenuated parent. In this approach, the genes that confer attenuation generally do not code for the HA and NA glycoproteins.

[0115] Viruses capable of reproducibly attenuating influenza viruses (donor influenza viruses) are available, and for example cold adapted (ca) donor viruses can be used for attenuated vaccine production. Attenuated live reassortant virus vaccines can be made by mating a ca donor virus with a virulent replicated virus. Reassortant progeny are then selected at 25°C in the presence of suitable antisera to inhibit replication of viruses bearing surface antigens of the attenuated ca donor virus (limiting replication of virulent viruses). Useful reassortants are: (a) infectious, (b) attenuated for seronegative non-adult mammals and immunologically primed adult mammals, (c) immunogenic, and (d) genetically stable. The immunogenicity of ca reassortants parallels their replication levels. Thus, acquisition of the six transferable genes of the ca donor viruses by the new wild-type viruses reproducibly attenuates these viruses for use in vaccination of both adult and non-adult compromised mammals.

[0116] Other attenuating mutations can be introduced into influenza virus genes by site-directed mutagenesis to rescue infectious viruses carrying these mutant genes. Attenuating mutations can be introduced into non-coding regions of the genome as well as into coding regions. Such attenuating mutations can also be introduced into genes other than HA or NA, such as the PB2 polymerase gene. Thus, novel donor viruses carrying attenuating mutations introduced by site-directed mutagenesis can also be generated, and such novel donor viruses can be used in the production of attenuated live reassortant vaccine candidates in a manner similar to that described above for the ca donor virus. Similarly, other known suitable attenuated donor strains can be reassorted with influenza virus to obtain attenuated vaccines suitable for use in vaccinating mammals.

[0117] In one embodiment, such attenuated viruses maintain genes from the virus that encode antigenic determinants substantially similar to those of the original clinical isolate, since the goal of an attenuated vaccine is to provide substantially the same antigenicity as the original clinical isolate of the virus, while at the same time lacking pathogenicity to the extent that the vaccine minimizes the chance of inducing severe pathology in the vaccinated mammal.

[0118] The viruses in this multivalent vaccine can then be attenuated or inactivated, formulated, and administered according to known methods as a vaccine to induce an immune response in animals, e.g., mammals. Methods for determining whether such attenuated or inactivated vaccines maintain antigenicity similar to that of clinical isolates or high growth strains derived therefrom are well known in the art. Such known methods include the use of antisera or antibodies that exclude viruses expressing antigenic determinants of the donor virus; chemical selection (e.g., amantadine or rimantadine); HA and NA activity and inhibition; and nucleic acid screening (such as probe hybridization or PCR) to identify donor genes that code for antigenic determinants (e.g., HA or NA genes) that are not present in the attenuated virus.

[0119] Pharmaceutical Compositions The pharmaceutical compositions of the present invention suitable for inoculation, e.g., nasal, parenteral or oral administration, contain one or more influenza virus isolates, e.g., one or more attenuated or inactivated influenza viruses, subunits thereof, isolated protein(s) thereof, and / or isolated nucleic acids encoding one or more proteins thereof, and optionally further comprise sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The compositions may further contain auxiliary agents or excipients known in the art. The compositions of the present invention are generally presented in the form of individual doses (unit doses).

[0120] Conventional vaccines generally contain about 0.1-200μg, for example 30-100μg, 0.1-2μg, 0.5-5μg, 1-10μg, 10μg-20μg, 15μg-30μg, or 10-30μg, of HA from each strain that goes into their composition. Vaccines which form the main component of the vaccine composition of the invention may contain a single influenza virus, or a combination of influenza viruses, for example at least two or three influenza viruses, including one or more reassortant(s).

[0121] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and / or emulsions, which may contain auxiliary agents or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration may generally include liposome solutions containing liquid dosage forms. Forms suitable for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs that contain inert diluents commonly used in the art, such as purified water. In addition to inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweeteners, flavoring agents, or perfuming agents.

[0122] When the composition of the present invention is used for administration to an individual, it can further contain salts, buffers, adjuvants, or other substances that are desirable to improve the effectiveness of the composition. For vaccines, adjuvants can be used, which are substances that can enhance specific immune responses. Usually, the adjuvant and the composition are mixed before being presented to the immune system, or presented separately, but at the same site of the organism to be immunized.

[0123] Heterogeneity in a vaccine can be provided by mixing replicated influenza viruses for at least two influenza virus strains, such as 2-20 strains, or any range or number therein. Vaccines can be provided for variation in a single strain of influenza virus using techniques known in the art.

[0124] The pharmaceutical compositions of the invention may further or additionally contain at least one chemotherapeutic compound, e.g., a gene therapy agent, an immunosuppressant, an anti-inflammatory agent, or an immune enhancing agent, and for vaccines, chemotherapeutic agents including, but not limited to, gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon-α, interferon-β, interferon-γ, tumor necrosis factor-α, thiosemicarbazone, methisazone, rifampin, ribavirin, pyrimidine analogs, purine analogs, foscarnet, phosphonoacetic acid, acyclovir, dideoxynucleosides, protease inhibitors, or ganciclovir.

[0125] The compositions may also contain variable but small amounts of endotoxin-free formaldehyde, and preservatives, which have been found to be safe and not to contribute to undesirable effects in the organism to which the compositions are administered.

[0126] Medical Purpose Administration of the composition (or the antisera it elicits) may be for either "prophylactic" or "therapeutic" purposes. When provided prophylactically, the composition of the invention, which is a vaccine, is provided prior to the appearance of any symptoms or clinical signs of pathogen infection. Prophylactic administration of the composition serves to prevent or attenuate subsequent infection. When provided prophylactically, the gene therapy composition of the invention is provided prior to the appearance of any symptoms or clinical signs of disease. Prophylactic administration of the composition serves to prevent or attenuate one or more symptoms or clinical signs associated with disease.

[0127] When provided therapeutically, a viral vaccine is provided upon detection of a symptom or clinical sign of an actual infection. The therapeutic administration of the compound(s) serves to attenuate any actual infection. When provided therapeutically, a gene therapy composition is provided upon detection of a symptom or clinical sign of the disease. The therapeutic administration of the compound(s) serves to attenuate the symptom or clinical sign of the disease.

[0128] Thus, the vaccine compositions of the invention may be administered either prior to the onset of infection (to prevent or attenuate anticipated infection) or after the onset of actual infection. Similarly, with respect to gene therapy, the compositions may be provided prior to the appearance of any symptoms or clinical signs of a disorder or disease, or after one or more symptoms have been detected.

[0129] A composition is said to be "pharmaceutical acceptable" if its administration can be tolerated by a recipient mammal. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. This is physiologically significant if the presence of the composition of the present invention results in a detectable change in the physiology of the recipient patient, for example, enhancing at least one primary or secondary humoral or cellular immune response against at least one strain of infectious influenza virus.

[0130] The "protection" provided need not be absolute, i.e., influenza infection need not be completely prevented or eradicated, provided there is a statistically significant improvement compared to a control population or set of mammals. Protection may be limited to reducing the severity or rapidity of onset of symptoms or clinical signs of influenza virus infection.

[0131] Medication Administration The compositions of the invention can provide resistance to one or more pathogens, e.g., one or more influenza virus strains, either by passive or active immunization. In active immunization, a live attenuated vaccine composition is administered prophylactically to a host (e.g., a mammal), and the host's immune response to this administration protects against infection and / or disease. For passive immunization, the induced antisera can be collected and administered to a recipient suspected of having an infection caused by at least one influenza virus strain. The gene therapy compositions of the invention can produce a desired gene product at prophylactic or therapeutic levels by active immunization.

[0132] In one embodiment, the vaccine is provided to a mammalian female (at or before the time of pregnancy or parturition) for a time and in an amount sufficient to cause the formation of an immune response that serves to protect both the female and the fetus or newborn (either across the placenta or via passive uptake of antibodies in the breast milk).

[0133] Thus, the present invention includes a method of preventing or attenuating a disorder or disease, such as an infectious disease, caused by at least one strain of a pathogen. As used herein, a vaccine is said to prevent or attenuate a disease if administration of the vaccine results in either a complete or partial attenuation (i.e., suppression) of clinical signs or conditions of the disease or results in complete or partial immunity of an individual to the disease. As used herein, a gene therapy composition is said to prevent or attenuate a disease if administration of the gene therapy composition results in either a complete or partial attenuation (i.e., suppression) of clinical signs or conditions of the disease or results in complete or partial immunity of an individual to the disease.

[0134] Compositions comprising at least one influenza virus of the present invention, including attenuated ones, and one or more other isolated viruses, one or more isolated viral proteins thereof, one or more isolated nucleic acid molecules encoding one or more viral proteins thereof, or combinations thereof, may be administered by any means that achieves the intended purpose.

[0135] For example, administration of such compositions can be by various parenteral routes, such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral or transdermal routes. Parenteral administration can be accomplished by bolus injection or by gradual infusion over time.

[0136] A typical regimen for preventing, suppressing or treating influenza virus associated pathology involves administration of an effective amount of the vaccine composition described herein administered as a single treatment or repeated in increasing or booster doses over a period of up to and including between one week and about 24 months, or any range or value therein.

[0137] An "effective amount" of a composition according to the present invention is an amount that is sufficient to achieve a desired effect. It is understood that the effective dose will depend on the species, age, sex, health condition, and weight of the recipient, the type of concomitant treatment, if any, frequency of treatment, and the nature of the desired effect. The ranges of effective doses provided below are not intended to limit the invention and represent dosage ranges.

[0138] The dose of a live attenuated or killed virus vaccine for an animal, such as an adult mammalian organism, is about 10 2 -10 20 , e.g., 10 3 -10 12 , 10 2 -10 10 , 10 5 -10 11 , 10 6 -10 15 , 10 2 -10 10 , or 10 15-10 20 The dose may be plaque forming units (PFU) / kg, or any range or value therein. The dose of a single virus isolate vaccine, e.g., an inactivated vaccine, may range from about 0.1 to 1000, e.g., 0.1 to 10 μg, 1 to 20 μg, 30 to 100 μg, 10 to 50 μg, 50 to 200 μg, or 150 to 300 μg of HA protein. However, this dose must be a safe and effective amount as determined by conventional methods using existing vaccines as a starting point.

[0139] The dose of immunoreactive HA in each dose of replicated viral vaccine may be standardized to include a suitable amount, such as 0.1 μg to 1 μg, 0.5 μg to 5 μg, 1 μg to 10 μg, 10 μg to 20 μg, 15 μg to 30 μg, or 30 μg to 100 μg, or any range or value therein, or an amount recommended by a government agency or recognized professional organization. The amount of NA may also be standardized, but this glycoprotein is unstable during purification and storage.

[0140] The dose of immunoreactive HA in each dose of replicated viral vaccine can be standardized to contain a suitable amount, for example, 1-50 μg or any range or value therein, or the amount recommended by the US Public Health Service (PHS), which is usually 15 μg per component for children older than 3 years of age, and 7.5 μg per component for children under 3 years of age. The amount of NA can also be standardized, but this glycoprotein is unstable during processing or purification and storage (Kendal et al., 1980; Kerr et al., 1975). Each 0.5 ml dose of vaccine can contain approximately 100-500 million virus particles, 500-2 billion virus particles, 1-50 billion virus particles, 1-10 billion virus particles, 20-40 billion virus particles, 1-5 billion virus particles, or 40-80 billion virus particles.

