High-titer recombinant influenza virus with enhanced replication in MDCK, Vero cells or eggs

Abstract

A composition useful for preparing high-titer influenza viruses in the absence of helper viruses is provided. [Solution] The present invention provides a composition useful for preparing high-titer influenza viruses, e.g., in the absence of helper virus, that provides growth promotion in cells in culture, such as MDCK cells, or in eggs, and that contains internal genes from influenza virus vaccine strains or isolates that are safe in humans, e.g., do not cause severe disease.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Patent Application No. 61 / 846,460, filed on July 15, 2013, and the disclosure of such application is incorporated herein by reference.

[0002] Statement Regarding Government Rights This invention was made with government support under grants AI070010 and HHSN266200700010C from the National Institutes of Health. The United States government has certain rights in this invention.

Background Art

[0003] Influenza is a major respiratory disease in some mammals, including horses, and is annually a cause of significant mortality and economic loss. Furthermore, influenza virus infection causes severe systemic disease in some species of birds and can lead to death. The segmented nature of the influenza virus genome allows for reassortment of segments during virus replication in cells infected with two or more influenza viruses. Reassortment of segments combined with genetic mutation and drift can give rise to innumerable influenza virus strains over time. Such strains exhibit antigenic variation in their hemagglutinin (HA) and / or neuraminidase (NA) proteins, and in particular, the gene encoding the HA protein has a high mutation rate. The current major practice for influenza prevention is vaccination. The influenza HA protein is the major target antigen for the host's protective immune response against the virus and is highly variable, so the isolation of influenza viruses and the identification and characterization of HA antigens in viruses associated with recent pandemics are important for vaccine production. Vaccines are designed to stimulate a protective immune response against the major and predicted influenza virus strains, based on spread and prediction (Park et al., 2004).

[0004] There are three general types of influenza viruses, types A, B, and C, which are defined by the lack of serological cross-reactivity among their internal proteins. Influenza A viruses are further classified into subtypes based on the antigenic and genetic differences in their glycoproteins, HA and NA proteins. All known HA and NA subtypes (H1 to H15 and N1 to N9) have been isolated from waterfowl, which are thought to function as the natural reservoir of influenza. The H1N1 pandemic virus caused a worldwide outbreak in 2009. Since the first vaccine candidate strain tested in 2009 did not grow at high titers, it clearly demonstrated the need to develop a vaccine virus backbone that would result in efficient replication in the vaccine virus candidate. Summary of the Invention Means for Solving the Problems

[0005] Mutations that enhance the replication ability of a virus in cell culture and / or in embryonated chicken eggs are useful for amplifying influenza viruses and for establishing a robust influenza vaccine platform. Currently, most influenza vaccines are produced in embryonated chicken eggs. Influenza vaccines produced in MDCK cells are currently approved for human use in the United States and in Europe, and influenza vaccines derived from Vero cells are approved for human use in Europe. A virus library with random mutations in the “internal” viral genes of a vaccine virus isolate (e.g., UW-PR8) (i.e., all viral genes other than those encoding the viral surface glycoproteins HA and NA) was generated and passaged in MDCK cells. The identified mutations led to higher virus titers in MDCK cells (and may also enhance virus titers in Vero cells and / or embryonated chicken eggs), enabling more efficient influenza virus growth and more economical vaccine production. Moreover, the mutations described above enhanced the replication ability of the UW-PR8 vaccine backbone virus. In addition to mutations in the coding regions of the six internal gene segments, mutations in non-coding regions, including promoter mutations, such as the C-to-U mutation at position 4 from the 3′ end of the PB2, PB1, and / or PA vRNA segments, were observed to enhance virus titers. Also, the resulting sequences can have codon usage optimized, e.g., for expression in mammalian cells such as canine cells, primate cells, or avian cells such as chicken embryos. The mutations can be used in various combinations depending on the cell line (or egg) being used, as well as the desired level of improvement in virus replication.

[0006] The present invention provides an isolated recombinant, e.g., reassortant influenza virus having a selected amino acid residue at one or more specific positions of one or more gene segments of PA, PB1, PB2, NP, M (encoding M1 and M2 proteins), and / or NS (encoding NS1 and NS2 proteins), e.g., having the selected amino acid residue at specific positions of PB1, PB2, and NS1; PA, PB1, PB2, NP, and NS1; PB1, PB2, NP, M, and NS1; and PA, PB1, PB2, NP, and NS1; or PA, PB1, PB2, NP, M, and NS1, and including, e.g., the HA and NA genes / proteins of interest from annual and worldwide pandemic strains, which virus is produced more efficiently and cost-effectively by cell culture (in MDCK or Vero cells) or in embryonated chicken eggs. In one embodiment, the recombinant reassortant influenza virus has an amino acid residue at position 142 of PA that results in enhanced growth in cells including MDCK cells, Vero cells, or eggs, i.e., the residue at position 142 of PA of the PA gene segment of the recombinant influenza virus is not lysine but is a residue correlated with enhanced replication in MDCK cells, Vero cells, or eggs, and is an amino acid residue optionally selected at one or more specific positions of PB1, PB2, NP, M1, and / or NS1. In one embodiment, the recombinant reassortant influenza virus has an amino acid residue at position 142 of PA that results in enhanced interaction with one or more host proteins in MDCK cells, Vero cells, or eggs as compared to the corresponding virus having lysine at position 142 of PA. In one embodiment, the recombinant reassortant influenza virus has asparagine or glutamine at position 142 of PA and has an amino acid residue optionally selected at one or more specific positions of PB1, PB2, NP, M1, and / or NS1.In one embodiment, the recombinant reassortant influenza virus has, at position 247 of PB1, an amino acid residue that confers enhanced growth in cells including, for example, MDCK cells, Vero cells, or eggs, as compared to the corresponding virus having glutamine at position 247 of PB1. That is, the residue at position 247 of PB1 of the recombinant influenza virus gene segment of PB1 is not glutamine, but is a residue that correlates with enhanced replication in MDCK cells, Vero cells, or eggs, and is an amino acid residue optionally selected at one or more specific positions of PA, PB2, NP, M1, and / or NS1 described herein. In one embodiment, the recombinant reassortant influenza virus has, at position 247 of PB1, an amino acid residue that confers enhanced interaction with one or more host proteins in MDCK cells, Vero cells, or eggs, as compared to the corresponding virus having glutamine at position 247 of PB1. In one embodiment, the recombinant reassortant influenza virus has histidine, arginine, or lysine at position 247 of PB1 and has an amino acid residue optionally selected at one or more specific positions of PA, PB2, NP, M1, and / or NS1 described herein. In one embodiment, for example, the recombinant reassortant influenza virus has, at positions 202 and / or 323 of PB2, amino acid residues that confer enhanced growth in cells including MDCK cells, Vero cells, or eggs, as compared to the corresponding virus having methionine at position 202 or phenylalanine at position 323 of PB2. That is, the residues at positions 202 and / or 323 of PB2 of the recombinant influenza virus gene segment of PB2 are not methionine or not phenylalanine, but are residues that correlate with enhanced replication in MDCK cells, Vero cells, or eggs, and are amino acid residues optionally selected at one or more specific positions of PA, PB1, NP, M1, and / or NS described herein. In one embodiment, the recombinant reassortant influenza virus has, at position 323 of PB2, an amino acid residue that confers an altered cap-binding interaction, as compared to the corresponding virus having phenylalanine at position 323 of PB2.In one embodiment, the recombinant reassortant influenza virus has leucine, alanine, threonine, valine, isoleucine, or glycine at position 202 and / or 323 of PB2, and optionally has a selected amino acid residue at one or more specific positions of PA, PB1, NP, M1, and / or NS described herein. In one embodiment, the recombinant reassortant influenza virus has, at position 74 of NP, an amino acid residue that confers enhanced growth in cells, including MDCK cells, Vero cells, or eggs, compared to the corresponding virus having arginine at position 74 of NP, i.e., the residue at position 74 of NP in the NP gene segment of the recombinant influenza virus is not arginine, but is a residue correlated with enhanced replication in MDCK cells, Vero cells, or eggs, and is an optionally selected amino acid residue at one or more specific positions of PA, PB1, PB2, M1, and / or NS described herein. In one embodiment, the recombinant reassortant influenza virus has, at position 74 of NP, an amino acid residue that can alter folding, stability, and / or interaction with other viral or host proteins compared to the corresponding virus having arginine at position 74 of NP. In one embodiment, the recombinant reassortant influenza virus has lysine or histidine at position 74 of NP, and optionally has a selected amino acid residue at one or more specific positions of PA, PB1, PB2, M1, and / or NS described herein. In one embodiment, the recombinant reassortant influenza virus has, at position 97 of M1 and / or 100 of M1, amino acid residues that confer enhanced growth in cells, including MDCK cells, Vero cells, or eggs, compared to the corresponding virus having valine at position 97 of M1 or tyrosine at position 100 of M1, i.e., the residues at positions 97 and / or 100 of M1 in the M gene segment of the recombinant influenza virus are not valine or tyrosine, respectively, but are residues correlated with enhanced replication in MDCK cells, Vero cells, or eggs, and are selected amino acid residues at one or more specific positions of PA, PB1, PB2, NP, and / or NS1 described herein.In one embodiment, the recombinant reassortant influenza virus has an amino acid residue at position 97 of M1 that can alter dimerization, compared to the corresponding virus having valine at position 97 of M1, for example. In one embodiment, the recombinant reassortant influenza virus has an amino acid residue at position 100 of M1 that can alter virus assembly, compared to the corresponding virus having tyrosine at position 100 of M1, for example. In one embodiment, the recombinant reassortant influenza virus has leucine, threonine, isoleucine, alanine, or glycine at position 97 of M1 and / or lysine, arginine, or histidine at position 100, and has the selected amino acid residues at one or more specific positions of PA, PB1, PB2, NP, and / or NS1 described herein. In one embodiment, the recombinant reassortant influenza virus has an amino acid residue at position 55 of NS1 that results in enhanced growth in cells including MDCK cells, Vero cells, or eggs, compared to the corresponding virus having lysine at position 55 of NS1, for example, and has the selected amino acid residues at one or more specific positions of PA, PB1, PB2, NP, and / or M1 described herein. In one embodiment, the recombinant reassortant influenza virus has asparagine, aspartic acid, glutamic acid, or glutamine at position 55 of NS1 and has the selected amino acid residues at one or more specific positions of PA, PB1, PB2, NP, and / or M1 described herein. In one embodiment, the present invention provides an isolated recombinant reassortant influenza virus having six "internal" gene segments from a vaccine influenza virus having two or more selected amino acid residues at the specific positions described herein, an NA gene segment selected from a first influenza virus isolate, and an HA gene segment from the same or a different isolate.

[0007] In one embodiment, the influenza virus of the present invention is a recombinant influenza virus having two or more selected amino acid residues at specific positions of one or more gene segments of PA, PB1, PB2, NP, M1, and / or NS1 that can be used together with the HA and NA genes of interest. In one embodiment, the recombinant reassortant influenza virus has, for example, an amino acid residue that promotes growth in MDCK cells, Vero cells, or in ovo at position 142 of PA, compared to the corresponding virus having lysine at position 142 of PA; has, for example, an amino acid residue that promotes growth in MDCK cells, Vero cells, or in ovo at position 247 of PB1, compared to the corresponding virus having glutamine at position 247 of PB1; has, for example, an amino acid residue that promotes growth in MDCK cells, Vero cells, or in ovo at position 202 and / or 323 of PB2, compared to the corresponding virus having methionine at position 202 or phenylalanine at position 323 of PB2; has, for example, an amino acid residue that promotes growth in MDCK cells, Vero cells, or in ovo at position 74 of NP, compared to the corresponding virus having arginine at position 74 of NP; has, for example, an amino acid residue that promotes growth in MDCK cells, Vero cells, or in ovo at position 97 and / or 100 of M1, compared to the corresponding virus having valine at position 97 or tyrosine at position 100 of M1; or has, for example, an amino acid residue that promotes growth in MDCK cells, Vero cells, or in ovo at position 55 of NS1, compared to the corresponding virus having lysine at position 55 of NS1; or a combination thereof.