[0141] Illustrative Viruses Described herein are useful modifications of influenza neuraminidase (NA) proteins that stabilize the hemagglutinin (HA) protein during egg passaging of influenza viruses expressing the modified neuraminidase proteins of influenza viruses. Modified nucleic acids encoding such modified neuraminidase proteins are also described. The modifications can include deletions, substitutions, and combinations thereof within the sequences of the neuraminidase proteins and nucleic acids. Viruses expressing such modified neuraminidase proteins show significantly reduced acquisition of antigenically compromising mutations in hemagglutinin (HA) during propagation of influenza in eggs.

[0142] For example, in some cases, the modified neuraminidase can have at least one, or at least two, or at least three modifications. Amino acid positions within the influenza neuraminidase protein that can be modified include, for example, based on the N2 numbering, one or more amino acids within positions 29-35, one or more amino acids within positions 44-52, one or more amino acids within positions 144-154, one or more amino acids within positions 240-250, one or more amino acids within positions 326-333, one or more amino acids within positions 344-350, one or more amino acids within positions 365-375, and combinations thereof. For example, the amino acid(s) can be any amino acid within these positions, such as any of the amino acids listed in the table below. [Table 1]

[0143] In some cases, selected amino acids within positions 29-35, 44-52, 144-154, 326-333, 344-350, and 365-375 can have conservative substitutions, but in other cases, selected amino acids within positions 29-35, 44-52, 144-150, 326-333, 344-350, and 365-375 can have non-conservative substitutions.

[0144] For example, the modified neuraminidase can have a deletion of at least one proline, asparagine, glutamine, valine, or a combination of proline, one or more asparagine(s), glutamine, and valine within positions 44-52 of the modified neuraminidase. The modified neuraminidase can have a substitution of threonine within positions 29-35, where the substitution is any amino acid. The modified neuraminidase can have a substitution of threonine or aspartic acid within positions 145-154 or 365 to 375, where the substitution is any amino acid. The modified neuraminidase can have a substitution of asparagine within positions 326-333, where the substitution is any amino acid. The modified neuraminidase can have a substitution of histidine within positions 345-350, where the substitution is any amino acid. Illustrative substitutions for various types of amino acids are provided in the table above.

[0145] An example of an influenza A virus (A / Yokohama / 2013 / 2003 (H3N2)) neuraminidase protein sequence is provided below. [Table 2]

[0146] Amino acids that may be modified to improve the stability of co-expressed HA are highlighted in bold and underlined in the sequence shown above. A nucleic acid encoding influenza A virus (A / Yokohama / 2013 / 2003(H3N2)) neuraminidase protein sequence is shown below. [Table 3]

[0147] Modifications at defined positions in neuraminidase can result in enhanced growth of the virus.

[0148] Another example of an influenza A virus (A / Yokohama / 47 / 2002 (H1N2)) neuraminidase sequence is shown below, with the positions of modifications highlighted in bold and underlined: [Table 4]

[0149] Amino acids that may be modified to improve the stability of co-expressed HA are highlighted in bold and underlined in the sequence shown above.

[0150] In some cases, one or more modifications can also be introduced into the HA, PA, PB1, PB2, NP, M1, M2, NS2, PB1-F2, PA-X, and / or NS1 proteins (and nucleic acids encoding such proteins).

[0151] Enhanced growth of the virus when passaged through embryonated chicken eggs or cultured cells is observed when the modified NA protein is expressed and such expression can result in significantly higher virus titers. Thus, the present invention provides a method for producing influenza viruses with enhanced replication in cell culture or embryonated eggs. The method includes providing cells suitable for influenza vaccine production; modifying a nucleic acid encoding neuraminidase; and isolating a virus strain with enhanced growth compared to one or more unmodified virus isolates. In some cases, the method for producing influenza viruses with enhanced replication in cell culture can involve continuously culturing one or more influenza virus isolates in embryonated chicken eggs; and isolating the continuously cultured viruses with enhanced growth compared to the one or more isolates prior to continuous culture. In some cases, these viruses can be grown or passaged in cells in culture, such as, for example, MDCK cells or Vero cells.

[0152] The modified neuraminidase can be expressed in various influenza strains. For example, the A / PuertoRico / 8 / 34 (H1N1), "PR8" virus often serves as the genetic backbone for the production of inactivated influenza vaccines. Some vaccine strains based on the PR8 backbone can replicate to relatively low titers in ovo and cell culture, resulting in delayed vaccine production and vaccine shortages. However, expression of the modified neuraminidase described herein can improve replication of PR8 (and other) influenza strains.

[0153] In one embodiment of the invention, vectors for vRNA production can include a vector comprising a promoter operably linked to a modified NA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to influenza virus NS DNA linked to a transcription termination sequence. In one embodiment, DNA for vRNA production of PB1, PB2, PA, NP, M, and NS has sequences from influenza viruses that replicate at high titers in cultured mammalian cells, such as MDCK cells, Vero cells, or PER.C6® cells, or in embryonated eggs, and / or sequences from vaccine viruses, such as those that do not cause significant disease in humans. DNA for vRNA production of NA can be from any NA, such as any of N1-N11, and DNA for vRNA production of HA can be from any HA, such as H1-H18. In one embodiment, DNA for vRNA production can be for influenza B or C viruses. DNA for vRNA production of NA and HA can be from different strains or isolates (6:1:1 reassortants) or from the same strain or isolate (6:2 reassortants), or NA can be from the same strain or isolate as for the internal genes (7:1 reassortants).Vectors for mRNA production can include a vector encoding a modified NA, a vector encoding influenza virus PA, a vector encoding influenza virus PB1, a vector encoding influenza virus PB2, and a vector encoding influenza virus NP, and optionally one or more vectors encoding NP, NS, M, e.g., M1 and M2, HA, or NA. Vectors encoding viral proteins may further include a transcription termination sequence.

[0154] Other reassortants with internal genes from other PR8 isolates or vaccine viruses may be utilized in the recombinant reassortant viruses of the invention. In particular, a 5:1:2 reassortant with PB1, PB2, PA, NP, and M ("5") of UW-PR8 and NS ("1") of PR8(Cam); a 6:1:1 reassortant with NA, PB1, PB2, PA, NP, and M ("6") of UW-PR8(modified) and NS ("1") of PR8(Cam); and a 7:1 reassortant with PB1, PB2, PA, NP, M, (modified) NA, and NS ("7") of UW-PR8 may be utilized.

[0155] A neuraminidase that may be modified may have a sequence different from that described herein. In some cases, however, the modified neuraminidase may have substantially the same activity as the corresponding polypeptide described by the sequence herein. As used herein, "substantially the same activity" includes an activity that is about 0.1%, 1%, 10%, 30%, 50%, 90%, e.g., up to 100% or greater activity, or a detectable protein level that is about 80%, 90% or greater protein level, of the corresponding protein described herein. In one embodiment, the nucleic acid encodes a polypeptide that is substantially the same, e.g., has at least 80%, e.g., 90%, 92%, 95%, 97%, 98%, or 99% contiguous amino acid sequence identity, including any integer between 80 and 99, to a polypeptide encoded by one of the sequences described herein. In one embodiment, an isolated and / or purified nucleic acid molecule comprises a nucleotide sequence that is substantially identical, e.g., at least 50%, e.g., 60%, 70%, 80%, or 90% or more contiguous nucleic acid sequence identity, including any integer between 50 and 100, to one of the nucleic acid sequences described herein. In one embodiment, the nucleic acid also encodes a polypeptide having at least 80%, e.g., 90%, 92%, 95%, 97%, 98%, or 99% contiguous amino acid sequence identity, including any integer between 80 and 99, to a polypeptide described herein.

[0156] In one embodiment, the modified influenza virus neuraminidase polypeptide has one or more, e.g., 2, 5, 10, 15, 20 or more conservative amino acid substitutions, e.g., 2, 5, 10, 15, 20 or more of a combination of conservative and non-conservative amino acid substitutions, up to 10% or 20% conservative substitutions, e.g., up to 10% or 20% conservative substitutions of residues or relative to a polypeptide with one of the sequences disclosed herein.

[0157] Thus, the present invention includes the use of isolated and purified vectors or plasmids expressing or encoding influenza virus proteins, or expressing or encoding influenza vRNA, both native and recombinant vRNA. These vectors can be used to express influenza cDNA, such as influenza A (e.g., any influenza A gene, including any of the 18 HA or 11 NA subtypes), B or C DNA (e.g., Fields, Virology (See Fields et al. (eds.), Lippincott, Williams and Wickens (2006), which is specifically incorporated herein by reference.) Any suitable promoter or transcription termination sequence may be utilized to express a protein or peptide, such as a viral protein or peptide, a protein or peptide of a non-viral pathogen, or a therapeutic protein or peptide.

[0158] The composition or vectors of the invention may also include a heterologous gene or open reading frame of interest, e.g., a foreign gene encoding an immunogenic peptide or protein useful as a vaccine or in gene replacement, e.g., an epitope useful in cancer therapy or vaccine, or a peptide or polypeptide useful in gene therapy. When preparing a virus, the vector or plasmid containing the gene or cDNA of interest can be replaced with a vector or plasmid of one influenza virus gene, or can be added to a vector or plasmid of all influenza virus genes. Thus, another embodiment of the invention includes a composition or vectors as described above, in which one of the vectors is replaced with or further includes a 5' influenza virus sequence, optionally including a 5' influenza virus coding sequence or a portion thereof, linked to a desired nucleic acid sequence, e.g., a desired cDNA, optionally including a 3' influenza virus coding sequence or a portion thereof. In one embodiment, the desired nucleic acid sequence, e.g., a cDNA, is in the antisense (antigenome) orientation. Introduction of such a vector, together with another vector described above, into a host cell permissive for influenza virus replication results in recombinant virus that contains vRNA corresponding to the heterologous sequence of the vector.

[0159] The promoter in the vector for vRNA production may be an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T7 promoter, or a T3 promoter, and optionally the vector includes a transcription termination sequence, such as an RNA polymerase I transcription termination sequence, an RNA polymerase II transcription termination sequence, an RNA polymerase III transcription termination sequence, or a ribozyme. Ribozymes within the scope of the present invention include, but are not limited to, Tetrahymena ribozyme, RNase P, hammerhead ribozyme, hairpin ribozyme, hepatitis ribozyme, and synthetic ribozymes. In one embodiment, the RNA polymerase I promoter is a human RNA polymerase I promoter.

[0160] The promoter or transcription termination sequence in a vRNA or viral protein expression vector may be the same or different from that promoter or any other vector. In one embodiment, a vector or plasmid expressing influenza vRNA contains a promoter suitable for expression in at least one particular host cell, e.g., an avian or mammalian host cell, e.g., a canine, feline, equine, bovine, ovine, or primate cell, including a human cell, or for expression in more than one host cell.

[0161] In one embodiment, at least one vector for vRNA comprises an RNA polymerase II promoter linked to another ribozyme sequence linked to a viral coding sequence linked to a ribozyme sequence, optionally linked to an RNA polymerase II transcription termination sequence. In one embodiment, at least two, for example 3, 4, 5, 6, 7 or 8 vectors for vRNA production comprise an RNA polymerase II promoter, a first ribozyme sequence, which is 5' to a sequence corresponding to a viral sequence including a viral coding sequence, which is 5' to a second ribozyme sequence, which is 5' to a transcription termination sequence. Each RNA polymerase II promoter in each vRNA vector may be the same or different from the RNA polymerase II promoter in any other vRNA vector. Similarly, each ribozyme sequence in each vRNA vector may be the same or different from the ribozyme sequence in any other vRNA vector. In one embodiment, the ribozyme sequences in a single vector are not identical.

[0162] In one embodiment, the invention provides multiple influenza virus vectors for reassortant, including a vector for vRNA production comprising a promoter operably linked to a modified influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operably linked to influenza virus NS DNA linked to a transcription termination sequence.Vectors for vRNA production comprising a promoter operably linked to a cDNA, wherein the DNA for modified NA, PB1, PB2, PA, NP, NS, and M is derived from one or more influenza vaccine seed viruses and comprises two or more signature residues at a specified position(s); and vectors for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NA, vectors for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA, vectors for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, vectors for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and vectors for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NP. The vectors include a vector for mRNA production comprising a promoter operably linked to a DNA segment, and optionally a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M2, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS1, or a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS2. In one embodiment, at least one vector comprises a sequence or a portion thereof corresponding to those encoding PB1, PB2, PA, NP, M, or NS that has substantially the same activity as the corresponding polypeptide described herein or the polypeptide encoded by the nucleic acid described herein. Optionally, two vectors comprise an influenza virus M linked to a transcription termination sequence.Alternatively, a vector containing a promoter operably linked to a cDNA may be used, e.g., a vector containing a promoter operably linked to an influenza virus M1 cDNA linked to a transcription termination sequence and a vector containing a promoter operably linked to an influenza virus M2 cDNA linked to a transcription termination sequence.

[0163] The vectors of the present invention may be physically linked, or each vector may be present on an individual plasmid or other, e.g., linear, nucleic acid delivery vehicle. In one embodiment, each vRNA producing vector is present on an individual plasmid. In one embodiment, each mRNA producing vector is present on an individual plasmid.

[0164] The present invention also provides a method for preparing influenza virus. The method includes contacting a cell with a plurality of vectors of the present invention, e.g., sequentially or simultaneously, in an amount effective to produce infectious influenza virus. The present invention also includes isolating the virus from a cell contacted with a plurality of vectors. Thus, the present invention further provides isolated viruses, as well as host cells contacted with a plurality of vectors or viruses of the present invention. In another embodiment, the present invention includes contacting a cell with one or more vectors, either a vRNA or a protein producing vector, before the other vector, either a vRNA or a protein producing vector. In one embodiment, the promoter for the vRNA vector utilized in the method is an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T3 promoter, or a T7 promoter. In one embodiment, the RNA polymerase I promoter is a human RNA polymerase I promoter. In one embodiment, each vRNA vector utilized in the method is on a separate plasmid. In one embodiment, the vRNA vector utilized in the method is on one plasmid, or on two or three different plasmids. In one embodiment, each mRNA vector utilized in the method is on a separate plasmid. In one embodiment, the mRNA vectors for PA, PB1, PB2 and NP utilized in the method are on one plasmid or on two or three different plasmids.

[0165] Illustrative viral sequence for the master vaccine strain (PR8UW). [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7]

[0166] Illustrative neuramidase modifications material Virus: Y2017: A / Yokohama / 2017 / 2003 (H3N2) HK4801:A / HongKong / 4801 / 2014(H3N2) Y2017-M3L4: Y2017 passaged 7 times in eggs HY-PR8: High-yield PR8 (H1N1).

[0167] result The Y2017 virus was passaged seven times in eggs (three times in the amniotic cavity and then four times in the allantoic cavity). The progeny virus, Y2017-M3L4, grew efficiently in the allantoic cavity (10 7 About 10 8 In contrast, the original Y2017 virus did not grow at all (<10 PFU / mL).

[0168] The mutations observed in the Y2017-M3L4 virus were: [Table 6]

[0169] Comparison of the growth ability of mutant Y2017 viruses generated by reverse genetics in allantoic fluid revealed that the NA mutation contributed to the high growth of Y2017-M3L4 virus (Figure 4). Plasmids with PB2-T147I were used for virus generation (PB2-T147I, V344L and PB2-T147I, V344L, E358K were not analyzed). No mutations were observed in the HA gene of the virus with the mutated NA segment and in other genes of that from wild-type Y2017 after replication in allantoic fluid (Figure 4).

[0170] Figure 5 shows the location of the NA mutation in Y2017-M3L4 in the 3D model.

[0171] Comparison of the growth ability of Y2017 viruses with NA mutations revealed that NA-D147N, N329D, and H347Q generally contributed to increased growth ability in allantoic fluid (Figure 6).

[0172] The NA of Y2017-M3L4 enabled the virus bearing the HK4801HA to replicate efficiently in the allantoic cavity, and the HY-PR8 backbone further enhanced the growth of this virus (Figure 7).

[0173] In summary, influenza virus mutations are described herein that inhibit (e.g., prevent) the acquisition of antigenic-impairing mutations in influenza hemagglutinin (HA) protein during growth in eggs and / or allow enhanced replication. In one embodiment, these mutations are in the neuraminidase (NA) viral segment of human influenza virus, and the mutant NA protein stabilizes the HA protein during egg-passage. Thus, in the presence of the mutant NA protein, the HA protein does not acquire egg-adaptation mutations. In some cases, the respective mutations in NA can also increase the yield of vaccine virus.

[0174] Analysis of the growth ability of NA mutant viruses revealed that NA-D147N, N329D, and H347Q contributed to the enhanced growth ability of the virus in allantoic fluid (Figure 12). After three passages in the allantoic cavity, no HA mutations were observed in viruses with HK4801HA, Y2017-M3L4NA, and HY-PR8 backbones (Figure 13).

[0175] By passaging HY-PR8 backbone viruses carrying HK4801NA (T148K and saturated mutations N329X and H347X) and HK4801HA in eggs, viruses carrying HK4801NA (T148K, D151E, H347G, and T369K) that replicated efficiently in the allantoic cavity emerged (Figure 14; 4M = T148K, D151E, H347G, and T369K). No HA mutations were observed during passage in eggs (once in the amniotic cavity, then five times in the allantoic cavity).

[0176] HK4801NA (T148K, D151E, H347G, and T369K) conferred efficient replication in the allantoic cavity to HY-PR8 backbone viruses carrying either HK4801HA or Singapore0019HA. Viral inoculation: 2 × 10 3 pfu / egg, 72 h incubation at 37°C (Figure 16).

[0177] The HA-encoding nucleic acid sequence and the NA-encoding nucleic acid and amino acid sequences for Singapore0019 are as follows: [Table 7] [Table 8] This codes for: [Table 9]

[0178] The NA mutations T153N, N329T, and T369K allowed efficient replication of A / Saitama / 102 / 2014 (H3N2) in the allantoic cavity (Kuwahara et al., 2018). Therefore, the effect of introducing NA-T153N, N329T (or D), T369K, and H347Q into HK4801NA (T148K) was tested. Figure 18 reports the virus titers for different combinations of NA residues identified in the screen. Figures 19 and 20 report the virus titers for viruses with different combinations of selected NA residues.

[0179] Alaska / 232 / 2015_HY-PR8(H3N2)WT / mutant viruses were passaged in eggs and the HA and NA segments were sequenced. The Alaska WT (WT is a relatively recent H3N2 virus with 245N; before 2015, H3N2 WT viruses had 245S), HA-R142S, -K189E viruses did not acquire mutations in HA even after three amniotic passages and ten allantoic passages. The HA-K189E / N158K / A212T mutant did not acquire mutations in HA, but had several mutations in NA that showed improved growth in eggs from p6 onwards (Figure 21). The difference in NA mutation between p4 (normal growth) (NA-N245S mutation, the virus grows 100-fold better than that with NA-245N) and p6 (better growth) was G346V (Figure 22). Therefore, G346V may also contribute to egg adaptation.

[0180] The NA of A / Alaska / 232 / 2015 has the following sequence: [Table 10]

[0181] NA pHH21 plasmids were constructed: Alaska NA-T148K / D151E / N245S (found in E4); Alaska NA-G346V; and Alaska NA-T148K / D151E / N245S / G346V (found in E6). Mutant NAs were combined with WT Alaska HA or HY-PR8 backbones. Eggs were inoculated with equal doses of WT / mutant Alaska virus and harvested virus was titered (FIG. 23). NA-T148K / D151E / N245S / G346V mutant virus grew to higher titers than WT virus, while the single mutation G346V did not increase virus growth compared to WT. These results suggest that the combination of G346V with one (or two to three) other mutations, such as T148K, D151E, and N245S, is important for the virus to grow efficiently in eggs. Collected virus samples with high titers (>5 Log10 PFU / mL) were sequenced but did not have additional mutations in HA and NA. EXAMPLES

[0182] The invention is illustrated by the following non-limiting examples.

[0183] Example I As shown in Figures 25-28, certain substitutions in N2 stabilized HA (e.g., did not result in substitutions in HA) for up to about 8 passages in eggs in various H3N2 isolates from different influenza seasons. However, HA substitutions were observed in some, but not all, isolates passaged in eggs for 10 passages (Figure 29). Unexpectedly, another change in NA (148I) correlated with HA stabilization even after 9 passages. Although the presence of this additional change in some cases did result in HA changes, the change in the stem region of HA (Figure 32) is unlikely to alter antigenicity.

[0184] 148I or 148K was introduced into NA along with other substitutions identified (Figures 33-35). The addition of 148I did not substantially alter virus titers in eggs, but 148K in some cases affected titers. Interestingly, viruses with NA with 148I in combination with other changes had reduced sialidase activity (Figure 36). For example, viruses with NA with 148I, 151E, 245S, 347G, and 369K, and viruses with NA with 148I, 150S, 151E, 245S, 347G, and 369K, and viruses with NA with 148K, 151E, 245S, 347G, and 369K had reduced sialidase activity.

[0185] Example II Mutations in the influenza surface glycoprotein neuraminidase (NA) confer efficient replication to modern human H3N2 viruses in eggs without acquiring mutations in the antigenic sites of the other surface glycoprotein (hemagglutinin) HA. The NA mutations allow these mutant NAs to recognize sialic acid linked to galactose via α2-3 linkages (Siaα2-3Gal) found on epithelial cells of the chorioallantoic membrane in chicken eggs. These NA mutations allow the virus to attach to and enter cells even under conditions in which the interaction between HA and its receptor is inhibited (Figure 38), suggesting that the mutant NAs act as alternative receptor-binding proteins to HA.

[0186] By having a HA protein with disrupted or no receptor-binding activity, the disclosed mutant NA can provide influenza viruses, such as H3N2 viruses, with efficient growth in embryonated chicken eggs without acquiring any egg-adapted HA mutations at the antigenic sites. Since the HA does not encounter the selective pressure to recognize the Siaα2-3Gal receptor in the presence of this mutant NA, the amino acid residues around the HA receptor-binding pocket remain unchanged during passage in embryonated chicken eggs.

[0187] For this purpose, HA proteins were constructed that lacked or had limited receptor-binding activity but maintained their antigenicity by introducing three mutations, Y98F, W153A, and H183F, located in the receptor-binding pocket that do not affect the antigenicity of HA (Figure 39A-B).