[0008] In one embodiment, the influenza virus of the present invention is a recombinant influenza virus having two or more selected amino acid residues at specific positions of one or more gene segments of PA, PB1, PB2, NP, M1, and / or NS1, which can be used together with the HA and NA genes of interest. In one embodiment, the recombinant reassortant influenza virus has two or more of lysine at position 142 of PA; glutamine at position 247 of PB1; leucine at position 202 and / or 323 of PB2; lysine at position 74 of NP; alanine at position 97 and histidine at position 100 of M1; or glutamic acid at position 55 of NS1.

[0009] The present invention provides an isolated recombinant, e.g., reassortant influenza virus having a selected amino acid residue at one or more specific positions of one or more gene segments of PA, PB1, PB2, NP, M1, and / or NS1, including one or more characteristic residues described herein, e.g., having a selected amino acid residue at specific positions of PB1, PB2, and NS; PB1, PB2, NP, and NS; PA, PB1, PB2, NP, and NS; PB1, PB2, NP, M, and NS; or PA, PB1, PB2, NP, M, and NS. In one embodiment, the recombinant reassortant influenza virus has an amino acid residue at position 105 and / or 401 of PA that confers enhanced growth in cells, e.g., MDCK cells, as compared to the corresponding virus having phenylalanine or arginine at positions 105 or 401 of PA, respectively. In one embodiment, the recombinant reassortant influenza virus has an amino acid residue at positions 40, 54, 59, 62, 63, 66(F2), 73(F2), 75, 76, 78, 79, 80, 112, 180, 327, 507, 624, 644, 667, 694, 695, 697, 699, 700, 701, 702, 705, 713, and / or 714 of PB1 that confers enhanced growth in cells, e.g., MDCK cells, as compared to the corresponding virus having methionine, arginine, threonine, glycine, alanine, asparagine, lysine, glutamic acid, aspartic acid, glutamic acid, proline, serine, glutamic acid, glycine, isoleucine, methionine, leucine, valine, isoleucine, asparagine, leucine, glutamic acid, phenylalanine, phenylalanine, proline, serine, tyrosine, serine or methionine at each of these positions, respectively.In one embodiment, the recombinant reassortant influenza virus has amino acid residues at positions 57, 58, 59, 61, 66, 202, 323, 368, 391, 504, 591, 677, 678, or 679 of PB2 that promote growth in cells, such as MDCK cells, compared to the corresponding virus having isoleucine, threonine, alanine, lysine, methionine, methionine, phenylalanine, arginine, glutamic acid, isoleucine, glutamine, glutamic acid, aspartic acid, or phenylalanine, respectively, at these positions. In one embodiment, the recombinant reassortant influenza virus has amino acid residues at positions 116, 224, 293, 371, 417, 422, or 442 of NP that promote growth in cells, such as MDCK cells, compared to the corresponding virus having leucine, asparagine, arginine, methionine, aspartic acid, arginine, or threonine, respectively, at these positions. In one embodiment, the recombinant reassortant influenza virus has an amino acid residue at position 90 of M1 that promotes growth in cells compared to the corresponding virus having serine at position 90 of M1. In one embodiment, the recombinant reassortant influenza virus has amino acid residues at positions 30, 49, 140, 161, or 223 of NS1 that promote growth in MDCK cells compared to the corresponding virus having proline, alanine, glutamine, threonine, or glutamic acid, respectively, at these positions. In one embodiment, the recombinant reassortant influenza virus does not have a valine residue at position 504 of PB2 and does not have a leucine residue at position 550 of PA.

[0010] In one embodiment, the influenza virus of the present invention has specific amino acid residues at one, two, three or more specific positions of PA, PB1, PB2, NP, M1, and / or NS1, and other than K142, S225, K356 or I550 of PA; other than E112, Q247, M507 or V644 of PB1; other than M202, F323 or I504 of PB2; other than R74, I112, I116, T442, or N417 of NP; other than V97 and / or Y100 of M1; and / or other than R140 or K55 of NS, and has at least 80% amino acid sequence identity with the corresponding polypeptide encoded by one of SEQ ID NOs: 1 to 6 or 10 to 15, such as any integer from 80 to 99, including 90%, 92%, 95%, 97%, 98%, or 99%. A recombinant influenza virus having an amino acid sequence. Residues other than the specified residues may be conservative substitutions. Conservative amino acid substitution means the interchangeability between residues having similar side chains. For example, the group of amino acids having aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine, the group of amino acids having aliphatic hydroxyl side chains are serine and threonine, the group of amino acids having amide-containing side chains are asparagine and glutamine, the group of amino acids having aromatic side chains are phenylalanine, tyrosine and tryptophan, the group of amino acids having basic side chains are lysine, arginine and histidine, and the group of 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, for example, 2, 3 or 4 non-conservative amino acid substitutions compared to the polypeptide encoded by one of SEQ ID NOs: 1 to 6 or 10 to 15.

[0011] In one embodiment, the influenza virus of the present invention has specific amino acid residues at one or more specific positions of PA, PB1, PB2, NP, M1, and / or NS1, and is a residue that is a conservative substitution for M202 of PB2, R74 of NP, and / or V97 of M1. It is a recombinant influenza virus having an amino acid sequence having at least 80%, for example, 90%, 92%, 95%, 97%, 98%, or 99% amino acid sequence identity to the corresponding polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15, including any integer from 80 to 99.

[0012] In one embodiment, the influenza virus of the present invention has specific amino acid residues at specific positions of PA, PB1, PB2, NP, M1, and / or NS1, and is, for example, a non-conservative substitution for K142 of PA, Q247 of PB1, M202, F323 or I504 of PB2, R74, I112, I116, J442 or N417 of NP, V97 and / or Y100 of M1, and / or K55 or R140 of NS1. It is a recombinant influenza virus having an amino acid sequence having at least 80%, for example, any integer from 80 to 99, 90%, 92%, 95%, 97%, 98%, or 99% amino acid sequence identity to the corresponding polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15.

[0013] In one embodiment, the influenza virus of the present invention has specific amino acid residues at specific positions of PA, PB1, PB2, NP, M1, and / or NS1, and has at least 80% amino acid sequence identity, for example, any integer from 80 to 99, including 90%, 92%, 95%, 97%, 98%, or 99%, to the corresponding polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15. For example, a PB2 gene segment having a residue that is a conservative substitution of isoleucine other than isoleucine at residue 504; a PB1 gene segment having a non-conservative substitution of E112; a PA gene segment having a substitution of S225; an NP gene segment having conservative substitutions of R74 and N417; an M gene segment having a conservative substitution of V97 and a non-conservative substitution of Y100; and an NS gene segment having a non-conservative substitution of K55. The recombinant influenza virus has these segments.

[0014] In one embodiment, the influenza virus of the present invention has specific amino acid residues at specific positions of PA, PB1, PB2, NP, M1, and / or NS1, and has at least 80% amino acid sequence identity, for example, any integer from 80 to 99, including 90%, 92%, 95%, 97%, 98%, or 99%, to the corresponding polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15. For example, a PB2 gene segment having non-conservative substitutions of M202 and F323; a PB1 gene segment having a non-conservative substitution of Q247; a PA gene segment having a non-conservative substitution of K142; an NP gene segment having a conservative substitution of R74; an M gene segment having a conservative substitution of V97 and a non-conservative substitution of Y100; and an NS gene segment having a conservative substitution of K55E. The recombinant influenza virus has these segments.

[0015] In one embodiment, the influenza virus of the present invention has specific amino acid residues at specific positions of PA, PB1, PB2, NP, M1, and / or NS1, and has at least 80% amino acid sequence identity to the corresponding polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15, for example, any integer from 80 to 99, 90%, 92%, 95%, 97%, 98%, or 99%, such as a PB2 segment having a conservative substitution of I504; a PB1 segment having a conservative substitution of M40L and a non-conservative substitution of G180; a PA segment having a conservative substitution of R401; an NP segment having a conservative substitution of I116; and an NS gene segment having a conservative substitution of A30 or R118, which is a recombinant influenza virus.

[0016] In one embodiment, the influenza virus of the present invention has specific amino acid residues at one or more specific positions of PA, PB1, PB2, NP, M1, and / or NS1, and has at least 80% amino acid sequence identity to the corresponding polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15, for example, including any integer from 80 to 99, 90%, 92%, 95%, 97%, or 99%, which is a recombinant influenza virus having an amino acid sequence having a non-conservative substitution residue such as a residue that is a non-conservative substitution for K142 of PA, Q247 of PB1, F323 of PB2, Y100 of M1, and / or K55 of NS1. In one embodiment, the substituted amino acid residue has an aliphatic side chain, an amide-containing side chain, a basic side chain, or a sulfur-containing side chain, so it is a substitution of an aromatic side chain or an acidic side chain (non-conservative substitution). In one embodiment, the recombinant influenza virus has a residue that is a neutral or positively charged residue substituted with a polar or negatively charged residue. Any combination of selected amino acid residues is also included at the specific positions described herein.

[0017] Gene segments of PA, PB1, PB2, NP, M, and / or NS having residues at specific positions can be used, to provide the reassortant vaccine virus of the present invention, in combination with gene segments of HA, such as H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, or H17, and gene segments of NA, such as N1, N2, N3, N4, N5, N6, N7, N8, N9, or N10, and can be any combination of HA and NA. In one embodiment, HA is H1, H5 or H7. In one embodiment, NA is N1 or N9. In one embodiment, the HA gene segment of the reassortant virus is heterologous to the gene segments of PA, PB1, PB2, NP, M, and NS. In one embodiment, the NA gene segment of the reassortant virus is heterologous to the gene segments of PA, PB1, PB2, NP, M, and NS. In one embodiment, the HA gene segment of the reassortant virus has gene segments of PA, PB1, PB2, NP, M, and NS that are derived from one influenza virus isolate or strain (“parent”), or a variant thereof, for example, having at least 95%, 96%, 97%, 98%, 99%, 99.5% amino acid sequence identity to the sequence of the parent influenza virus isolate or strain, or encoding an influenza virus protein having 1, 2, 5, 10, or 20 substitutions.

[0018] In one embodiment, the parental strain has a gene segment having a sequence corresponding to SEQ ID NOs: 1-6 or 10-15. In one embodiment, the HA gene segment of the reassembled virus is a chimeric HA gene segment, e.g., a chimeric of an HA signal peptide sequence and / or an HA transmembrane domain sequence derived from the HA gene segment of a parental isolate or strain, or a variant thereof, and a heterologous HA ectodomain sequence linked thereto. In one embodiment, the NA gene segment of the isolated recombinant virus is a chimeric NA gene segment, e.g., a chimeric of an NA transmembrane domain sequence derived from the NA gene segment of a parental isolate or strain, or a variant thereof, and / or a stalk sequence derived from a parental isolate or strain, or a variant thereof, and a heterologous NA ectodomain sequence linked thereto. In one embodiment, the NA gene segment of the isolated recombinant virus is a chimeric NA gene, e.g., a chimeric of a heterologous NA ectodomain sequence linked to an NA transmembrane domain sequence derived from the NA gene segment of a parental isolate or strain, or a variant thereof, and / or a stalk sequence derived from a second isolate or strain, or a variant thereof. In one embodiment, the isolated recombinant virus has a heterologous HA gene segment, a heterologous NA gene segment, a chimeric HA gene segment, a chimeric NA gene segment, or any combination thereof. The nucleic acid sequence utilized to prepare the vRNA may introduce residues at specific positions by recombinant methods or may be selected as having residues at specific positions.