[0188] Example III An illustrative NA residue was found in egg-grown A / HongKong / 4801 / 2014 and A / Alaska / 232 / 2015 ("6M"). Introduction of the 6M mutation into the NA of A / Yokohama / 48 / 2018 and A / Yokohama / 147 / 2017 enhanced the growth of HY-PR8-backbone viruses. Therefore, the effect of introducing the 6M mutation into other strains and the effect of carrying Yokohama147NA(6M) on the growth of viruses carrying HA from other strains was tested (Figures 43-44). Harvested viruses carrying the NA(6M) of each strain or the Yokohama / 147 / 2017NA(6M) were sequenced. No additional mutations in HA or NA were found.

[0189] Viruses carrying A / Yokohama / 147 / 2017 NA(6M) acquired HA-D225G and K27E after 10 passages. HA-K27E was located in the stem region of the HA protein, suggesting that K27E is unlikely to alter HA antigenicity. HA-D225G was located near the receptor binding site. However, the reactivity of H3N2 viruses carrying HA-D225N with ferret antisera was only two-fold different from that of wild-type viruses, suggesting that HA-D225G alone is unlikely to substantially alter HA antigenicity (Chambers et al., Cell Rep. 2015).

[0190] We also analyzed whether viruses with HA from Yokohama147NA(6M) and other strains could replicate without acquiring HA mutations at the major antigenic site during egg passage. Although many of the tested viruses acquired HA-D225G during egg passage, none of them acquired HA mutations at the major antigenic site. G479E, K453N, E484G (located in the stem region) and R545K (located at the cytoplasmic tail) were unlikely to alter HA antigenicity (Figure 45).

[0191] The mechanism of how the NA(6M) mutant virus can replicate efficiently in eggs was investigated. VP40-induced VLPs carrying FLAG-tagged Yokohama147NA or Yokohama147NA(6M) were prepared. Immunoblotting analysis with anti-FLAG and anti-VP40 antibodies showed a reduced molecular weight of the Yokohama147NA(6M) protein compared with that of the wild-type Yokohama147NA protein (Figure 48). Figure 49 shows another Western blot analysis, suggesting that the glycosylation site of the mutant NA protein was lost due to the introduction of the 6M mutation.

[0192] Next, the receptor-binding specificity of Yokohama147HA, Yokohama147NA, and Yokohama147NA(6M) was analyzed using a glycan microarray containing a library of α2-3 and α2-6 sialosides containing N-linked glycans representative of those found on the egg chorioallantoic membrane. This analysis showed that Yokohama147NA(6M) bound to α2-3 sialosides found on the egg chorioallantoic membrane (Figure 50).

[0193] We determined whether the 6M mutation altered NA sialidase activity (Figure 51). Ebola VP40-based VLPs carrying Yokohama147NA or NA(6M) were serially diluted and incubated with the sialidase substrate 4-MUNANA, and the released 4-MU was quantified to assess sialidase activity. This analysis revealed that introduction of 6M into Yokohama147NA reduced its sialidase activity.

[0194] To identify additional NA mutations that may enable efficient replication of the virus in eggs independent of HA receptor binding activity, HY-PR8 backbone viruses with HA (del RBS) and A / Kansas / 14 / 2017 NA (6M) were generated and then passaged in eggs. Upon passage, mutant NA was obtained (T148I, D151E, N245S, T329S, K344E, G346V, H347G and T369K) (=6M+T148I+T329S+K344E). HY-PR8 backbone viruses with wild-type HA and NA (6M+T148I+T329S+K344E) from A / Kansas / 14 / 2017 were prepared and then analyzed to determine whether the virus acquired HA mutations upon passage in eggs. The virus with NA(6M+T148I+T329S+K344E) did not acquire any HA or NA mutations upon 10 egg passages (FIG. 52).

[0195] Example IV In one embodiment, an isolated recombinant influenza virus is provided that includes a selected NA viral segment encoding a plurality of selected residues or residue deletions in NA, wherein the selected NA viral segment does not encode an NA having a threonine (T) or lysine (K) at residue 148, and does not encode a threonine at residue 32, an aspartic acid (D) at position 151, an asparagine (N) at position 245, an asparagine at residue 329, a glycine (G) at position 346, a histidine at residue 347, or includes an NA having one or more deletions at residues 46 to 50, or any combination thereof. and wherein the numbering is based on N2, and wherein the recombinant influenza virus has enhanced replication in avian eggs or has reduced HA mutations when propagated in avian eggs compared to a corresponding influenza virus having an NA that encodes a threonine or lysine at residue 148 and one or more threonines at residue 32, or a deletion of residues 46 to 50, an aspartic acid at position 147, an aspartic acid at residue 151, an asparagine at residue 245, an asparagine at residue 329, a glycine at residue 346, or no histidine at residue 347, or any combination thereof. In one embodiment, the selected NA viral segment does not encode an NA having a threonine at residue 148 and does not encode an NA having an aspartic acid at position 151, an asparagine at position 245, a histidine at residue 347, or a threonine at residue 369, or any combination thereof, where the numbering is based on N2, and wherein the recombinant influenza virus has enhanced replication in avian eggs or has reduced HA mutations when propagated in avian eggs compared to a corresponding influenza virus having an NA encoding a threonine at residue 148, an aspartic acid at residue 151, an asparagine at residue 245, a histidine at residue 347, and a threonine at residue 369, or any combination thereof.In one embodiment, the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, and does not encode an NA having an aspartic acid at position 151, an asparagine at position 245, a histidine at residue 347, or a threonine at residue 369, or any combination thereof, where the numbering is based on N2, and wherein the recombinant influenza virus when propagated in avian eggs has enhanced replication in avian eggs or has reduced HA mutations compared to a corresponding influenza virus having an NA encoding a threonine or lysine at residue 148, an aspartic acid at residue 151, an asparagine at residue 245, a histidine at residue 347, and a threonine at residue 369, or any combination thereof. In one embodiment, the selected NA viral segment encodes an NA having an isoleucine (I), leucine (L), glycine, or alanine (A) at residue 148. In one embodiment, the isolated recombinant influenza virus is a reassortant. In one embodiment, the NA viral segment encodes an NA having at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50. In one embodiment, the NA viral segment encodes an NA having at least 90% amino acid sequence identity to SEQ ID NO:2. In one embodiment, the NA viral segment encodes N2, N3, N7, or N9. In one embodiment, the NA viral segment encodes N1, N4, N5, N6, N8, N10, or N11. In one embodiment, the residue at position 32 is A, I, G, or L, wherein the deletion is a deletion of residues 46 to 50, wherein the residue at position 147 is N or glutamine (Q), wherein the residue at position 329 is D or glutamic acid E, or wherein the residue at position 346 is serine (S), T, proline (P), tyrosine (Y), tryptophan (W), A, N, I, or L. In one embodiment, the residue at position 346 is V, S, I, or L. In one embodiment, the residue at position 148 is I. In one embodiment, the residue at position 151 is E, N, or Q.In one embodiment, the residue at position 245 is S, T, I, L, A, N, W, Y, P, V, or G. In one embodiment, the residue at position 347 is G, Q, S, T, Y, C, or W. In one embodiment, the residue at position 369 is K, H, R, E, P, or D. In one embodiment, the residue at position 147 is N or Q, the residue at position 329 is D or E, and the residue at position 347 is G, Q, S, T, Y, C, or W, or any combination thereof. In one embodiment, the residue at position 147 is N or Q, the residue at position 329 is D or E, and the residue at position 347 is G or Q, or any combination thereof. In one embodiment, the residue at position 148 is K, R or H, the residue at position 151 is E, N or Q, and the residue at position 245 is S, T, I, L, A, W, Y, P, V, or G, or any combination thereof. In one embodiment, the residue at position 148 is K, R or H, the residue at position 151 is E, N or Q, the residue at position 245 is S, T, I, L, A, or V, and / or the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V, or any combination thereof. In one embodiment, the selected NA viral segment does not encode an NA with a threonine at position 148, does not encode an NA with an aspartic acid at position 151, does not encode an NA with an asparagine at position 245, does not encode an NA with a histidine, arginine or asparagine at residue 347, or any combination thereof. In one embodiment, the selected NA viral segment does not encode an NA with an aspartic acid at position 147, does not encode an NA with an asparagine at residue 329, does not encode an NA with a histidine, arginine or asparagine at residue 347, or any combination thereof. In one embodiment, the selected NA viral segment does not encode an NA with a threonine at position 148, does not encode an NA with an aspartic acid at position 151, does not encode an NA with an asparagine at position 245, does not encode an NA with a glycine at position 346, or any combination thereof. In one embodiment, the HA is H1, H3, H5, H7, or H9.In one embodiment, the virus is an influenza A virus. In one embodiment, the PA, PB1, PB2, NP, M, and NS viral segments have at least 85% nucleic acid sequence identity to SEQ ID NO:24-29 or 39-44, or encode polypeptides having at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID NO:24-29 or 39-44. In one embodiment, PB2 has an I, A, L, or G at residue 147.

[0196] Also provided in one embodiment is an isolated recombinant nucleic acid comprising a nucleic acid sequence for an influenza virus NA viral segment encoding an NA having a plurality of selected residues or residue deletions, wherein the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, and does not encode a threonine at residue 32, an aspartic acid at position 151, an asparagine at position 245, an asparagine at residue 329, a glycine at position 346, a histidine at residue 347, or comprises an NA having one or more deletions from residues 46 to 50, or any combination thereof, wherein the numbering is based on N2. In one embodiment, the selected NA viral segment does not encode an NA having a threonine at residue 148 and does not encode an NA having an aspartic acid at position 151, an asparagine at position 245, a histidine at residue 347, or a threonine at residue 369, or any combination thereof, where the numbering is based on N2, and wherein the recombinant influenza virus has enhanced replication in avian eggs or has reduced HA mutations when propagated in avian eggs compared to a corresponding influenza virus having an NA encoding a threonine at residue 148, an aspartic acid at residue 151, an asparagine at residue 245, a histidine at residue 347, and a threonine at residue 369, or any combination thereof. In one embodiment, the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, and does encode an NA having an aspartic acid at position 151, an asparagine at position 245, a histidine at residue 347, or a threonine at residue 369, or any combination thereof, where the numbering is based on N2, and wherein the recombinant influenza virus has enhanced replication in avian eggs or has reduced HA mutations when propagated in avian eggs, compared to a corresponding influenza virus having an NA encoding a threonine or lysine at residue 148, an aspartic acid at residue 151, an asparagine at residue 245, a histidine at residue 347, and a threonine at residue 369, or any combination thereof.In one embodiment, the selected NA viral segment encodes an NA having an isoleucine (I), leucine (L), glycine (G) or alanine (A) at residue 148. In one embodiment, the NA has at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. In one embodiment, the NA is N2, N3, N7 or N9. In one embodiment, the NA is N1, N4, N5, N6, N8, N10 or N11. In one embodiment, the HA is H1, H2, H3, H5, H7 or H9. In one embodiment, the residue at position 32 is A, I, G, or L, the residue at position 147 is N or Q, the residue at position 329 is D or E, the residue at position 151 is E, N or Q, the residue at position 148 is I, L, V, A, or G, the residue at position 245 is S, T, I, L, A, W, Y, P, V, or G, the residue at position 347 is G, Q, S, or T, the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V, and the residue at position 369 is K, H, R, E, P, or D, or any combination thereof. In one embodiment, the residue at position 151 is E, N or Q, the residue at position 148 is I, L, V, A or G, the residue at position 245 is S, T, I, L, A, W, Y, P, V or G, the residue at position 329 is S, I, L, A, W, Y, P, V or G, the residue at position 347 is G, Q, S or T, the residue at position 346 is S, T, P, Y, W, A, N, I, L or V, the residue at position 369 is K, H, R, E, P or D, or any combination thereof. In one embodiment, the NA has at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49, or has at least 90% amino acid sequence identity to a NA encoded by one of SEQ ID NOs:51-59 or 69-70.

[0197] In one embodiment, a method of preparing influenza virus is provided, comprising administering to a subject a vector for vRNA production comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operably linked to influenza virus NS DNA linked to a transcription termination sequence, wherein the PB1, PB2, PA, NP, NS, and M in the vector for vRNA production are present in an amount effective to produce an infectious influenza virus. a vector, the DNA being derived from one or more influenza vaccine virus isolates, wherein the NA DNA in the vector for vRNA production encodes an NA having a plurality of selected residues or residue deletions, wherein the selected NA viral segment does not encode one or more of: a threonine or lysine at residue 148, a threonine at residue 32, an aspartic acid at position 151, an asparagine at position 245, an asparagine or threonine at residue 329, a lysine at residue 344, a glycine at position 346, a histidine at residue 347, or comprises an NA having one or more deletions from residues 46 to 50, or any combination thereof, wherein the numbering is based on N2; and a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA.The method includes contacting the cell with a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M2, or a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS2. In one embodiment, the NA has at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49. In one embodiment, NA is N2, N3, N7, or N9. In one embodiment, HA is H1, H3, H7, or H9. In one embodiment, HA is H2, H4, H5, H6, H8, or any of H10-H18. In one embodiment, the residue at position 147 is N or Q, the residue at position 329 is D or E, the residue at position 347 is G, Q, N, S, T, Y, C, or W, or the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V. In one embodiment, the residue at position 151 is E, N or Q, the residue at position 148 is I, L, V, A, or G, the residue at position 245 is S, T, I, L, A, W, Y, P, V, or G, the residue at position 347 is G, Q, S, or T, the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V, and the residue at position 369 is K, H, D, E, or R, or any combination thereof.The PB1, PB2, NP, M, and NS viral segments have at least 85% nucleic acid sequence identity to SEQ ID NOs: 24 to 29 or 39 to 44, or encode polypeptides having at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID NOs: 24 to 29 or 39 to 44.

[0198] Further provided is a method of immunizing a bird or mammal, comprising: administering to the bird or mammal a composition having an effective amount of a virus described herein. In one embodiment, the composition contains at least one other, different influenza virus. In one embodiment, the mammal is a human. In one embodiment, the composition is administered intranasally or by injection.

[0199] The viruses described herein may be passaged in eggs or other cells.

[0200] Illustrative backbone viral segments include, but are not limited to, PB2, M202L, F323L; PB1, Q247H; PA, K142N; NP, R74K; M, V97A, Y100H; and NS, K55E, or PB2, I504V; PB1, M40L / G180W; PA, R401K; NP, I116L, and NS1, A30P / R118K.

[0201] Example V In one embodiment, a method is provided for decreasing influenza HA binding to a cell, comprising altering one or more residues in the HA binding pocket of HA that binds to sialic acid on the allantois. In one embodiment, the nucleic acid encoding HA is altered. In one embodiment, the HA is H1, H3, H7, or H9. In one embodiment, the HA is any of H2, H4, H5, H6, H8, or H10-H18. In one embodiment, the residue at position 98, 153, or 183 of HA is altered based on the numbering of H3 HA. In one embodiment, the residue at position 98 is not Y. In one embodiment, the residue at position 153 is not W. In one embodiment, the residue at position 183 is not H. In one embodiment, the residue at position 98 is F, G, I, V, T, H, W, or L. In one embodiment, the residue at position 153 is A, G, I, V, T, or L. In one embodiment, the residue at position 183 is F, A, G, I, L, V, Y, W, P, or T.

[0202] In one embodiment, a method is provided for preparing an influenza virus that binds to a cell via influenza neuraminidase, comprising providing a vector comprising a recombinant nucleic acid molecule comprising the sequence of an influenza virus HA segment from a first influenza virus isolate, wherein the segment encodes an HA with an amino acid other than tyrosine at position 98 of HA1, other than tryptophan at position 153 of HA1, other than histidine at position 183 of HA1, or any combination thereof, wherein the numbering for the HA1 residues is for H3; modifying the HA segment such that at position 98 it encodes F, G, I, V, T, H, W, or L, or at position 153 it encodes A, G, I, V, T, or L, or at position 183 it encodes F, A, G, I, L, V, Y, W, P, or T, or any combination thereof, thereby resulting in a modified HA segment;and a vector comprising a promoter that produces a full-length genomic influenza virus RNA or its complement operably linked to an influenza virus PA segment DNA linked to a transcription termination sequence, a vector comprising a promoter that produces a full-length genomic influenza virus RNA or its complement operably linked to an influenza virus PB1 segment DNA linked to a transcription termination sequence, a vector comprising a promoter that produces a full-length genomic influenza virus RNA or its complement operably linked to an influenza virus PB2 segment DNA linked to a transcription termination sequence, a vector comprising a promoter that produces a full-length genomic influenza virus RNA or its complement operably linked to a modified HA segment linked to a transcription termination sequence, in an amount effective to produce an infectious influenza virus that binds to a cell via NA. vectors comprising a promoter which produces a full-length genomic influenza virus RNA or its complement operably linked to an influenza virus NP segment DNA linked to a transcription termination sequence; vectors comprising a promoter which produces a full-length genomic influenza virus RNA or its complement operably linked to an influenza virus NA segment DNA linked to a transcription termination sequence; vectors comprising a promoter which produces a full-length genomic influenza virus RNA or its complement operably linked to an influenza virus M segment DNA linked to a transcription termination sequence; and vectors comprising a promoter which produces a full-length genomic influenza virus RNA or its complement operably linked to an influenza virus NS segment DNA linked to a transcription termination sequence;and contacting the cell with a vector comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus PB2, and a vector comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus NP, and optionally a vector comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus M1, a vector comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus M2, a vector comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus NS1, or a vector comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus NS2;

[0203] Example VI In one embodiment, the isolated recombinant influenza virus comprises a selected NA viral segment, an HA viral segment, and one or more of a PB1, PB2, PA, NP, M, and NS viral segment encoding a plurality of selected residues. In one embodiment, the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, does not encode an aspartic acid (D) at position 151, does not encode an asparagine at position 245, does not encode a threonine at position 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and / or does not encode a threonine at position 369, where numbering is based on N2, and where the recombinant influenza virus, when propagated in avian eggs, encodes a threonine or lysine at residue 148, The NA segment of the recombinant virus has enhanced replication in avian eggs, reduced sialidase activity, enhanced binding to specific sialic acid residues, and / or reduced HA mutations compared to a corresponding influenza virus having an NA encoding an aspartic acid at residue 151, an asparagine at residue 245, a threonine at residue 329, a lysine at residue 344, a glycine at residue 346, a histidine at residue 347, or a threonine at position 369, or any combination thereof. In one embodiment, the NA segment of the recombinant virus has serine (S), valine (V), alanine (A), G, cysteine ​​(C), methionine (M), isoleucine (I), or leucine (L) at position 329, or V, S, T, proline (P), tyrosine (Y), tryptophan (W), A, N, I, or L at position 346. In one embodiment, the NA segment of the recombinant virus has an I at position 148. In one embodiment, the NA segment of the recombinant virus has an E, N, or Q at position 151. In one embodiment, the NA segment of the recombinant virus has an S, T, I, L, A, W, Y, P, V, or G at position 245. In one embodiment, the NA segment of the recombinant virus has an S, I, L, A, W, Y, P, V, or G at position 329.In one embodiment, the NA segment of the recombinant virus has an E, H, D, N or Q at position 344. In one embodiment, the NA segment of the recombinant virus has a V, S, T, I, L, A, W, Y or P at position 346. In one embodiment, the NA segment of the recombinant virus has a G, Q, S, T, Y, C or W at position 347. In one embodiment, the NA segment of the recombinant virus has a K, H, R, E, P or D at position 369. In one embodiment, the recombinant virus is a reassortant. In one embodiment, the NA viral segment encodes an NA having at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49, or having at least 90% amino acid sequence identity to an NA encoded by any one of SEQ ID NOs:51-59. In one embodiment, the NA viral segment encodes an NA having at least 90% amino acid sequence identity to SEQ ID NO:2. In one embodiment, the NA viral segment encodes N2, N3, N7, or N9. In one embodiment, the NA viral segment encodes N1, N4, N5, N6, N8, N10, or N11. In one embodiment, the HA is H2 or H3. In one embodiment, the virus is an influenza A virus. In one embodiment, the PA, PB1, PB2, NP, M, and NS viral segments encode polypeptides having at least 85% nucleic acid sequence identity to SEQ ID NO: 24-29 or 39-44, or at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID NO: 24-29 or 39-44. In one embodiment, PB2 has an I, A, L, or G at residue 147.

[0204] Further provided is an isolated recombinant nucleic acid comprising a nucleic acid sequence of an influenza virus NA viral segment encoding an NA having a plurality of selected residues, wherein the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, does not encode an aspartic acid at position 151, does not encode an asparagine at position 245, does not encode a threonine at position 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and / or does not encode a threonine at position 369, wherein the numbering is based on N2. In one embodiment, the NA has at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49, or has at least 90% amino acid sequence identity to an NA encoded by one of SEQ ID NOs:51-59. In one embodiment, the NA is N2, N3, N7, or N9. In one embodiment, NA is N1, N4, N5, N6, N8, N10 or N11. In one embodiment, the residue at position 148 is I, the residue at position 329 is S, the residue at position 151 is E, N or Q, the residue at position 245 is S, T, I, L, A, V or G, the residue at position 347 is G, Q, S or T, the residue at position 346 is S, T, P, Y, W, A, N, I, L or V, and the residue at position 369 is K, H, R, E, P or D, or any combination thereof. In one embodiment, the residue at position 151 is E, N or Q, the residue at position 148 is I or K, the residue at position 245 is S, T, I, L, A, W, Y, P, V, or G, the residue at position 347 is G, Q, S, or T, the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V, and the residue at position 369 is K, H, R, E, P, or D, or any combination thereof.

[0205] In one embodiment, a method for preparing influenza virus is provided, which comprises administering to a subject a vector for vRNA production comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NS DNA linked to a transcription termination sequence, in an amount effective to produce an infectious influenza virus. a vector in which the DNA is derived from one or more influenza vaccine virus isolates, wherein the NA DNA in the vector for vRNA production encodes an NA having a plurality of selected residues, wherein the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, does not encode an aspartic acid at position 151, does not encode an asparagine at position 245, does not encode a threonine at position 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and / or does not encode a threonine at position 369, wherein the numbering is based on N2; and a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding an influenza virus PA.contacting the cell with a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus M2, or a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NS2. In one embodiment, the NA has at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48 or SEQ ID NO:49, or has at least 90% amino acid sequence identity to the NA encoded by one of SEQ ID NOs:51-59. In one embodiment, the NA is N2, N3, N7, or N9. In one embodiment, the HA is H1, H2, H3, H7, or H9. In one embodiment, the HA is H2, H4, H5, H6, H8, or any of H10-H18. In one embodiment, the residue at position 329 is S, A, I, L, or G, the residue at position 347 is G, Q, N, S, T, Y, C, or W, or the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V. In one embodiment, the residue at position 151 is E, N or Q, the residue at position 148 is K, H, D or E, the residue at position 245 is S, T, I, L, A, W, Y, P, V or G, the residue at position 347 is G, Q, S or T, and the residue at position 346 is V, S, T, P, Y, W, A, N, I or L;The residue at position 369 is K, H, D, E, or R, or any combination thereof. In one embodiment, the PA, PB1, PB2, NP, M, and NS viral segments have at least 85% nucleic acid sequence identity to SEQ ID NOs: 24-29 or 39-44, or encode polypeptides having at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID NOs: 24-29 or 39-44. Also provided are isolated viruses prepared by this method.