[0019] A / Puerto Rico / 8 / 34 (H1N1), "PR8", serves as a genetic backbone for the production of inactivated influenza vaccines. Occasionally, PR8 backbone-based vaccine strains replicate at relatively low titers in eggs and cell cultures, leading to slow vaccine production and vaccine shortages. Various mutagenesis strategies were used to determine whether a high-yield vaccine strain backbone for propagation in MDCK cells, chicken eggs, and Vero cells could be prepared to meet the requirements of seasonal influenza and highly pathogenic pandemic viruses. For example, a PR8 backbone random mutation library was screened for high-replicating mutant strains by introducing random mutations into the internal PR8 genes by error-prone PCR, introducing mutations that confer high replication and high polymerase activity, and optimizing the PR8 internal genes by codon bias. In another approach, the HA gene was optimized to enhance virus replication and HA content, for example, by optimizing the HA promoter to create a strong promoter, optimizing the HA non-coding region, and / or optimizing the HA signal peptide.

[0020] As described herein, influenza virus isolates useful as vaccine viruses carrying heterologous gene segments of NA and / or HA (e.g., A / Puerto Rico / 8 / 34, "PR8", including certain isolates such as UW-PR8) can be serially passaged in MDCK cells, for example, 10 to 12 times, or fewer passages may be utilized to obtain a virus with enhanced replication in those cells. In one embodiment, the virus obtained after serial passage with enhanced replication has a titer at least 1 or 2 logs higher than the virus that was not serially passaged. In one embodiment, the virus obtained after serial passage has substitutions in two or more internal gene segments compared to the parental virus.

[0021] Therefore, when utilized with the HA and NA sequences of interest for cells in culture, such as MDCK or Vero cells or for vaccine viruses to be grown or passaged in ovo, selection of a sequence having the residues disclosed at one or more specific positions of PA, PB1, PB2, NP, M1, and / or NS1 or substitution of the disclosed residues confers growth promotion of the virus in the cultured cells and can result in a significantly higher virus titer. Thereby, the present invention provides a method of selecting influenza viruses having replication promotion in cell culture. The method comprises providing cells suitable for influenza vaccine production; continuously culturing one or more influenza virus isolates in the cells; and isolating the continuously cultured virus having growth promotion as compared to the one or more isolates before continuous culture. In one embodiment, the cells are canine or primate cells, such as human or monkey cells.

[0022] In one embodiment, the influenza virus of the present invention is a recombinant influenza virus having two or more selected amino acid residues at one or more specific positions of PA, PB1, PB2, NP, M1, and / or NS1 that can be used together with the HA and NA genes of interest. In one embodiment, the recombinant reassortant influenza virus has asparagine or glutamine at position 142 of PA, cysteine at position 225, arginine or histidine at position 356 of PA, or leucine, valine, threonine, or glycine at position 550 of PA; histidine, arginine, or lysine at position 247 of PB1, valine, leucine, isoleucine, threonine, alanine, or glycine at position 507 of PB1, and / or alanine, glycine, leucine, or isoleucine at position 644 of PB1; leucine, alanine, valine, isoleucine, glycine, or threonine at position 202 and / or 323 of PB2, or valine, leucine, glycine, threonine, or alanine at position 504 of PB2; lysine or histidine at position 74 of NP, leucine, valine, glycine, or alanine at positions 112, 116, or 442 of NP; leucine, isoleucine, alanine, glycine, or threonine at position 97 of M1, and / or lysine, arginine, or histidine at position 100 of M1; or asparagine, aspartic acid, glutamic acid, or glutamine at position 55 of NS1, or glutamine or asparagine at position 140 of NS1.

[0023] The present invention provides a plurality of influenza virus vectors of the present invention that are useful for the preparation of reassortant viruses, including, for example, 6:1:1 reassortants, 6:2 reassortants, and 7:1 reassortants. A 6:1:1 reassortant within the scope of the present invention is an influenza virus having six internal gene segments from a vaccine virus, an NA gene segment from a different (second) virus isolate, and an HA gene segment from a third isolate. A 6:2 reassortant within the scope of the present invention is an influenza virus having six internal gene segments from a vaccine virus, and an NA gene segment and an HA gene segment from a different (second) virus isolate; and a 7:1 reassortant within the scope of the present invention is an influenza virus having six internal gene segments, an NA gene segment from a vaccine virus, and an HA gene segment from a virus origin different from the vaccine virus, or an influenza virus having six internal gene segments and an HA gene segment from a vaccine virus, wherein the NA gene segment is from a virus origin different from the vaccine virus.

[0024] In one embodiment of the present invention, a plurality thereof includes vectors containing a promoter operably linked to influenza virus PA DNA bound to a transcription termination sequence, a vector containing a promoter operably linked to influenza virus PB1 DNA bound to a transcription termination sequence, a vector containing a promoter operably linked to influenza virus PB2 DNA bound to a transcription termination sequence, a vector containing a promoter operably linked to influenza virus HA DNA bound to a transcription termination sequence, a vector containing a promoter operably linked to influenza virus NP DNA bound to a transcription termination sequence, a vector containing a promoter operably linked to influenza virus NA DNA bound to a transcription termination sequence, a vector containing a promoter operably linked to influenza virus M DNA bound to a transcription termination sequence, and a vector containing a promoter operably linked to influenza virus NS DNA bound to a transcription termination sequence, and includes vectors for vRNA production selected therefrom. In one embodiment, the DNA for vRNA production of PB1, PB2, PA, NP, M, and NS replicates at high titers in cultured mammalian cells such as, for example, MDCK cells, Vero cells or PER.C6® cells, or optionally in embryonated eggs, and / or has sequences derived from a vaccine virus that does not cause severe disease in humans. The DNA for vRNA production of NA can be derived from any NA, for example, any of N1 to N10, and the DNA for vRNA production of HA can be derived from any HA, for example, any of H1 to H17. In one embodiment, the DNA for vRNA production can be for influenza B or C virus. The DNA for vRNA production of NA and HA can be derived from different strains or different isolates (6: 1: 1 reassortant) or the same strain or the same isolate (6: 2 reassortant), or NA can be derived from the same strain or the same isolate as the internal gene (7: 1 reassortant).Also included are vectors for mRNA production selected from among a number of vectors encoding influenza virus PA, vectors encoding influenza virus PB1, vectors encoding influenza virus PB2, vectors encoding influenza virus NP, and optionally one or more vectors encoding NP, NS, M, such as M1 and M2, HA or NA. Vectors encoding viral proteins can further include transcription termination sequences.

[0025] Viruses that can provide internal genes for reassortants within the scope of the present invention have a high titer in MDCK cells, for example, at least about 10 5 PFU / mL, for example, at least 10 6 PFU / mL, 10 7 PFU / mL or 10 8 PFU / mL titer; a high titer in embryonated eggs, for example, at least about 10 7 EID 50 / mL, for example, at least 10 8 EID 50 / mL, 10 9 EID 50 / mL or 10 10 EID 50 / mL titer; a high titer in cells such as MDCK cells, for example, at least about 10 7 PFU / mL, for example, at least 10 8 PFU / mL titer, or viruses having a high titer in two or more of these host cells.

[0026] In one embodiment, the titer of the reassortant virus of the present invention in cells such as MDCK cells or Vero cells can be 1 log, 2 logs, 3 logs, or more than the titer of the corresponding virus lacking specific residues at specific positions.

[0027] Other reassortants having internal genes from other PR8 isolates or vaccine viruses are available for use in the recombinant reassortant viruses of the present invention. In particular, 5:1:2 reassortants having UW-PR8 PB1, PB2, PA, NP, and M ("5") and PR8(Cam) NS ("1"); 6:1:1 reassortants having UW-PR8 NA, PB1, PB2, PA, NP, and M ("6") and PR8(Cam) NS ("1"); and 7:1 reassortants having UW-PR8 PB1, PB2, PA, NP, M, NA, and NS ("7") are available.

[0028] In one embodiment, the DNA related to the internal genes of PB1, PB2, PA, NP, M, and NS encodes a protein having substantially the same activity as the corresponding polypeptide encoded by one of SEQ ID NOs: 1 to 6 or 10 to 15. As used herein, "substantially the same activity" means, for each of the activity or protein level, about 0.1%, 1%, 10%, 30%, 50%, 90%, for example, 100% or more of the activity of the corresponding full-length polypeptide, or about 80%, 90% or more of the detectable protein level. In one embodiment, the nucleic acid sequence is substantially identical to a polypeptide having a continuous amino acid sequence identity of, for example, any integer% from 80 to 99, at least 80%, for example, 90%, 92%, 95%, 97%, 98% or 99% with the polypeptide encoded by one of SEQ ID NOs: 1 to 6 or 10 to 15, and encodes a polypeptide. In one embodiment, the isolated and / or purified nucleic acid molecule comprises a nucleotide sequence that is substantially identical to having a continuous nucleic acid sequence identity of, for example, any integer% from 50 to 100, at least 50%, for example, 60%, 70%, 80% or 90%, or more with one of SEQ ID NOs: 1 to 6 or 33 - 38.In one embodiment, the influenza virus polypeptide has one or more, for example, 2, 5, 10, 15, 20 or more conservative amino acid substitutions, for example, a combination of conservative and non-conservative amino acid substitutions, 2, 5, 10, 15, 20 or more up to a maximum of 10% or 20% conservative substitutions, for example, having up to a maximum of 10% or 20% conservative substitutions of residues compared to the polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15, having characteristic residues in two or more of PA, PB1, PB2, NP, M1, and / or NS1, and having characteristic residues in two or more of the gene segments of PA, PB1, PB2, NP, M1, and / or NS1, for example, asparagine or glutamine at position 142 of PA; histidine, arginine or lysine at position 247 of PB1; leucine, alanine, valine, isoleucine, glycine, or serine at positions 202 and / or 323 of PB2; lysine or histidine at position 74 of NP; leucine, isoleucine, alanine, glycine, or serine at position 202 and / or lysine, arginine, or histidine at position 100 of M1; or asparagine, aspartic acid, glutamic acid or glutamine at position 44 of NS1. In one embodiment, the influenza virus polypeptide has one or more, for example, 2, 3, 4, 5, 6, 7 or 8 conservative amino acid substitutions and / or non-conservative amino acid substitutions compared to the polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15, for example, of the virus isolates of 1, 4, 36, 38, P17, P25 or P61 in Table 4.

[0029] Accordingly, the present invention includes the use of an isolated and purified vector or plasmid that expresses or encodes a protein of an influenza virus, or that expresses or encodes both influenza vRNA, native and recombinant vRNA. The vector includes influenza cDNA, for example, influenza A (any influenza A gene including, for example, any of the 16 HA or 9 NA subtypes), B or C DNA (see Fields Virology (Fields et al. (eds.), Lippincott, Williams and Wickens (2006), which are hereby incorporated by reference in their entirety). Any suitable promoter or transcription termination sequence can be utilized to express a protein or peptide, for example, a viral protein or peptide, a protein or peptide of a non-viral pathogen, or a therapeutic protein or peptide).

[0030] The vector composition of the present invention or a plurality of vectors can also include, for example, a heterologous gene or a translation region that can encode an immunogenic peptide or protein useful as a vaccine or in gene replacement, such as an epitope useful in cancer treatment or a 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 related to an influenza virus gene, or added to a vector or plasmid related to all influenza virus genes. Accordingly, another embodiment of the present invention is a composition of the above vectors in which one of the vectors is replaced or a plurality of such vectors, further comprising a 5' influenza virus sequence. Optionally, the 5' influenza virus sequence is bound to a nucleic acid sequence of interest, for example, a cDNA of interest, optionally bound to a 3' influenza virus sequence or a portion thereof containing a 3' influenza virus coding sequence, and contains a 5' influenza virus coding sequence or a portion thereof. In one embodiment, the nucleic acid sequence of interest, such as cDNA, is in the antisense (antigenomic) orientation. Introduction of these vectors, in combination with the other vectors described above, into a host cell permissive for influenza virus replication results in a recombinant virus containing vRNA corresponding to the heterologous sequences of such vectors.