[0206] The recombinant virus may be utilized in a method of immunizing a bird or mammal, which comprises administering to the bird or mammal a composition having an effective amount of the virus. In one embodiment, the composition comprises at least one other, different influenza virus. In one embodiment, the mammal is a human. In one embodiment, the composition is administered intranasally or by injection.

[0207] Example VII In one embodiment, an isolated recombinant influenza virus is provided that includes a selected NA viral segment encoding a plurality of selected residues or residue deletions in NA. The virus includes a selected NA viral segment encoding a plurality of selected residues, an HA viral segment, and one or more of a PB1 viral segment, a PB2 viral segment, a PA viral segment, a NP viral segment, a M viral segment, and a NS viral segment. In one embodiment, the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, does not encode an aspartic acid at position 151, does not encode an asparagine at position 245, does not encode a threonine at position 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and does not encode a threonine at position 369, where the numbering is based on N2, and where the recombinant influenza virus has no threonine or lysine at residue 148 when propagated in avian eggs. The influenza viruses have enhanced replication in avian eggs, have reduced sialidase activity, have enhanced binding to α2-3 sialosides, or have reduced HA mutations compared to the corresponding influenza virus having an NA encoding an onine or lysine at residue 151, an aspartic acid at residue 151, an asparagine at residue 245, a threonine at residue 329, a lysine at residue 344, a glycine at residue 346, a histidine at residue 347, and a threonine at residue 369.In one embodiment, the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, does not encode a threonine at residue 32, does not encode an aspartic acid at position 151, does not encode an asparagine at position 245, does not encode an asparagine or threonine at residue 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and / or does not encode a threonine at residue 369, or comprises an NA having one or more deletions of residues 46 to 50, or any combination thereof, wherein the numbering is based on N2, and wherein the recombinant influenza virus is capable of expressing NA when propagated in avian eggs. and has enhanced replication in avian eggs, reduced sialidase activity, enhanced binding to α2-3 sialosides, or reduced HA mutations, compared to a corresponding influenza virus having an NA that encodes a threonine or lysine at residue 148 and a threonine at residue 32, has no deletion from residues 46 to 50, has an aspartic acid at position 147, has an aspartic acid at residue 151, has an asparagine at residue 245, has an asparagine or threonine at residue 329, has a glycine at residue 346, has a histidine at residue 347, has a threonine at residue 369, or any combination thereof. In one embodiment, the selected NA segment encodes two or more of positions 148, 151, 245, 329, 344, 347, or 369 having a lysine or isoleucine at residue 148, a glutamic acid at residue 151, a serine, threonine, glycine, alanine, leucine, or isoleucine at residue 245, or a serine, glycine, alanine, leucine, or isoleucine at residue 329, a glutamic acid, aspartic acid, glutamine, asparagine, or histidine at residue 344, a valine, leucine, isoleucine, threonine, or serine at residue 346, a glycine, alanine, valine, leucine, isoleucine, or threonine at residue 347, or a lysine, histidine, aspartic acid, or glutamic acid at residue 369.In one embodiment, the selected NA segment encodes two or more of positions 148, 151, 245, 329, 344, 347, or 369 having an isoleucine (I) at residue 148, a glutamic acid at residue 151, a serine, threonine, leucine, or isoleucine at residue 245, or a serine, leucine, or isoleucine at residue 329, a glutamic acid, aspartic acid, or histidine at residue 344, a valine, leucine, or isoleucine at residue 346, a glycine, alanine, valine, leucine, or isoleucine at residue 347, or a lysine, aspartic acid, or glutamic acid at residue 369. In one embodiment, the selected NA segment does not encode a threonine at residue 148, does not encode an asparagine at residue 245, does not encode a threonine at residue 369, does not encode an aspartic acid at residue 151, does not encode a lysine at residue 344, does not encode a glycine at residue 346, does not encode a histidine at residue 347, and does not encode a threonine at residue 369. In one embodiment, the selected NA segment encodes lysine or isoleucine (I) at residue 148, glutamic acid (E) at residue 151, serine (S), threonine, glycine, alanine (A), leucine (L) or isoleucine at residue 245, serine, glycine, alanine, leucine or isoleucine at residue 329, glutamic acid, arginine (R), aspartic acid (D) or histidine at residue 344, valine, leucine, isoleucine, threonine or serine at residue 346, glycine, alanine, valine, leucine, isoleucine or threonine at residue 347, or lysine, histidine, aspartic acid or glutamic acid at residue 369.In one embodiment, the selected NA viral segment does not encode an NA having a threonine at residue 148 and does not encode an NA having an aspartic acid at position 151, an asparagine at position 245, a valine, serine, isoleucine or leucine at residue 346, a histidine at residue 347, or a threonine at residue 369, or any combination thereof, where the numbering is based on N2, and wherein the recombinant influenza virus, when propagated in avian eggs, has enhanced replication in avian eggs or has reduced HA mutations compared to a corresponding influenza virus having an NA encoding a threonine at residue 148, an aspartic acid at residue 151, an asparagine at residue 245, a histidine at residue 347, and a threonine at residue 369, or any combination thereof; or wherein The selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, and does not encode an NA having an aspartic acid at position 151, an asparagine at position 245, a valine, serine, isoleucine or leucine at residue 346, a histidine at residue 347, or a threonine at residue 369, or any combination thereof, where the numbering is based on N2, and wherein the recombinant influenza virus, when propagated in avian eggs, has enhanced replication in avian eggs or has reduced HA mutations compared to a corresponding influenza virus having an NA encoding a threonine or lysine at residue 148, an aspartic acid at residue 151, an asparagine at residue 245, a glycine at residue 346, a histidine at residue 347, and a threonine at residue 369, or any combination thereof. In one embodiment, the selected NA viral segment encodes an NA having an isoleucine, leucine, glycine or alanine at residue 148. In one embodiment, the residue at position 32 is A, I, G, or L, and the deletion is a deletion of residues 46 to 50, wherein the residue at position 147 is N or glutamine (Q), wherein the residue at position 329 is D or glutamic acid, or wherein the residue at position 346 is serine, T, proline (P), tyrosine (Y), tryptophan (W), A, N, I, or L.In one embodiment, the residue at position 148 is I, the residue at position 151 is E, N or Q, the residue at position 245 is S, T, I, L, A, N, W, Y, P, V, or G, the residue at position 347 is G, Q, S, T, Y, C or W, and the residue at position 369 is K, H, R, E, P, or D, or any combination thereof. In one embodiment, the residue at position 329 is serine, valine, alanine, G, cysteine ​​(C), methionine (M), isoleucine or leucine, or wherein the residue at position 346 is V, S, T, proline (P), tyrosine (Y), tryptophan (W), A, N, I, or L. In one embodiment, the residue at position 148 is I, the residue at position 151 is E, N or Q, the residue at position 245 is S, T, I, L, A, W, Y, P, V or G, the residue at position 329 is S, I, L, A, W, Y, P, V or G, the residue at position 344 is E, H, D, N or Q, the residue at position 346 is V, S, T, I, L, A, W, Y or P, the residue at position 347 is G, Q, S, T, Y, C or W, or the residue at position 369 is K, H, R, E, P or D. In one embodiment, the isolated recombinant influenza virus is a reassortant. In one embodiment, the NA viral segment encodes an NA having at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49, or having at least 90% amino acid sequence identity to an NA encoded by any one of SEQ ID NOs:51-59. In one embodiment, the NA viral segment encodes an N2, N3, N7, or N9 NA. In one embodiment, the NA viral segment encodes an N1, N4, N5, N6, N8, N10, or N11 NA. In one embodiment, the recombinant virus has an H1, H2, H3, H5, H7, or H9 HA. In one embodiment, the isolated recombinant influenza virus is an influenza A virus.In one embodiment, the PA, PB1, PB2, NP, M, and NS viral segments have at least 85% nucleic acid sequence identity to SEQ ID NO: 24-29 or 39-44, or encode a polypeptide having at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID NO: 24-29 or 39-44. In one embodiment, PB2 has I, A, L, or G. In one embodiment, the virus has one or more of PB2-I504V, PB1-M40L / G180W, PA-R401K, NP-I116L, or NS1-A30P / R118K. In one embodiment, the virus has PB2-I504V, PB1-M40L / G180W, PA-R401K, NP-I116L, and NS1-A30P / R118K.

[0208] In one embodiment, an isolated recombinant nucleic acid is provided that includes a nucleic acid sequence of an influenza virus NA viral segment encoding an NA having a plurality of selected residues or residue deletions, wherein the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, and does not encode an NA having a threonine at residue 32, an aspartic acid at position 151, an asparagine at position 245, an asparagine or threonine at residue 329, a glycine at position 346, a histidine at residue 347, or an NA having a threonine at residue 329, an aspartic acid at position 151, an asparagine at position 245, an asparagine or threonine at residue 329, a glycine at position 346, a histidine at residue 347, or an NA having a threonine at residue 329, an aspartic acid at position 151, an asparagine at position 245, an asparagine or threonine at residue 329, an asparagine or threonine at residue ...7, a glycine at position 346, a histidine at 50, or any combination thereof, or wherein the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, does not encode an aspartic acid at position 151, does not encode an asparagine at position 245, does not encode a threonine at position 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and does not encode a threonine at position 369, where the numbering is based on N2. In one embodiment, the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, does not encode an NA having an aspartic acid at position 151, does not encode an NA having an asparagine at position 245, does not encode an NA having a glycine at residue 346, does not encode an NA having a histidine at residue 347, or does not encode an NA having a threonine at residue 369, or any combination thereof. In one embodiment, the residue at position 151 is E, N, or Q. In one embodiment, the residue at position 148 is I, L, V, A, or G. In one embodiment, the residue at position 245 is S, T, I, L, A, W, Y, P, V, or G, or position 329 is S, I, L, A, W, Y, P, V, or G. In one embodiment, the residue at position 347 is G, Q, S, or T. In one embodiment, the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V. In one embodiment, the residue at position 369 is K, H, R, E, P, or D.In one embodiment, the residue at position 32 is A, I, G, or L, the residue at position 147 is N or Q, the residue at position 329 is D or E, the residue at position 151 is E, N or Q, the residue at position 148 is I, L, V, A, or G, the residue at position 245 is S, T, I, L, A, W, Y, P, V, or G, the residue at position 347 is G, Q, S, or T, the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V, and the residue at position 369 is K, H, R, E, P, or D, or any combination thereof. In one embodiment, the residue at position 151 is E, N or Q, the residue at position 148 is I, L, V, A or G, the residue at position 245 is S, T, I, L, A, W, Y, P, V or G, the residue at position 347 is G, Q, S or T, the residue at position 346 is S, T, P, Y, W, A, N, I, L or V, the residue at position 369 is K, H, R, E, P or D, or any combination thereof. In one embodiment, the NA has at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49, or has at least 90% amino acid sequence identity to the NA encoded by SEQ ID NO:51-59. In one embodiment, the NA is N2, N3, N7, or N9. In one embodiment, NA is N1, N4, N5, N6, N8, N10 or N11.