[0031] The promoter in the vector for vRNA production can be an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T7 promoter, or a T3 promoter. Optionally, the vector includes a transcription termination sequence such as, for example, 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, ribonuclease 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.

[0032] The promoter or transcription termination sequence in the vRNA or viral protein expression vector can be the same as or different from the promoter or any other vector. In one embodiment, the vector or plasmid that expresses influenza vRNA includes a promoter suitable for expression in at least one specific host cell, such as a bird, or a mammalian host cell, such as a primate cell including, for example, dog, cat, horse, cow, or human cells, or expression in one or more hosts.

[0033] In one embodiment, at least one vector for vRNA comprises an RNA polymerase II promoter optionally linked to an RNA polymerase II transcription termination sequence, linked to a ribozyme sequence linked to a viral coding sequence linked to another ribozyme sequence. In one embodiment, at least 2, such as 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 comprising a viral coding sequence, 5' to a second ribozyme sequence, and 5' to a transcription termination sequence. Each RNA polymerase II promoter in each vRNA vector can be the same as or different from the RNA polymerase II promoter in any other vRNA vector. Similarly, each ribozyme sequence in each vRNA vector can be the same as or different from the ribozyme sequence in any other vRNA vector. In one embodiment, the ribozyme sequences in a single vector are not identical.

[0034] In one embodiment, the present invention provides a plurality of influenza virus vectors for a reassembly, such vectors including 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 influenza virus NS linked to a transcription termination sequenceA vector for vRNA production comprising a promoter operably linked to cDNA, wherein the DNAs of PB1, PB2, PA, NP, NS, and M are derived from one or more influenza vaccine seed viruses and contain two or more characteristic residues at specific positions; a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA, 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, 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, at least one vector comprises a sequence corresponding to a sequence encoding PB1, PB2, PA, NP, M, or NS, or a portion thereof, and has substantially the same activity as the corresponding polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15. For example, it comprises a sequence encoding a polypeptide having 80-100% amino acid identity, at least 80% amino acid identity, such as 85%, 90%, 92%, 95%, 98%, 99% or 100% amino acid identity, with the polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15. Optionally, influenza virus M bound to a transcription termination sequenceInstead of a vector containing a promoter operably linked to cDNA, for example, two vectors such as a vector containing a promoter operably linked to influenza virus M1 cDNA linked to a transcription termination sequence, and a vector containing a promoter operably linked to influenza virus M2 cDNA linked to a transcription termination sequence are available.

[0035] A number of vectors of the present invention can be physically bound, or each vector can be present on an individual plasmid or other, for example, linear, nucleic acid delivery vehicle. In one embodiment, each vRNA production vector is on a separate plasmid. In one embodiment, each mRNA production vector is on a separate plasmid.

[0036] The present invention also provides a method for preparing influenza virus. Such a method includes, for example, contacting a cell with a number of vectors of the present invention in an amount effective to obtain an infectious influenza virus, either continuously or simultaneously. The present invention also includes the step of isolating the virus from the cells bound to a number of vectors. Accordingly, the present invention further provides an isolated virus and host cells bound to a number of vectors or viruses of the present invention. In other embodiments, the present invention includes the step of contacting a cell with one or more vectors, vRNAs or protein production vectors prior to other vectors, vRNAs or protein production vectors. 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 present on a separate plasmid. In one embodiment, the vRNA vectors utilized in the method are present on one plasmid or two or three different plasmids. In one embodiment, each mRNA vector utilized in the method is present on a separate plasmid. In one embodiment, the mRNA vectors for PA, PB1, PB2 and NP utilized in the method are present on one plasmid or two or three different plasmids.

[0037] In one embodiment, the present invention provides a method for selecting an influenza virus having enhanced replication in cell culture. Such a method includes providing a cell suitable for influenza vaccine production; continuously culturing one or more influenza virus isolates in said cell; and isolating the continuously cultured virus having enhanced growth compared to one or more isolates prior to continuous culture. In one embodiment, the cell is a rodent or primate cell.

[0038] The method for producing a virus that does not require helper virus infection described in this specification is useful in virus mutagenicity testing, production of vaccines (e.g., for AIDS, influenza, hepatitis B, hepatitis C, rhinovirus, filovirus, malaria, herpes, and foot and mouth diseases), and production of gene therapy vectors (e.g., for cancer, AIDS, adenosine deaminase, muscular dystrophy, ornithine transcarbamylase deficiency, and central nervous system tumors). Accordingly, it provides a virus for use in medical therapy (e.g., vaccine or gene therapy).

[0039] The present invention also provides an isolated viral polypeptide, and methods for preparing and using the recombinant virus of the present invention. Such methods include administering to a host organism, e.g., a mammal, an effective amount of the influenza virus of the present invention, e.g., an inactivated virus preparation, optionally in combination with an adjuvant and / or a carrier, in an amount effective to prevent or alleviate infection of an animal, e.g., a mammal, by the virus or a virus closely related antigenically thereto. In one embodiment, the virus is administered intramuscularly, while in other embodiments, the virus is administered intranasally. In some dosing protocols, all doses can be administered intramuscularly or intranasally, while in other protocols, a combination of intramuscular and intranasal administration can be utilized. The vaccine can further comprise, for example, other isolates of influenza virus, other pathogens, additional biological substances or microbial components from other organisms, including recombinant influenza virus, to form, for example, a multivalent vaccine. In one embodiment, for example, intranasal vaccination with an inactivated influenza virus and a mucosal adjuvant can induce virus-specific IgA and neutralizing antibodies in the nasopharynx, as well as serum IgG.

[0040] The influenza virus of the present invention can be used in combination with other antiviral agents, e.g., amantadine, rimantadine, and / or neuraminidase inhibitors, and can be administered separately, for example, in combination with those antiviral agents, before, during, and / or after administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0041]

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Mode for Carrying Out the Invention

[0042] Detailed Description of the Invention Definitions As used herein, the term "isolated" means the in vitro preparation and / or isolation of a nucleic acid molecule of the present invention, such as a vector or plasmid, peptide or polypeptide (protein), or virus, and thus is not associated with in vivo substances or is substantially purified from in vitro substances. The preparation of an isolated virus is usually obtained by in vitro culture and propagation and / or through passage in eggs and is substantially free of other infectious pathogens.

[0043] As used herein, "substantially purified" means that the species of interest is the dominant species in the composition, for example, on a molar basis, more abundant than any other individual species, preferably at least about 80%, optionally 90% or more, for example, 95%, 98%, 99% or more of the species present in the composition.

[0044] As used herein, "substantially free of" means below the detection level for a particular infectious pathogen using standard detection methods for that pathogen.

[0045] A "recombinant" virus is a virus that has been manipulated in vitro, for example, using recombinant DNA technology, to introduce changes into the viral genome. Reassortant viruses can be prepared by recombinant or non-recombinant techniques.

[0046] As used herein, the term "recombinant nucleic acid" or "recombinant DNA sequence or segment" refers to a nucleic acid that is derived from or isolated from a particular origin and can then be chemically altered in vitro, e.g., DNA, the sequence of which is not of natural origin or corresponds to a sequence of natural origin that does not exist in the native genome. Examples of DNA "derived from" a particular origin can be recognized as useful fragments and can then be chemically synthesized in essentially pure form. Examples of such DNA "isolated" from a particular origin can be useful DNA sequences excised or removed from such origin by chemical means, e.g., the use of restriction enzymes, and can be further manipulated, e.g., amplified, by genetic engineering methods for use in the present invention.

[0047] As used herein, a "heterologous" influenza virus gene or gene segment is one that is derived from an influenza virus source that is different from most other influenza virus genes or gene segments in a recombinant, e.g., reassortant, influenza virus.

[0048] The terms "isolated polypeptide," "isolated peptide," or "isolated protein" include polypeptides, peptides, or proteins encoded by cDNA or recombinant RNA, including one of synthetic origin, or combinations thereof.

[0049] As used herein, the term "recombinant protein" or "recombinant polypeptide" means a protein molecule expressed from a recombinant DNA molecule. In contrast, the term "native protein" is used herein to refer to a protein isolated from a natural (i.e., non-recombinant) origin. Molecular biological techniques can be used to produce recombinant forms of proteins that have the same properties as the native form of the protein.

[0050] Methods for sequence comparison are well known in the art. Thus, determination of the percent identity between any two sequences can be accomplished using mathematical algorithms.

[0051] The computer execution of these mathematical algorithms can be utilized for array comparison that measures array identity. Array comparison using these programs can be performed using default parameters. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves the initial identification of high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, such identification determining whether or not the word in the database sequence aligns with a word of the same length and matches or meets a positive-valued threshold T. T is referred to as the neighborhood word score threshold. These initial neighboring word hits function as seeds to initiate a search for longer HSPs that contain them. Subsequently, word hits are extended in both directions along each sequence as long as the cumulative array comparison score can be increased. The cumulative score is calculated for nucleotide sequences using parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for a mismatched residue; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of word hits in each direction stops when the cumulative array comparison score becomes zero or less due to the accumulation of one or more negative-scoring residue sequence comparisons such that the cumulative score decreases from its maximum achieved value by an amount X, or when the end of one of the sequences is reached.

[0052] Furthermore, for calculating percent sequence identity, the BLAST algorithm can also perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm can be the minimum total probability (P(N)) that a match between two nucleotide or amino acid sequences could occur by chance. For example, when the minimum total probability of comparison of a test nucleic acid sequence to a 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, the test nucleic acid sequence is considered to be similar to the reference sequence.

[0053] The BLASTN program (for nucleotide sequences) can use, as defaults, an 11 wordlength (W), an expectation value (E) of 10, a cutoff of 100, M = 5, N = 4, and comparison of both strands. For amino acid sequences, the BLASTP program can use, as defaults, a 3 wordlength (W), an expectation value (E) of 10, and the BLOSUM62 scoring matrix. See http: / / www.ncbi.nlm.nih.gov. Sequence comparisons can also be performed manually by observation.

[0054] For sequence comparison, typically one sequence functions as the reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. Subsequently, the sequence comparison algorithm calculates the percent sequence identity for the test sequence compared to the reference sequence based on the designated program parameters.

[0055] Structure and Propagation of Influenza Virus Influenza A virus has a genome of eight single-stranded negative-sense viral RNAs (vRNAs) that encode at least 10 proteins. The life cycle of the influenza virus begins with the binding of hemagglutinin (HA) to sialic acid-containing receptors on the surface of host cells, followed by receptor-mediated endocytosis. The low pH in the late endosome causes a conformational change in HA, resulting in the exposure of the N-terminus of the HA2 subunit (the so-called fusion peptide). The fusion peptide induces the fusion of the viral and endosomal membranes, releasing the matrix protein (M1) and the RNP complex into the cytoplasm. The RNP consists of the nucleoprotein (NP) that capsidizes the vRNA and the viral polymerase complex formed by the PA, PB1, and PB2 proteins. The RNP is transported into the nucleus where transcription and replication occur. The RNA polymerase complex catalyzes three different reactions: the synthesis of mRNA with a 5' cap and a 3' polyA structure, the synthesis of full-length complementary RNA (cRNA), and the synthesis of genomic vRNA using cRNA as a template. The newly synthesized vRNA, NP, and polymerase proteins are subsequently assembled into RNP, exported from the nucleus, transported to the cell membrane where budding of progeny virus particles occurs. The neuraminidase (NA) protein plays an important role at the end stage of infection by removing sialic acid from sialyl oligosaccharides, thereby releasing newly assembled virus particles from the cell surface and preventing self-aggregation of virus particles. Virus assembly involves protein-protein and protein-vRNA interactions, but the nature of these interactions is largely unknown.

[0056] Influenza B and C viruses are structurally and functionally similar to influenza A virus, but there are some differences. For example, influenza B virus does not have an M2 protein with ion channel activity, but has BM2 and has a gene segment with both NA and NB sequences. Influenza C virus has only seven gene segments.

[0057] Cell lines that can be used in the present invention For example, any mutant cells, such as any avian cells, or mammalian cells such as human cells, for example, 293T or PER.C6® cells, or canine cells such as MDCK, bovine, equine, feline, porcine, ovine, rodent cells such as mink cells, for example, MvLu1 cells, or hamster cells such as CHO cells, or non-human primate cells such as Vero cells, etc., which assist in the efficient replication of influenza virus, can be used to isolate and / or propagate influenza virus. The isolated virus can be used to prepare reassortant viruses. In one embodiment, the host cells for vaccine production are maintained mammalian or avian established cells or cell lines. The complete characterization of the cells used can be carried out such that it is possible to include appropriate tests regarding the purity of the final product. Data that can be used for cell characterization includes: (a) information regarding its origin, derivation, and passage history; (b) information regarding its growth and morphological characteristics; (c) test results for adventitious agents; (d) distinct characteristics such as biochemical, immunological, and cytogenetic patterns that can be clearly recognized among other established cells; and (e) test results regarding tumorigenicity. In one embodiment, the passage level or population doublings of the host cells used are kept as low as possible.

[0058] In one embodiment, the cells are continuously established cells that are WHO-approved or can be approved. The conditions required for the authentication of such established cells include characterization regarding at least one of lineage, growth characteristics, immune markers, virus susceptibility, tumorigenicity, and storage conditions, and tests in animals, eggs, and cell culture. Such characterization is used to confirm that the cells do not contain detectable adventitious agents. In some countries, karyology may also be required. Furthermore, tumorigenicity can be tested in cells at the same passage level as those used for vaccine production. The virus can be purified by a process that has been shown to yield consistent results prior to vaccine production (see, for example, World Health Organization, 1982).

[0059] Viruses produced by host cells can be highly purified prior to formulation into vaccines or gene therapies. Generally, purification methods result in extensive removal of cellular DNA and other cellular components, as well as adventitious agents. Methods that extensively degrade or denature DNA can also be used.

[0060] Influenza vaccine The vaccines of the invention include the isolated recombinant influenza virus of the invention, and optionally one or more other isolated viruses including one or more other isolated influenza viruses, one or more isolated influenza viruses or one or more other pathogens, such as one or more immunogenic proteins or glycoproteins from one or more bacteria, non-influenza viruses, yeast or fungi, or an isolated nucleic acid encoding one or more viral proteins including one or more immunogenic proteins of the isolated influenza virus of the invention (e.g., a DNA vaccine). In one embodiment, the influenza virus of the invention can be a vaccine vector for influenza virus or other pathogens.

[0061] Whole virus particle vaccines can be concentrated by ultrafiltration and subsequently purified by zone centrifugation or chromatography. Viruses other than the virus of the invention, such as those included in a multivalent vaccine, can be inactivated, for example, using formalin or beta-propiolactone, before or after purification.

[0062] Subunit vaccines contain purified glycoproteins. Such vaccines can be prepared as follows: Using a virus suspension fragmented by treatment with a surfactant, for example, surface antigens are purified by ultracentrifugation. Thus, subunit vaccines mainly contain HA protein and also contain NA. The surfactant used can be, for example, a cationic surfactant such as hexadecyltrimethylammonium bromide (Bachmeyer, 1975), an anionic surfactant such as ammonium deoxycholate (Laver & Webster, 1976), or a nonionic surfactant such as the commercially available Triton X100. Hemagglutinin can also be isolated and subsequently purified with a protease such as bromelin after treatment of virus particles. Subunit vaccines can also be combined with the attenuated virus of the present invention in a multivalent vaccine.

[0063] Split vaccines contain virus particles that have been subjected to treatment with a lipid-dissolving agent. Split vaccines can be prepared as follows: The above-mentioned aqueous suspension of the obtained purified virus, whether inactivated or not, is combined with a surfactant and treated under stirring with a lipid solvent such as ethyl ether or chloroform. The dissolution of the viral envelope lipid results in fragmentation of the virus particles. The extracted aqueous phase contains a split vaccine mainly composed of hemagglutinin and neuraminidase, together with the removed lipid environment of viral origin and the core or its degradation products. Subsequently, the residual infectious particles are inactivated if they have not yet been inactivated. Split vaccines can be combined with the attenuated virus of the present invention in a multivalent vaccine.

[0064] Inactivated vaccine Inactivated influenza virus vaccines are provided by inactivation of the replicated virus using well-known methods such as, but not limited to, formalin or β-propiolactone treatment. Inactivated vaccine types that can be used in the present invention can include whole virus (WV) vaccines or subvillon particle (SV) (split) vaccines. WV vaccines contain untreated inactivated virus, while SV vaccines contain purified virus that has been disrupted by solubilization of the lipid-containing viral envelope with a surfactant followed by chemical inactivation of the residual virus.

[0065] Furthermore, vaccines that can be used include vaccines containing isolated HA and NA surface proteins, which are referred to as surface antigen or subunit vaccines.

[0066] Live attenuated influenza virus vaccine For example, live attenuated influenza virus vaccines, such as vaccines containing the recombinant virus of the present invention, can be used to prevent or treat influenza virus infection. Attenuation can be achieved in one step according to well-known methods by transfer of an attenuation gene derived from an attenuated donor virus to a replication isolate or reassorted virus. Resistance to influenza A virus is first mediated by the development of an immune response to the HA and / or NA glycoproteins, so the genes encoding these surface antibodies are derived from reassorted viruses or clinical isolates. The attenuation gene is derived from the attenuated parent. In this approach, the gene conferring attenuation usually does not encode HA and NA glycoproteins.

[0067] A virus (donor influenza virus) that can reproducibly attenuate influenza virus can be used. For example, a (ca) donor virus acclimated to low temperature can be used for the production of an attenuated vaccine. A live, attenuated reassortant virus vaccine can be produced by conjugating the ca donor virus with a pathogenic replicating virus. The progeny of the reassortant are subsequently selected at 25°C (where replication of the pathogenic virus is restricted) in the presence of a suitable antiserum, which inhibits the replication of viruses having surface antibodies of the attenuated ca donor virus. 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 the ca reassortants is comparable to their replication levels. Thus, the acquisition of six transmissible genes of the ca donor virus by a novel wild - type virus has reproducibly attenuated these viruses for use in vaccination of both adult and non - adult susceptible mammals.

[0068] Other attenuating mutations can be introduced into the influenza virus gene by site - directed mutagenesis and infectious viruses having these mutant genes can be rescued. The attenuating mutations can be introduced into the untranslated regions and the translated regions of the genome. These attenuating mutations can also be introduced into genes other than HA or NA, for example, into the PB2 polymerase gene. Thus, a new donor virus having attenuating mutations introduced by site - directed mutagenesis can also be produced and, in a manner similar to the above - described method for the ca donor virus, such a potential new donor virus can be used in the production of an attenuated reassortant vaccine. Similarly, other well - known and suitable attenuated donor strains can be reassorted with influenza virus to obtain an attenuated vaccine suitable for use in vaccination of mammals.

[0069] In one embodiment, such an attenuated virus maintains a gene derived from a virus encoding antigenic determinants substantially similar to those of the original clinical isolate. This is because the attenuated vaccine provides substantially the same antigenicity as the original clinical isolate of the virus while at the same time lacking pathogenicity to the extent that it minimizes the possibility of inducing a severe disease state of the vaccine in the vaccinated mammal.

[0070] Thus, the virus in the multivalent vaccine can be attenuated or inactivated, formulated and administered according to methods well known as vaccines, in order to introduce an immune response in animals, such as mammals. Methods for measuring whether such an attenuated or inactivated vaccine retains antigenicity similar to that of a clinical isolate or a fast-growing strain derived therefrom are well known in the art. Such well-known methods include the use of antiserum or antibodies to remove 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 (e.g., probe hybridization or PCR, etc.) to confirm that the donor gene encoding the antigenic determinant (e.g., the HA or NA gene) is not present in the attenuated virus.

[0071] Pharmaceutical composition The pharmaceutical composition of the present invention suitable for inoculation, such as intranasal, parenteral or oral administration, comprises one or more influenza virus isolates, such as one or more attenuated or inactivated influenza viruses, their subunits, their isolated proteins, and / or one or more isolated nucleic acids encoding such proteins, which optionally further comprise sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Such compositions can further comprise adjuvants or excipients well known in the art. Such compositions of the present invention are generally presented in individual dosage forms (unit doses).

[0072] Conventional vaccines generally contain from about 0.1 to 200 μg, for example, 30 to 100 μg of HA from each strain added to their compositions. The vaccines that form the main component of the vaccine composition of the present invention can include a combination of influenza viruses, such as one influenza virus, or one or more reassortants, for example, at least 2 or 3 influenza viruses.

[0073] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and / or emulsions, and can contain adjuvants or excipients well known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate that are injectable. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration can generally include liposome solutions containing the liquid dosage form. Forms suitable for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art such as purified water. In addition to such inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or aromatic agents.

[0074] When the composition of the present invention is used for administration to an individual, it can further contain salts, buffers, adjuvants, or other substances desirable for improving the effectiveness of the composition. For vaccines, adjuvants, substances that can enhance the specific immune response, can be used. Usually, the adjuvant and the composition are mixed before presentation to the immune system or presented separately in the same site of the immunized organism.

[0075] The heterogenicity of the vaccine can be provided by mixing at least two influenza virus strains, such as from 2 to 20 strains or any range or value thereof, of the replicated influenza virus. The vaccine can be subjected to variations in a single strain of influenza virus using techniques well known in the art.

[0076] The pharmaceutical composition of the present invention can further contain or additionally contain at least one, for example, a chemotherapeutic compound for gene therapy, an immunosuppressant, an anti-inflammatory agent or an immunopotentiator, and a chemotherapeutic agent for vaccines, including but not limited to gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon-α, interferon-β, interferon-γ, tumor necrosis factor alpha, thiosemicarbazone, methisazone, rifampin, ribavirin, pyrimidine analogs, purine analogs, foscarnet, phosphonoacetic acid, acyclovir, dideoxynucleosides, protease inhibitors, or ganciclovir.

[0077] Such a composition can also contain a variable but small amount of formaldehyde free of endotoxin and a preservative, which has been found to be safe in the organism to which the composition is administered and not to contribute to undesirable effects.

[0078] Medical purposes The administration of such a composition (or the antiserum it induces) can be for "prophylactic" or "therapeutic" purposes. When used prophylactically, such a composition of the present invention that is a vaccine is provided before any symptoms or clinical signs of pathogen infection become apparent. Prophylactic administration of such a composition helps prevent or attenuate subsequent infections. When used prophylactically, the gene therapy composition of the present invention is provided before any symptoms or clinical signs of the disease become apparent. Prophylactic administration of such a composition helps prevent or attenuate one or more symptoms or clinical signs associated with the disease.

[0079] When provided therapeutically, the viral vaccine serves for the detection of the symptoms or clinical signs of an actual infection. The therapeutic administration of the compound serves to attenuate an actual infection. When provided therapeutically, the gene therapy composition serves for the detection of the symptoms or clinical signs of a disease. The therapeutic administration of the compound serves to attenuate the symptoms or clinical signs of that disease.