[0209] In one embodiment, a method for preparing influenza virus is provided, comprising administering to a human an effective amount of a vector for vRNA production comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operably linked to influenza virus NS DNA linked to a transcription termination sequence, wherein PB1, PB2, PA, NP, NS, and M in the vector for vRNA production are operably linked to an influenza virus PA DNA linked to a transcription termination sequence, in an amount effective to produce an infectious influenza virus. a vector for vRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA; a vector for vRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA; a vector for vRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA; a vector for vRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA; a vector for vRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA;contacting the cell with a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus M2, or a vector for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NS2. In one embodiment, the NA has at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48 or SEQ ID NO:49, or has at least 90% amino acid sequence identity to the NA encoded by one of SEQ ID NOs:51-59. In one embodiment, the NA is N2, N3, N7 or N9. In one embodiment, the HA is H2 or H3. In one embodiment, the residue at position 329 is S, the residue at position 347 is G, and the residue at position 346 is V. In one embodiment, the residue at position 151 is E, N or Q, the residue at position 148 is I, L, V, A, or G, the residue at position 245 is S, T, I, L, A, V or G, the residue at position 344 is E, D, N, H or Q, the residue at position 347 is G, L, I, V, A, S or T, the residue at position 346 is V, S, T, A, N, I, L or V, and the residue at position 369 is K, H, D, E, or R, or any combination thereof.or encodes a polypeptide having at least 85% nucleic acid sequence identity to SEQ ID NO:24 to 29 or 39 to 44, or at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID NO:24 to 29 or 39 to 44. Also provided is an isolated virus prepared by this method.

[0210] In one embodiment, a method of immunizing a bird or mammal is provided, comprising: administering to the bird or mammal a composition having an effective amount of the virus. In one embodiment, the composition comprises at least one other, different influenza virus. In one embodiment, the mammal is a human. In one embodiment, the composition is administered intranasally or by injection.

[0211] Further provided is a method of passaging the virus in eggs.

[0212] References Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics , 3rd ed., ADIS Press, Ltd., Williams and Wilkins, Baltimore, MD (1987). Aymard-Henry et al. Virology: A Practical Approach , Oxford IRL Press, Oxford, 119-150 (1985). Bachmeyer, Intervirology , 5 :260 (1975). Edited by Berkow et al. The Merck Manual , 16th edition, Merck & Co., Rahway, NJ (1992). Hatta et al. Science , 293 :1840 (2001). Horimoto et al. J. Virol. , 68 :3120 (1994). Horimoto et al. Vaccine , 24 :3669 (2006). Keitel et al., in Textbook of Influenza, eds. Nickolson, KG, Webster, RG, and Hay, A. (Blackwell, Oxford), pp. 373-390 (1998). Kuwahara et al. Jpn. J. Infect. Dis. , 71 :234 (2018). Laver & Webster, Virology , 69:511 (1976). Neumann et al. Adv. Virus Res. , 53 :265 (1999). Neumann et al. J. Gen. Virol. , 83 :2635 (2002). Neumann et al. J. Virol. , 71 :9690 (1997). Neumann et al. Proc. Natl. Acad. Sci. USA , 96 :9345 (1999). Neumann et al. Virology , 287 :243 (2001). Osol (Editor), Remington's Pharmaceutical Sciences , Mack Publishing Co., Easton, PA 1324-1341 (1980). Sugawara et al. Biological , 30 :303 (2002). Webby & Webster et al. Science , 302 :1519 (2003). Wood & Robertson, Nat. Rev. Microbiol. , 2 :842 (2004). World Health Organization TSR No. 673 (1982). World Health Organization. Confirmed human cases of avian influenza A (H5N1). http: / / www.who.int / csr / disease / avian_influenza / country / en / index.html

[0213] All publications, patents, and patent applications are incorporated herein by reference. In the foregoing specification, the invention has been described in connection with certain preferred embodiments thereof, and numerous details have been set forth for purposes of illustration, but it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments, and that the specific details described herein may be varied considerably without departing from the basic spirit of the invention.

Claims

1. 1. An isolated recombinant influenza virus comprising a selected NA viral segment encoding a plurality of selected residues or residue deletions in NA: wherein the selected NA viral segment does not encode an NA having a threonine (T) or lysine (K) at residue 148, does not encode an aspartic acid (D) at position 151, does not encode an asparagine (N) at position 245, does not encode a threonine at position 329, does not encode a lysine at position 344, does not encode a glycine (G) at position 346, does not encode a histidine (H) at residue 347, and does not encode a threonine at position 369, where the numbering is based on N2, and wherein the recombinant influenza virus, when propagated in avian eggs, does not encode an NA having a threonine (T) or lysine (K) at residue 148, does not encode an aspartic acid (D) at position 151, does not encode an asparagine (N) at position 245, does not encode a threonine at position 329, does not encode a lysine at position 344, does not encode a glycine (G) at position 346, does not encode a histidine (H) at residue 347, and does not encode a threonine at position 369, where the numbering is based on N2, has enhanced replication in avian eggs, has decreased sialidase activity, has increased binding to α2-3 sialosides, or has decreased HA mutations, compared to a corresponding influenza virus having a NA encoding a threonine or lysine at residue 148, an aspartic acid at residue 151, an asparagine at residue 245, a threonine at residue 329, a lysine at residue 344, a glycine at residue 346, a histidine at residue 347, and a threonine at position 369; wherein the selected NA viral segment encodes an NA having an isoleucine (I) at residue 148, and does not encode an aspartic acid at position 151, does not encode an asparagine (N) at position 245, does not encode a threonine at position 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and does not encode a threonine at position 369, where the numbering is based on N2, and wherein the recombinant influenza virus, when propagated in avian eggs, does not encode a threonine at residue 148. has enhanced replication in avian eggs, has decreased sialidase activity, has increased binding to α2-3 sialosides, or has decreased HA mutations, compared to a corresponding influenza virus having a NA encoding a cysteine ​​or lysine at residue 151, an aspartic acid at residue 152, an asparagine at residue 245, a threonine at residue 329, a lysine at residue 344, a glycine at residue 346, a histidine at residue 347, and a threonine at residue 369; wherein the selected NA viral segment encodes an NA having a serine at position 329 and a glutamic acid at position 344, where numbering is based on N2, and wherein the recombinant influenza virus has enhanced replication in avian eggs, has reduced sialidase activity, has enhanced binding to α2-3 sialosides, or has reduced HA mutations, when propagated in avian eggs, compared to a corresponding influenza virus having an NA encoding a threonine or asparagine at residue 329, or a lysine at residue 344; or wherein the selected NA viral segment does not encode an NA with a threonine or lysine at residue 148, does not encode a threonine at residue 32, does not encode an aspartic acid at position 151, does not encode an asparagine at position 245, does not encode an asparagine or threonine at residue 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and / or does not encode a threonine at residue 369, or comprises an NA with one or more deletions from residues 46 to 50, or any combination thereof, wherein the numbering is based on N2, and wherein the recombinant influenza virus, when propagated in avian eggs, 23. An isolated recombinant influenza virus having an HA encoding a nucleotide sequence ...

2. The recombinant virus of claim 1, wherein the selected NA segment does not encode a threonine at residue 148, does not encode an asparagine at residue 245, does not encode a threonine at residue 369, does not encode an aspartic acid at residue 151, does not encode a lysine at residue 344, does not encode a glycine at residue 346, does not encode a histidine at residue 347, and does not encode a threonine at residue 369.

3. 2. The recombinant virus of claim 1, wherein the selected NA segment encodes two or more of positions 148, 151, 245, 329, 344, 347, or 369 having a lysine or isoleucine at residue 148; a glutamic acid (E) at residue 151; a serine (S), threonine, glycine, alanine (A), leucine, or isoleucine at residue 245; a serine, glycine, alanine, leucine, or isoleucine at residue 329; a glutamic acid, aspartic acid, or histidine at residue 344; a valine, leucine, isoleucine, threonine, or serine at residue 346; a glycine, alanine, valine, leucine, isoleucine, or threonine at residue 347; or a lysine, histidine, aspartic acid, or glutamic acid at residue 369.

4. or wherein the selected NA viral segment does not encode an NA having a threonine at residue 148 and does not encode an NA having an aspartic acid at position 151, an asparagine at position 245, a valine, serine, isoleucine or leucine at residue 346, a histidine at residue 347, or a threonine at residue 369, or any combination thereof, where the numbering is based on N2, and wherein the recombinant influenza virus, when propagated in avian eggs, has enhanced replication in avian eggs or has reduced HA mutations compared to a corresponding influenza virus having an NA encoding a threonine at residue 148, an aspartic acid at residue 151, an asparagine at residue 245, a histidine at residue 347, and a threonine at residue 369, or any combination thereof; 2. The recombinant influenza virus of claim 1, wherein the recombinant influenza virus does not encode an NA having a threonine or lysine at residue 148, and does not encode an NA having an aspartic acid at position 151, an asparagine at position 245, a valine, serine, isoleucine or leucine at residue 346, a histidine at residue 347, or a threonine at residue 369, or any combination thereof, wherein the numbering is based on N2, and wherein the recombinant influenza virus has enhanced replication in avian eggs or has reduced HA mutations when propagated in avian eggs compared to a corresponding influenza virus having an NA encoding a threonine or lysine at residue 148, an aspartic acid at residue 151, an asparagine at residue 245, a glycine at residue 346, a histidine at residue 347, and a threonine at residue 369, or any combination thereof.

5. 2. The recombinant influenza virus of claim 1, wherein the selected NA viral segment encodes an NA having an isoleucine, leucine, glycine, or alanine at residue 148.

6. 2. The isolated recombinant influenza virus of claim 1, wherein the residue at position 32 is A, I, G, or L; the deletion is a deletion of residues 46 to 50; the residue at position 147 is N or glutamine (Q); the residue at position 329 is D or glutamic acid; or the residue at position 346 is serine, T, proline (P), tyrosine (Y), tryptophan (W), A, N, I, or L.

7. 2. The isolated recombinant influenza virus of claim 1, wherein the residue at position 148 is I; the residue at position 151 is E, N or Q; the residue at position 245 is S, T, I, L, A, W, Y, P, V or G; the residue at position 347 is G, Q, S, T, Y, C or W; and the residue at position 369 is K, H, R, E, P or D, or any combination thereof.

8. 2. The isolated recombinant influenza virus of claim 1, wherein the residue at position 329 is serine, valine, alanine, G, cysteine ​​(C), methionine (M), isoleucine or leucine, or the residue at position 346 is V, S, T, proline, tyrosine, tryptophan, A, N, I, or L.

9. 2. The isolated recombinant influenza virus of claim 1, wherein the residue at position 148 is I; the residue at position 151 is E, N or Q; the residue at position 245 is S, T, I, L, A, W, Y, P, V or G; the residue at position 329 is S, I, L, A, W, Y, P, V or G; the residue at position 344 is E, H, D, N or Q; the residue at position 346 is V, S, T, I, L, A, W, Y or P; the residue at position 347 is G, Q, S, T, Y, C or W; or the residue at position 369 is K, H, R, E, P or D.