[0080] Accordingly, the vaccine composition of the present invention can be provided before the occurrence of an infection (to prevent or attenuate a predicted infection) or after the induction of an actual infection. Similarly, such a composition can be provided for gene therapy either before the manifestation of any symptoms or clinical signs of a disease or disorder or after one or more symptoms have been detected.

[0081] A composition can be “pharmaceutically acceptable” if the recipient mammal can tolerate its administration. Such an agent is administered in a “therapeutically effective amount” if the amount administered is physiologically significant. The composition of the present invention is physiologically effective if its presence results in a detectable change in the physiological function of the recipient patient, for example, by enhancing at least one primary or secondary humoral or cellular immune response against at least one strain of an infectious influenza virus.

[0082] The “prevention” provided need not be absolute, i.e., there need only be a statistically significant improvement as compared to a control group or population of mammals, and it is not necessary for influenza infection to be completely prevented or eradicated. Prevention can be limited to reducing the severity or rate of occurrence of the symptoms or clinical signs of influenza virus infection.

[0083] Pharmaceutical administration The composition of the present invention can confer resistance to one or more pathogens, such as one or more influenza virus strains, 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 the administration prevents infection and / or disease. With respect to passive immunization, the induced antiserum can be recovered and administered to a recipient suspected of having an infection caused by at least one influenza virus strain. The gene therapy composition of the present invention can obtain a gene product of interest at a prophylactic or therapeutic level by active immunization.

[0084] In one embodiment, the vaccine is provided to female mammals (during or prior to pregnancy or parturition) under conditions of time and amount sufficient to generate an immune response, thereby serving to protect both the female and the fetus or neonate (either through passive uptake of antibodies across the placenta or via breast milk).

[0085] Accordingly, the present invention includes methods for preventing or reducing a disorder or disease, such as an infection by at least one strain of a pathogen. As used herein, a vaccine prevents or reduces a disease if its administration results in the total or partial alleviation (i.e., suppression) of the clinical signs or condition of the disease, or the total or partial immunization of an individual against the disease. As used herein, a gene therapy composition prevents or reduces a disease if its administration results in the total or partial alleviation (i.e., suppression) of the clinical signs or condition of the disease, or the total or partial immunization of an individual against the disease.

[0086] A composition of the present invention having at least one influenza virus, including an attenuated virus 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 of those viral proteins, or combinations thereof, can be administered by any means for achieving the purpose.

[0087] For example, administration of these compositions can be carried out by various parenteral routes such as, for example, subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral or transdermal routes. Parenteral administration can be achieved by rapid intravenous injection or by infusion over time.

[0088] A typical dosing regimen for preventing, suppressing, or treating influenza virus-related symptoms includes administration of an effective amount of the vaccine composition of the present specification, administered as a single treatment, or repeated as a booster or additional dose, for a period of 1 week to about 24 months, or any range or value therein, and during that period.

[0089] According to the present invention, an "effective amount" of a composition is an amount sufficient to achieve the desired effect. It is understood that the effective dose can depend on the species, age, sex, health, and weight of the recipient, the type of co-treatment if any, the frequency of treatment, and the nature of the desired effect. The ranges of effective amounts provided below are not intended to limit the dosage ranges of the present invention.

[0090] For example, the dosage of a live attenuated or inactivated virus vaccine for an animal such as an adult mammalian organism can be about 10 2 ~10 15 , for example, 10 3 ~10 12 plaque forming units (PFU) / kg, or any range therein. The dosage of an inactivated vaccine can be about 0.1 - 1000, for example, 30 - 100 μg of HA protein. However, the dosage should be a safe and effective amount determined by conventional methods starting from existing vaccines.

[0091] The dosage of immunoreactive HA in each dose of the replicated virus vaccine can be standardized to a suitable dosage, for example, 30 - 100 μg or any range or value therein, or to an amount recommended by a government agency or an approved professional body. The amount of NA can also be standardized, although this glycoprotein can be unstable during purification and storage.

[0092] The dose of immunogenic HA in each dose of the replicated viral vaccine can be standardized to contain an appropriate amount, such as 1-50 μg or any range or value therein, the amount recommended by the U.S. Public Heath Service (PHS), usually 15 μg per component for older children (3 years and above), and 7.5 μg for children under 3 years. The amount of NA can also be standardized, but this glycoprotein can become unstable during processor purification and storage (Kendal et al., 1980; Kerr et al., 1975). Each 0.5-ml dose of the vaccine can contain approximately 10-50 billion virus particles, preferably 10 billion particles.

[0093] The present invention will be described by the following non-limiting examples.

Example

[0094] Example 1 Method Cells and Viruses 293T human embryonic kidney cells are maintained in Dulbecco's modified Eagle's minimum essential medium (DMEM) with 10% fetal bovine serum and antibiotics. Madin-Darby canine kidney (MDCK) cells are grown in MEM with 5% newborn calf serum and antibiotics. African green monkey vero WCB cells (Sugawara et al., 2002) established after biosafety testing for use in human vaccine production are maintained in serum-free VP-SFM medium (GIBCO-BRL) containing antibiotics. The cells are maintained at 37 °C in 5% CO2. The vaccine seed virus recommended by the WHO is NIBRG-14.

[0095] Plasmid Preparation and Reverse Genetics To generate reassortants of influenza A virus, reverse genetics based on plasmids (Neumann et al., 1999) is used. Full-length cDNAs were cloned into plasmids under the control of the human polymerase I promoter and the mouse RNA polymerase I terminator (PolI plasmid).

[0096] A series of pre-generated PolI constructs derived from the A / WSN / 33 (H5N1; WSN) or PR8 strains are used for reverse genetics (Horimoto et al., 2006; Neumann et al., 1999). The World Health Organization (WHO) recommends A / Puerto Rico / 8 / 34 (H1N1; PR8) as a donor virus for its safety in humans (Wood & Robertson, 2004; Webby & Webster, 2003).

[0097] Plasmids expressing WSN or PR8 NP, PA, PB1, or PB2 under the control of the chicken actin, e.g., beta-actin, promoter are used for all reverse genetics experiments (Horimoto et al., 2006; Neumann et al., 1999). That is, the PolI plasmid and the protein expression plasmid are mixed with the transfection reagent, Trans-IT 293T (Panvera), incubated at room temperature for 15 minutes, and then added to 293T cells. The transfected cells are incubated in Opti-MEM I (GIBCO-BRL) for 48 hours. For reverse genetics in Vero WCB cells, a plasmid mixture is transfected using an electroporator (Amaxa) according to the manufacturer's instructions. Sixteen hours after transfection, freshly prepared Vero WCB cells are added to the transfected cells, and 6 hours later, TPCK-trypsin (1 μg / mL) is added to the culture. The transfected cells are incubated in serum-free VP-SFM for a total of 4 days. Supernatants containing infectious virus can be recovered and biologically cloned by limiting dilution.

[0098] The HA and NA genes from A / Hong Kong / 213 / 2003 (H5N1) and the other remnants were used to prepare a recombinant virus having the genes of an influenza A virus derived from UW-PR8. The titer of the recombinant virus was 1010.67 EID 50 / mL, and the titer of HA was 1:1600.

[0099]

Table 1

[0100] The sequence of the PR8 (UW) gene is as follows:

Chemical formula

[0101]

Chemical formula

[0102]

Chemical formula

[0103]

Chemical formula

[0104]

Chemical formula

[0105]

Chemical formula

[0106]

Chemical formula

[0107] [Chemical]

[0108] The high - titer A / PR / 8 / 34 (H1N1, UW - PR8) virus grows 10 - fold better in eggs than other A / PR / 8 / 34 PR8 strains (1010 EID 50 / mL; HA titer: 1:8,000). Therefore, the substitution of the HA and NA genes of UW - PR8 with those of currently circulating strains of influenza virus results in a vaccine strain that can be safely produced, confirming the use of UW - PR8 as a master vaccine strain.

[0109] Genes contributing to the different growth characteristics between UW - PR8 and PR8 (Cambridge) that provide the non - HA and non - NA genes of the NIBRG - 14 vaccine strain (Figure 1) were measured. High titers were obtained in eggs when most of the internal genes were derived from UW - PR8. The highest titers were those with the M gene segment of UW - PR8 and the NS gene of PR8 (Cambridge). The NS gene of UW - PR8 has K (lysine) at residue 55, while the NS gene of PR8 (Cam) has E (glutamic acid). The polymerase subunits (PA, PB1, and PB2) and the NP gene of UW - PR8 enhanced the growth of the H5N1 vaccine seed virus in chicken embryonated eggs, and the NS gene of PR8 (Cambridge) enhanced the growth of the H5N1 vaccine seed virus in chicken embryonated eggs. The tyrosine (Y) at position 360 in PB2 of UW - PR8 probably contributes to the high growth rate of the virus in MDCK cells.

[0110] Example 2 To develop a high-yield A / PR / 8 / 34 (H1N1; PR8) virus backbone for the growth of vaccine virus in a specific host cell, random mutagenesis of the internal genes of PR8 (HG) (PR8UW) was performed. Random mutations were introduced into the internal genes of UW-PR8 (Example 1) by error-prone PCR, and then a plasmid library having the random mutations in the individual UW-PR8 internal genes was prepared. Next, a virus library (PR8 / H5N1) having random mutations in the individual UW-PR8 internal genes was created together with other wild-type internal genes, NA, and a "detoxified" HA gene of A / chicken / Indonesia / NC / 09 (H5N1) virus (Table 1) to create a "6+2" recombinant virus. Serial passage of the virus in MDCK cells was utilized to select mutant strains having high growth characteristics.

[0111]

Table 2

[0112] The virus library was passaged 12 times in MDCK cells, or after 2 passages, the library was mixed and another 10 passages were performed (Figure 2). After 10 to about 12 consecutive passages in MDCK cells, a plaque assay was performed and more than 1400 individual plaques were selected. Figure 3 shows the number of clones having various HA titers. Growth-promoting mutations included: PB2: M202L, F323L, I504V, PB1: E112G, V644A, NP: R74K, N417D, I116L, and NS: S161T. Figure 4 shows the titers of recombinant viruses created from the selected mutations. The sequences of 36 viruses having the highest HA titers from the random mutagenesis library were determined (Table 2).

[0113]

Table 3

Table 4

[0114] In a second approach, potential mutations promoting growth described in the literature were introduced into the background of the UW-PR8 virus (see Table 3 regarding virus stock titers), and then tested for replication ability. Figures 5A - D show the growth curves of various viruses.

[0115] [Table 5]

[0116] In a third approach, candidate strains from Approaches 1 and 2 were combined, and HA titers and PFU / mL were measured (Table 4).

[0117] [Table 6] [Table 7]

[0118] As shown in Table 4, several recombinant viruses that replicated better than the wild type, such as #1, #4, #36, #38, P17, P16, and P61, were identified. To identify the growth characteristics of these viruses, the growth rate in MDCK cells was measured (Figure 7). For one candidate strain, the virus was purified by sucrose gradient, and the HA content and total viral protein were evaluated. Figure 8A shows the HA titers for the wild type (UW-PR8) and #4, Figure 8B shows the viral proteins for the wild type (UW-PR8) and #4, and Figure 8C is the SDS-PAGE analysis of the viral proteins for the wild type (UW-PR8) and #4. Further analysis demonstrated that the virus with the V97A / Y100H mutation in M1 produced higher HA titers than the parental virus but had a lower virus titer (see Figures 9A - B). The V97A / Y100H mutation in M1 may result in particles with a larger surface area into which more HA proteins can be incorporated. Since inactivated influenza viruses are administered based on their HA content, mutant strains with a high HA content are attractive vaccine candidates.

[0119] To identify mutations in the influenza promoter region that result in enhanced replication, viruses having a "U" at position 4 at the 3' end of all eight vRNA segments were generated with the internal genes of PA, PB1, and PB2 of UW-PR8 (the PB2, PB1, and PA segments of UW-PR8 have a "C" at position 4). The growth curves of the resulting viruses are shown in Figure 11C. Viruses having combinations of promoter mutations and amino acid changes were generated and titers were measured (Table 5).

[0120] [Table 8]

[0121] Codon optimization was also performed. Modifying codons can enhance protein expression but may also alter the RNA structure and stability. For example, codon optimization of the PB2 gene segment was performed to reflect the codon usage in canine cells (since MDCK cells are of canine origin) (Figure 10A), but the remaining packaging signals (located at the 5' and 3' termini of vRNA) remained unchanged. In one approach, codon optimization was performed for all codons in the "internal" region of the PB2 gene (Figure 10C), and in another approach, codon optimization was performed for so-called "rare" codons (which are used at a significantly lower frequency compared to the codons most frequently used for a given amino acid) (Figure 10B) (see SEQ ID NO: 25 in Figure 10F). Analysis was performed using "Graphical Codon Usage Analyser" (www.gcua.de). The titers of those viruses are shown in Table 6 (see also Figures 10B - C).

[0122]

Table 9

[0123] Optimization of the rare codons of PB2 resulted in an enhanced titer compared to the wild - type virus (UW - PR8) (see Figure 10D). The other gene segments were codon - optimized, and the titers of the viruses having those segments or combinations of the optimized segments were measured (Figure 10E). In another approach to enhancing the virus titer in MDCK cells, chimeric HA and NA genes were prepared (Figure 13A), and the titers of the viruses having those genes were measured (Figure 13B).

[0124] Viruses having combinations of the above mutations (high-growth backbone mutations, promoter mutations, chimeric HA and NA genes, and canine codon optimization) were prepared, and the growth rates, PFUs, and HA titers of those viruses were measured (see Figure 14). Representative backbone mutations are canine codon opti-PB2+C4U+M202L, F323L; PB1:C4U+Q247H; PA:C4U+K142N; NP: canine codon opti-NP+R74K; M:V97A, Y100H; and NS:K55E. Any mutations described herein, or any combination thereof, may be combined, for example, with seasonal H1N1 and H3N2, H3N2 variants, PdmH1N1, H5N1, H7N9 or H9N2, or other clades or candidate vaccine strains. For example, the HA and NA genes from A / California / 04 / 2009 (pdm H1N1) were combined with the six internal genes of UW-PR / 8 to create a "6+2" recombinant virus. Eleven virus libraries were generated and passaged 10 times in eggs. Three rounds of limiting dilution were performed to screen for high-growth mutants (Figure 15). In one embodiment, mutants with high-growth characteristics in MDCK cells have a PB2 gene segment with a promoter mutation (C4U) and a mutation that results in I504V (relative to the parental virus); a PB1 gene segment with a promoter mutation (C4U) and a mutation that results in E112G; a PA gene segment with a promoter mutation (C4U) and a mutation that results in S225C; an NP gene segment with mutations that result in R74K and N417D; an M gene segment with mutations that result in V97A and Y100H; and an NS gene segment with a mutation that results in K55E, where optionally the sequence of one or more gene segments, e.g., the NP gene segment, is modified to include optimized canine codons. In one embodiment, mutants with high-growth characteristics in MDCK cells have a canine codon-optimized PB2 gene segment with a mutant promoter mutation (C4U) and mutations that result in M202L and F323L; a PB1 gene segment with a promoter mutation (C4U) and a mutation that results in Q247H; a PA gene segment with a promoter mutation (C4U) and a mutation that results in K142N; a canine codon-optimized NP gene segment with a mutation that results in R74K; an M gene segment with mutations that result in V97A and Y100H; and an NS gene segment with a mutation that results in K55E.

[0125] Similar experiments were performed in Vero cells using clones with high replication characteristics in MDCK cells, for example, after about 3 to 5 passages in Vero cells (see Figure 16). Figure 17 shows five viruses that may have high replication characteristics in Vero cells. In one embodiment, the PR8(UW) mutant strain with high growth characteristics in Vero cells has the following mutations that can be used in various combinations to enhance the replication ability of the PR8(UW) virus: PB2 segment: C4U (promoter mutation), I504V (amino acid change); PB1 segment: C4U (promoter mutation); M40L (amino acid change), G180W (amino acid change); PA segment: C4U (promoter mutation), R401K (amino acid change); NP segment: I116L (amino acid change); NS segment: A30P (amino acid change in NS1), or R118K (amino acid change in NS1).

[0126] Cited References Avery’s Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, 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). Berkow et al., eds., 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, K. G., Webster, R. G., and Hay, A. (Blackwell, Oxford), pp. 373-390 (1998). 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 (ed.), Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 1324-1341 (1980). Sugawara et al., Biologicals, 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

[0127] All publications, patents and patent applications are hereby incorporated by reference. In the foregoing specification, the invention has been described with reference to specific preferred embodiments and many details have been set forth for purposes of illustration, but the invention is applicable to further embodiments and it will be apparent to those skilled in the art that some of the details described herein may be considerably modified without departing from the basic principles of the invention. [1] An isolated recombinant influenza virus having PA, PB1, PB2, NP, NS, and M gene segments derived from a first influenza vaccine virus isolate, a heterologous or chimeric influenza virus NA gene segment, and a heterologous or chimeric HA gene segment, wherein two or more of said PA, PB1, PB2, NP, NS, and M gene segments have selected amino acid residues at positions 30, 31, 105, 142, 149, 225, 356, 357, 401, and / or 550 of PA; positions 40, 54, 59, 62, 63, 75, 76, 78, 79, 80, 112, 180, 247, 327, 507, 624, 644, 667, 694, 695, 697, 699, 700, 701, 702, 705, 713, and / or 714 of PB1; positions 57, 58, 59, 61, 66, 202, 323, 368, 391, 504, 591, 677, 678, and / or 679 of PB2; positions 74, 112, 116, 224, 293, 371, 377, 417, 422 or 442 of NP; positions 90, 97, and / or 100 of M1; or positions 30, 49, 55, 118, 140, 161, and / or 223 of NS1 of the isolated recombinant influenza virus. [2] having 142N, 225C, 356R, or 550L in PA; having one or more of 112G, 247H, 507V, or 644A in PB1; having one or more of 202L, 323L or 504V in PB2; having one or more of 74K, 112L, 116L, 417D, or 442A in NP; having 97A and / or 100H in M1; and / or having 55E and / or 140Q in NS1, or having a combination thereof, the isolated virus according to claim 1. [3] having at least one of 202L and / or 323L in PB2, 247H in PB1, or 74K in NP, and optionally having at least one of 142N in PA1, 55K in NS1 or 97A and / or 100H in M1, the isolated virus according to claim 1 or 2. [4] having at least one of 202L and / or 323L in PB2, 247H in PB1 or 74K in NP, and having at least one of 142N in PA1, 55K in NS1 or 97A and / or 100H in M1, the isolated virus according to claim 1 or 2. [5] having 202L and / or 323L in PB2, the isolated virus according to claim 1. [6] having 247H in PB1, the isolated virus according to claim 1 or 5. [7] having 74K in NP, the isolated virus according to claim 1, 5 or 6. [8] having U at position 4 of the gene segment of any one of PB1, PB2 or PA, the isolated virus according to any one of claims 1 to 7. [9] having 40I, 40L, 112G, 180W, 247H, 507V, or 644A in PB1, the isolated virus according to claim 1 or 2.

[10] having 202L and / or 323L in PB2, the isolated virus according to any one of claims 1 to 2 or 9.

[11] having 74K, 112L, 116L, 377N, 417D, or 422L in NP, the isolated virus according to any one of claims 1 to 2 or 9 to 10.

[12] The isolated virus according to any one of claims 1-2 or 9-11, having 30P, 118K, 161T or 140Q in NS1.

[13] The isolated virus according to any one of claims 1-2 or 9-12, having 142N, 225C, 356R, 401K, or 550L in PA.

[14] The isolated virus according to claim 1, wherein the selected amino acid residue at a specific position of the PA is present at position 97, 105, 142, 149, 225, 356, 357, 401, 404, and / or 421 (in singular or plural).

[15] The isolated virus according to claim 1 or 14, wherein the selected amino acid residue at a specific position of the PB1 is present at position 12, 40, 54, 59, 62, 63, 66, 75, 76, 78, 79, 80, 180, 247, 507, 624, 644, 694, 695, 697, 699, 700, 701, 705, 713, 714, and / or 762 (in singular or plural).

[16] The isolated virus according to any one of claims 1 or 14-15, wherein the selected amino acid residue at a specific position of the PB2 is present at position 57, 58, 59, 61, 66, 202, 243, 323, 504, 677, 678, and / or 679 (in singular or plural).

[17] The isolated virus according to any one of claims 1 or 14-16, wherein the selected amino acid residue at a specific position of the NP is present at position 74, 112, 116, 224, 293, 417, and / or 442 (in singular or plural).

[18] The isolated virus according to any one of claims 1-17, wherein the selected amino acid residue at a specific position of the M1 is present at position 90, 97, and / or 100 (in singular or plural).

[19] The isolated virus according to any one of claims 1-18, wherein the selected amino acid residue at a specific position of the NS1 is present at position 49, 30, 55, 161, and / or 223 (in singular or plural).

[20] The isolated virus according to any one of claims 1 to 19, wherein at least one of the PA, PB1, PB2, NP, NS, and M gene segments has a promoter mutation from C to U.

[21] The isolated virus according to any one of claims 1 to 20, wherein the NA gene segment and the HA gene segment are derived from the same influenza virus isolate.

[22] The PA, PB1, PB2, NP, NS, and M gene segments include at least one sequence of the following: PB1 having an amino acid sequence encoded by SEQ ID NO: 2 or having at least 95% amino acid sequence identity to PB1 encoded by SEQ ID NO: 2; PB2 having an amino acid sequence encoded by SEQ ID NO: 3 or having at least 95% amino acid sequence identity to PB2 encoded by SEQ ID NO: 3; PA having an amino acid sequence encoded by SEQ ID NO: 1 or having at least 95% amino acid sequence identity to PA encoded by SEQ ID NO: 1; NP having an amino acid sequence encoded by SEQ ID NO: 4 or having at least 95% amino acid sequence identity to NP encoded by SEQ ID NO: 4; M having an amino acid sequence encoded by SEQ ID NO: 5 or having at least 95% amino acid sequence identity to M encoded by SEQ ID NO: 5; or NS having an amino acid sequence encoded by SEQ ID NO: 6 or having at least 95% amino acid sequence identity to NS encoded by SEQ ID NO: 6; or the PA, PB1, PB2, NP, NS, and M gene segments are the following: PB1 having an amino acid sequence encoded by SEQ ID NO: 10 or having at least 95% amino acid sequence identity to PB1 encoded by SEQ ID NO: 10; PB2 having an amino acid sequence encoded by SEQ ID NO: 11 or having at least 95% amino acid sequence identity to PB2 encoded by SEQ ID NO: 11; PA having an amino acid sequence encoded by SEQ ID NO: 12 or having at least 95% amino acid sequence identity to PA encoded by SEQ ID NO: 12; NP having an amino acid sequence encoded by SEQ ID NO: 13 or having at least 95% amino acid sequence identity to NP encoded by SEQ ID NO: 13; M having an amino acid sequence encoded by SEQ ID NO: 14 or having at least 95% amino acid sequence identity to M encoded by SEQ ID NO: 14;Or, the isolated virus according to any one of claims 1 to 21, comprising at least one sequence of NS having an amino acid sequence encoded by SEQ ID NO: 15 or NS having at least 95% amino acid sequence identity to NS encoded by SEQ ID NO: 15.;

[23] The isolated virus according to any one of claims 1 to 22, having a heterologous HA gene segment, a heterologous NA gene segment, a chimeric HA gene segment, a chimeric NA gene segment, or any combination thereof.