10. The isolated recombinant influenza virus of any one of claims 1 to 9, which is a reassortant.

11. 11. The isolated recombinant influenza virus of any one of claims 1 to 10, wherein the NA viral segment encodes an NA having at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49, or having at least 90% amino acid sequence identity to an NA encoded by any one of SEQ ID NOs:51-59 or 69-70.

12. The isolated recombinant influenza virus of any one of claims 1 to 11, wherein the NA viral segment encodes N2, N3, N7, or N9.

13. 11. The isolated recombinant influenza virus of any one of claims 1 to 10, wherein the NA viral segment encodes N1, N4, N5, N6, N8, N10 or N11.

14. The isolated recombinant influenza virus of any one of claims 1 to 13, wherein the HA is H1, H2, H3, H5, H7, or H9.

15. The isolated recombinant influenza virus of any one of claims 1 to 14, wherein the virus is an influenza A virus.

16. 16. The isolated recombinant influenza virus of any one of claims 1 to 15, wherein the PA, PB1, PB2, NP, M, and NS viral segments have at least 85% nucleic acid sequence identity to SEQ ID NO:24 to 29 or 39 to 44, or encode a polypeptide having at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID NO:24 to 29 or 39 to 44.

17. 17. The isolated recombinant influenza virus of any one of claims 1 to 16, wherein PB2 has an I, A, L, or G at residue 147.

18. 18. The isolated recombinant influenza virus of any one of claims 1 to 17, comprising one or more of PB2-I504V, PB1-M40L / G180W, PA-R401K, NP-I116L, or NS1-A30P / R118K.

19. 18. The isolated recombinant influenza virus of any one of claims 1 to 17, having PB2-I504V, PB1-M40L / G180W, PA-R401K, NP-I116L, and NS1-A30P / R118K.

20. An isolated recombinant nucleic acid comprising a nucleic acid sequence of an influenza virus NA viral segment encoding an NA having a plurality of selected residues or residue deletions, wherein the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, and does not encode a threonine at residue 32, an aspartic acid at position 151, an asparagine at position 245, an asparagine or threonine at residue 329, a glycine at position 346, a histidine at residue 347, or one or more deletions at residues 46 through 50. or any combination thereof, or wherein the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, does not encode an aspartic acid at position 151, does not encode an asparagine at position 245, does not encode a threonine at position 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and does not encode a threonine at position 369, wherein the numbering is based on N2.

21. 21. The recombinant nucleic acid of claim 20, wherein the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, does not encode an NA having an aspartic acid at position 151, does not encode an NA having an asparagine at position 245, does not encode an NA having a glycine at residue 346, does not encode an NA having a histidine at residue 347, or does not encode an NA having a threonine at residue 369, or any combination thereof.

22. 22. The isolated recombinant nucleic acid of claim 20 or 21, wherein the residue at position 151 is E, N or Q.

23. 23. The recombinant nucleic acid of any one of claims 20 to 22, wherein the residue at position 148 is I, L, V, A, or G.

24. 24. The recombinant nucleic acid of any one of claims 20 to 23, wherein the residue at position 245 is S, T, I, L, A, W, Y, P, V, or G, or the residue at position 329 is S, I, L, A, W, Y, P, V, or G.

25. The recombinant nucleic acid of any one of claims 20 to 24, wherein the residue at position 347 is G, Q, S, or T.

26. 26. The recombinant nucleic acid of any one of claims 20 to 25, wherein the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V.

27. 27. The recombinant nucleic acid of any one of claims 20 to 26, wherein the residue at position 369 is K, H, R, E, P, or D.

28. 21. The isolated recombinant nucleic acid of claim 20, wherein the residue at position 32 is A, I, G, or L, the residue at position 147 is N or Q, the residue at position 329 is D or E, the residue at position 151 is E, N or Q, the residue at position 148 is I, L, V, A, or G, the residue at position 245 is S, T, I, L, A, W, Y, P, V, or G, the residue at position 347 is G, Q, S, or T, the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V, the residue at position 369 is K, H, R, E, P, or D, or any combination thereof.

29. 21. The isolated recombinant nucleic acid of claim 20, wherein the residue at position 151 is E, N or Q; the residue at position 148 is I, L, V, A, or G; the residue at position 245 is S, T, I, L, A, W, Y, P, V, or G; the residue at position 347 is G, Q, S, or T; the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V; the residue at position 369 is K, H, R, E, P, or D, or any combination thereof.

30. The recombinant nucleic acid of claims 20 to 29, wherein the NA has at least 90% amino acid sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49, or has at least 90% amino acid sequence identity with a NA encoded by one of SEQ ID NOs:51-59 or 69-70.

31. The recombinant nucleic acid of any one of claims 20 to 29, wherein the NA is N2, N3, N7, or N9.

32. The recombinant nucleic acid according to any one of claims 20 to 29, wherein the NA is N1, N4, N5, N6, N8, N10 or N11.

33. 1. A method for preparing an influenza virus comprising administering to the patient a virion ... Vectors for vRNA production comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence, vectors for vRNA production comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence, vectors for vRNA production comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence, vectors for vRNA production comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence, vectors for vRNA production comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence, vectors for vRNA production comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence, vectors for vRNA production comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence, and vectors for vRNA production comprising a promoter operably linked to influenza virus NS DNA linked to a transcription termination sequence, wherein PB1, PB2, PA, NP, NS, and M in the vectors for vRNA production a vector, the DNA being derived from one or more influenza vaccine virus isolates, wherein the NA DNA in the vector for vRNA production encodes an NA having a plurality of selected residues or residue deletions, wherein the selected NA viral segment does not encode an NA having a threonine or lysine at residue 148, does not encode an aspartic acid at position 151, does not encode an asparagine at position 245, does not encode an asparagine or threonine at residue 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and does not encode a threonine at residue 369, or any combination thereof, wherein the numbering is based on N2; and Vectors for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PA, vectors for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, vectors for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and vectors for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally vectors for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus HA, vectors for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NA, vectors for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus M1, vectors for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus M2, or vectors for the production of mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NS2. The method comprises contacting a cell with

34. The method of claim 33, wherein the NA has at least 90% amino acid sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48 or SEQ ID NO:49, or has at least 90% amino acid sequence identity with a NA encoded by one of SEQ ID NOs:51-59 or 69-70.

35. 34. The method of claim 33, wherein the NA is N2, N3, N7, or N9.

36. The method of any one of claims 33 to 35, wherein the HA is H2 or H3.

37. 37. The method of any one of claims 33 to 36, wherein the residue at position 329 is S, the residue at position 347 is G, and the residue at position 346 is V.

38. 38. The method of any one of claims 33-37, wherein the residue at position 151 is E, N or Q, the residue at position 148 is I, L, V, A or G, the residue at position 245 is S, T, I, L, A, V or G, the residue at position 344 is E, D, N, H or Q, the residue at position 347 is G, L, I, V, A, S or T, the residue at position 346 is V, S, T, A, N, I, L or V, the residue at position 369 is K, H, D, E or R, or any combination thereof.

39. 39. The method of any one of claims 33 to 38, wherein the PA, PB1, PB2, NP, M, and NS viral segments have at least 85% nucleic acid sequence identity to SEQ ID NO:24 to 29 or 39 to 44, or encode a polypeptide having at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID NO:24 to 29 or 39 to 44.

40. 40. An isolated virus prepared by the method of any one of claims 33 to 39.

41. A method of immunizing a bird or mammal comprising administering to the bird or mammal a composition having an effective amount of the virus of any one of claims 1 to 19 or 40.

42. 42. The method of claim 41, wherein the composition contains at least one other different influenza virus.

43. 43. The method of claim 41 or 42, wherein the mammal is a human.

44. The method of any one of claims 41 to 44, wherein the composition is administered intranasally or by injection.

45. A method comprising passaging a virus according to any one of claims 1 to 19 and 40 in eggs.

46. A method of reducing influenza HA binding to a cell comprising altering one or more residues within the HA binding pocket of HA that binds to sialic acid on the allantois.

47. The method of claim 46, wherein the nucleic acid encoding HA is altered.

48. 48. The method of claim 46 or 47, wherein the HA is H1, H3, H7, or H9.

49. The method of claim 46 or 47, wherein the HA is any of H2, H4, H5, H6, H8, or H10-H18.

50. The method of any one of claims 46 to 49, wherein residues at positions 98, 153, or 183 of the HA are altered based on the numbering of H3 HA.

51. 51. The method of claim 50, wherein the residue at position 98 is not Y.

52. 52. The method of claim 50 or 51, wherein the residue at position 153 is not W.

53. 53. The method of claim 50, 51 or 52, wherein the residue at position 183 is not H.

54. 51. The method of claim 50, wherein the residue at position 98 is F, G, I, V, T, H, W, or L.

55. 55. The method of claim 54, wherein the residue at position 153 is A, G, I, V, T, or L.

56. 56. The method of claim 54 or 55, wherein the residue at position 183 is F, A, G, I, L, V, Y, W, P, or T.

57. 51. The method of claim 50, wherein the residue at position 98 is F, H, P or W.

58. 58. The method of claim 57, wherein the residue at position 153 is A, G, I, V, or L.

59. 59. The method of claim 57 or 58, wherein the residue at position 183 is F, Y, W or P.

60. 1. A method for preparing an influenza virus that binds to a cell via influenza neuraminidase, comprising: providing a vector comprising a recombinant nucleic acid molecule comprising the sequence of an influenza virus HA segment from a first influenza virus isolate, said segment encoding an HA with an amino acid other than tyrosine at position 98 of HA1, other than tryptophan at position 153 of HA1, other than histidine at position 183 of HA1, or any combination thereof, wherein the numbering of the HA1 residues is with respect to H3; modifying the HA segment to encode F, G, I, V, T, H, W, or L at position 98, A, G, I, V, T, or L at position 153, F, A, G, I, L, V, Y, W, P, or T at position 183, or any combination thereof, thereby resulting in a modified HA segment; and in an amount effective to cause infectious influenza virus to bind to cells via NA, Vectors comprising a promoter that produces a full-length genomic influenza virus RNA or its complement operably linked to an influenza virus PA segment DNA linked to a transcription termination sequence, vectors comprising a promoter that produces a full-length genomic influenza virus RNA or its complement operably linked to an influenza virus PB1 segment DNA linked to a transcription termination sequence, vectors comprising a promoter that produces a full-length genomic influenza virus RNA or its complement operably linked to an influenza virus PB2 segment DNA linked to a transcription termination sequence, vectors comprising a promoter that produces a full-length genomic influenza virus RNA or its complement operably linked to a modified HA segment ... vectors comprising a promoter that produces a full-length genomic influenza virus RNA or its complement operably linked to influenza virus NP segment DNA linked in sequence, vectors comprising a promoter that produces a full-length genomic influenza virus RNA or its complement operably linked to influenza virus NA segment DNA linked to a transcription termination sequence, vectors comprising a promoter that produces a full-length genomic influenza virus RNA or its complement operably linked to influenza virus M segment DNA linked to a transcription termination sequence, and vectors comprising a promoter that produces a full-length genomic influenza virus RNA or its complement operably linked to influenza virus NS segment DNA linked to a transcription termination sequence; and Vectors comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus PA, vectors comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus PB1, vectors comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus PB2, and vectors comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus NP, and optionally vectors comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus HA, vectors comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus NA, vectors comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus M1, vectors comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus M2, or vectors comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus NS1, or vectors comprising a promoter that produces an mRNA operably linked to a DNA segment encoding influenza virus NS2. The method comprises contacting a cell with

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