[24] A vaccine comprising the isolated recombinant virus according to any one of claims 1 to 23.

[25] A plurality of influenza virus vectors for preparing reassemblants, a) 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 cDNA linked to a transcription termination sequence, wherein the PB1, PB2, PA, NP, NS, and M DNA in the vector for vRNA production are derived from one or more influenza vaccine virus isolates, and the NA DNA in the vector for NA vRNA production has a sequence related to a heterologous NA, and the HA DNA in the vector for HA vRNA production is 30, 31, 105, 142, 149, 225, 356, 357, 401, and / or 550 of PA; 40, 54, 59, 62, 63, 75, 76, 78, 79, 80, 112, 180, 247, 327, 507, 624, 644, 667, 694, 695, 697, 699, 700, 701, 702, 705, 713, or 714, and / or 247 of PB1; 57, 58, 59, 61, 66, 202, 323, 368, 391, 504, 591, 677, 678, 679, 202, and / or 323 of PB2; 74, 112, 116, 224, 293, 371, 377, 417, 422, and / or 442 of NP; 90, 97, and / or 100 of M1;Alternatively, a vector having the sequences of heterologous HA of NS 30, 49, 55, 118, 140, 161, and / or 223; and; b) a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA, 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, A vector comprising.

[26] The vector according to claim 25, wherein the PB1, PB2, PA, NP, NS, and M DNA in the vector for vRNA production have sequences corresponding to DNA encoding a polypeptide having at least 95% amino acid sequence identity with the corresponding polypeptide encoded by SEQ ID NOs: 1-6 or 10-15.

[27] The vector according to claim 25 or 26, wherein the promoter for the vRNA vector is an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T3 promoter or a T7 promoter.

[28] The vector according to any one of claims 25 to 27, wherein the NA is N9.

[29] The vector according to any one of claims 25 to 28, wherein the HA is H7.

[30] The vector according to any one of claims 25 to 29, wherein the PA, PB1, PB2, NP, NS, and M gene segments have a mutation with respect to C of the promoter.

[31] A method for preparing an influenza virus, An amount effective for obtaining an infectious influenza virus, 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, The PB1, PB2, PA, NP, NS, and M DNAs in the vector for vRNA production are derived from one or more influenza vaccine virus isolates, the NA DNA in the vector for NA vRNA production has a sequence related to a heterologous NA, and the HA DNA in the vector for HA vRNA production has a heterologous HA sequence of 30, 31, 105, 142, 149, 225, 356, 357, 401, and / or 550 of PA; 40, 54, 59, 62, 63, 75, 76, 78, 79, 80, 112, 180, 247, 327, 507, 624, 644, 667, 694, 695, 697, 699, 700, 701, 702, 705, 713, 714, and / or 247 of PB1; 57, 58, 59, 61, 66, 202, 323, 368, 391, 504, 591, 677, 678, 679, 202, and / or 323 of PB2; 74, 112, 116, 224, 293, 371, 377, 417, 422, and / or 442 of NP; 90, 97, and / or 100 of M1; or 30, 49, 55, 118, 140, 161, or 223 of NS, and 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, vectors for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NP, optionally vectors for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus HA, 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 M1, vectors for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M2, or vectors for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS2. A method for preparing an influenza virus, comprising contacting the virus with a cell.

[32] The method according to claim 31, wherein the cell is a chicken cell.

[33] The method according to claim 31, wherein the cell is a mammalian cell.

[34] The method according to claim 31, wherein the cell is a Vero cell, a human cell or an MDCK cell.

[35] The method according to any one of claims 31 to 34, wherein the PB1, PB2, PA, NP, NS, and M DNA in the vector for vRNA production has a sequence corresponding to a DNA encoding a polypeptide having at least 95% amino acid sequence identity with the corresponding polypeptide encoded by SEQ ID NOs: 1 to 6 or 10 to 15.

[36] The method according to any one of claims 31 to 35, further comprising a method for isolating the virus.

[37] The method according to any one of claims 31 to 36, wherein at least one of the PA, PB1, or PB2 gene segments has a promoter mutation from C to U.

[38] A virus obtained by the method according to any one of claims 31 to 37.

[39] A vector for vRNA or mRNA expression of influenza virus PA, having at least 95% amino acid sequence identity to the polypeptide encoded by SEQ ID NO: 1 and having threonine at position 30, lysine at position 31, cysteine at position 105, or lysine at position 401.

[40] A vector for vRNA or mRNA expression of influenza virus PB1, having at least 95% amino acid sequence identity to the polypeptide encoded by SEQ ID NO: 2 and having leucine at position 40, alanine or isoleucine at position 54, glycine at position 112, histidine at position 247, valine at position 507, alanine at position 644, or cysteine at position 713.

[41] A vector for vRNA or mRNA expression of PB2, having at least 95% amino acid sequence identity to the polypeptide encoded by SEQ ID NO: 3 and having leucine at position 202 and / or 323.

[42] A vector for vRNA or mRNA expression of influenza virus NP, having at least 95% amino acid sequence identity to the polypeptide encoded by SEQ ID NO: 4 and having lysine at position 74, leucine at position 116, isoleucine at position 224, lysine at position 293, asparagine at position 377, or aspartic acid at position 417.

[43] A vector for vRNA or mRNA expression of influenza virus NS1, having at least 95% amino acid sequence identity to the NS1 polypeptide encoded by SEQ ID NO: 6 and having proline at position 30, alanine at position 49, lysine at position 118, glutamine at position 140, threonine at position 161, or glutamic acid at position 223. A vector for influenza virus M1 vRNA or mRNA expression, having at least 95% amino acid sequence identity to the M1 polypeptide encoded by accession number 5 and having serine at position 90.

Claims

**Claim 1** An isolated recombinant influenza A virus having PA, PB1, PB2, NP, NS, and M virus segments, a heterologous influenza virus NA virus segment, and a heterologous HA virus segment, wherein the PA virus segment encodes a PA having 401K; and the PB1 virus segment encodes a PB1 having 40L and 180W; And here, the recombinant virus has a titer exceeding 3×10 ７ PFU / ml and has an HA titer higher than 27, the recombinant influenza A virus. **Claim 2** The isolated virus according to claim 1, further comprising having 644A in PB1; having 74K and 417D, or 116L in NP; or having 97A and / or 100H in M1. **Claim 3** The isolated virus according to claim 1 or 2, having U at position 4 of any one of the virus segments of PB1, PB2 or PA. **Claim 4** The isolated virus according to claim 1, further comprising having 40L, or 644A in PB1; having 74K and 417D, or 116L in NP; or having 30P, or 161T in NS1. **Claim 5** The isolated virus according to claim 1, having 401K in PA; 40L and 180W in PB1, 202L and 323L in PB2; or 116L in NP. **Claim 6** The isolated virus according to any one of claims 1 to 5, wherein at least one of the virus segments of PA, PB1, PB2, NP, NS, and M has a promoter mutation from C to U. **Claim 7** The isolated virus according to any one of claims 1 to 6, wherein the NA virus segment and the HA virus segment are derived from the same influenza virus isolate. **Claim 8** The PA, PB1, PB2, NP, NS and M virus segments in the recombinant virus are as follows: PB1 having the amino acid sequence encoded by SEQ ID NO: 2, or PB1 having at least 95% amino acid sequence identity with the PB1 encoded by SEQ ID NO: 2; PB2 having the amino acid sequence encoded by SEQ ID NO: 3, or PB2 having at least 95% identity with the PB2 encoded by SEQ ID NO: 3; PA having the amino acid sequence encoded by SEQ ID NO: 1, or PA having at least 95% identity with the PA encoded by SEQ ID NO: 3; NP having the amino acid sequence encoded by SEQ ID NO: 4, or NP having at least 95% identity to the NP encoded by SEQ ID NO: 4; M having the amino acid sequence encoded by SEQ ID NO: 5, or M having at least 95% identity to the M encoded by SEQ ID NO: 5; or NS having the amino acid sequence encoded by SEQ ID NO: 6, or NS having at least 95% identity to the NS encoded by SEQ ID NO: 6 comprising at least one of the sequences of, wherein said PA, PB1, PB2, NP, NS, and M viral segments are the following: PB1 having the amino acid sequence encoded by SEQ ID NO: 10, or PB1 having at least 95% amino acid sequence identity to the PB1 encoded by SEQ ID NO: 10; PB2 having the amino acid sequence encoded by SEQ ID NO: 11, or PB2 having at least 95% identity to the PB2 encoded by SEQ ID NO: 11; PA having the amino acid sequence encoded by SEQ ID NO: 12, or PA having at least 95% identity to the PA encoded by SEQ ID NO: 12; NP having the amino acid sequence encoded by SEQ ID NO: 13, or NP having at least 95% identity to the NP encoded by SEQ ID NO: 13; M having the amino acid sequence encoded by SEQ ID NO: 14, or M having at least 95% identity to the M encoded by SEQ ID NO: 14; or NS having the amino acid sequence encoded by SEQ ID NO: 15, or NS having at least 95% identity to the NS encoded by SEQ ID NO: 15 The isolated virus according to any one of claims 1 to 7, comprising at least one of the sequences of.

9. The isolated virus has a titer exceeding 10 8 PFU / ml, and is the isolated virus according to any one of claims 1 to 8.

10. A vaccine comprising the isolated recombinant virus according to any one of claims 1 to 9.

11. A method for preparing an influenza A virus, comprising the following: A vRNA production vector comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence, A vRNA production vector comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence, A vRNA production vector 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 DNA of the vRNA production vector are from one or more influenza virus isolates, the NA DNA in the vRNA production vector for NA has a sequence of a heterologous NA, and the HA DNA in the vRNA production vector for HA has a sequence of a heterologous HA; wherein the PA DNA is encoded as PA having 401K, and wherein the PB1 DNA is encoded as PB1 having 40L and 180W; and An mRNA production vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, An mRNA production vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, An mRNA production vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, An mRNA production vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, An mRNA production vector comprising a promoter operably linked to a DNA segment encoding influenza virus HA, An mRNA production vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, An mRNA production vector comprising a promoter operably linked to a DNA segment encoding influenza virus M1, An mRNA production vector comprising a promoter operably linked to a DNA segment encoding influenza virus M2 An mRNA production vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS2, contacting the cells in an amount effective to produce an infectious influenza virus The method comprising the above.

12. The method according to claim 11, wherein the cells are avian cells or mammalian cells.

13. The method according to claim 12, wherein the cells are Vero cells, human cells or MDCK cells.

14. The method according to any one of claims 11 to 13, wherein the PB1, PB2, PA, NP, NS and M DNA in the vRNA production vector have sequences corresponding to sequences encoding polypeptides having at least 95% amino acid sequence identity to the corresponding polypeptides encoded by SEQ ID NOs: 1 to 6 or 10 to 15.

15. The method according to any one of claims 11 to 14, further comprising isolating the virus.

16. The method according to any one of claims 11 to 15, wherein at least one of the PA, PB1, or PB2 virus segments has a promoter mutation from C to U.

17. PB2 contains C4U and 504V; PB1 contains C4U, 40L and 180W; PA contains C4U and 401K; NP contains 116L; and / or NS1 contains 30P or 118K, the virus according to claim 2.

18. PB2 has 504V, and PB1 has 112G; or PB2 has 504V, PB1 has 40L and 180W, PA has 401K, NS1 has 30P or R118K; or PB2 has 202L and 323L, and PB1 has 247H, The isolated virus according to claim 2.

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