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

Abstract

To provide a composition useful to prepare high titer influenza viruses, in the absence of helper virus.SOLUTION: The invention provides a composition useful to prepare high titer influenza viruses, e.g., in the absence of helper virus, which includes internal genes from an influenza virus vaccine strain or isolate, e.g., one that is safe in humans, for instance, one that does not result in significant disease, that confer enhanced growth in cells in culture, such as MDCK cells, or in eggs.SELECTED DRAWING: Figure 17

Description

[Technical Field]

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

[0002] Matters concerning the rights of the government This invention was completed with government support received from the U.S. National Institutes of Health as AI070010 and HHSN266200700010C. The U.S. Government reserves certain rights to this invention. [Background technology]

[0003] Influenza is a major respiratory disease in some mammals, including horses, and annually causes significant mortality and economic losses. Furthermore, infection with the influenza virus can lead to severe systemic disease and death in some bird species. The segmentation properties of the influenza virus genome allow for segment reassortment during viral replication in cells infected with two or more influenza viruses. Segment reassortment, coupled with genetic mutations and drift, can give rise to countless influenza virus strains over time. Such strains exhibit antigenic variation in their hemagglutinin (HA) and / or neuraminidase (NA) proteins, with the gene encoding the HA protein having a particularly high mutation rate. The current primary practice for influenza prevention is vaccination. Since the influenza HA protein is the primary target antigen for the host's protective immune response to the virus and is highly variable, the isolation of influenza viruses and the identification and characterization of HA antigens in viruses associated with recent pandemics are crucial for vaccine production. Vaccines are designed to stimulate a protective immune response against the major and anticipated influenza virus strains, based on prevalence and forecasts (Park et al., 2004).

[0004] Influenza viruses exist in three general types: A, B, and C, defined by the lack of serocross-reactivity between their internal proteins. Influenza A viruses are further classified into subtypes based on antigenic and genetic differences in their glycoproteins, HA, and NA proteins. All known HA and NA subtypes (H1-H15 and N1-N9) have been isolated from waterfowl, which are thought to function as a natural reservoir of influenza. The H1N1 generalized virus caused a global pandemic in 2009. The first vaccine candidate strains tested in 2009 did not grow to high titers, clearly demonstrating the need to develop a vaccine virus backbone that would allow vaccine virus candidates to replicate efficiently. [Overview of the Initiative] [Means for solving the problem]

[0005] Mutations that enhance viral replication ability in cell culture and / or hatched eggs are useful for amplifying influenza viruses and establishing robust influenza vaccine platforms. Currently, most influenza vaccines are produced in hatched eggs. Influenza vaccines produced in MDCK cells are currently approved for human use in the United States and Europe, and influenza vaccines derived from Vero cells are approved for human use in Europe. Viral libraries with random mutations in the "internal" viral genes (i.e., all viral genes other than those encoding the viral surface glycoproteins HA and NA) of vaccine virus isolates (e.g., UW-PR8) were constructed in MDCK cells and passaged. The identified mutations resulted in higher viral titers in MDCK cells (and may also enhance viral titers in Vero cells and / or hatched eggs), enabling more effective influenza virus replication and more economical vaccine production. Furthermore, 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 C-to-U mutations at positions 3-4 of the PB2, PB1, and / or PA vRNA segments, were observed to enhance viral titers. Furthermore, the resulting sequences can be optimized for codon utilization and may be optimized for expression in mammalian cells such as canine, primate, or chicken cells, e.g., chicken embryos. The mutations can be used in various combinations that have the desired level of improvement in viral replication, as the results are influenced by the cell line (or egg) being used.

[0006] The present invention provides isolated recombinant, for example, reassembled influenza viruses having selected amino acid residues at specific positions in one or more gene segments of PA, PB1, PB2, NP, M (encoding M1 and M2 proteins), and / or NS (encoding NS1 and NS2 proteins), for example, having selected amino acid residues at specific positions in 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 comprising, for example, HA and NA genes / proteins of interest derived from annual and global epidemic strains, the viruses being produced more efficiently and cost-effectively by cell culture (in MDCK or Vero cells) or in hatched chicken eggs. In one embodiment, the recombinant reassembled 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, compared to a corresponding virus having lysine at position 142 of PA, i.e., the residue at position 142 of PA in 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 arbitrarily selected at one or more specific positions of PB1, PB2, NP, M1, and / or NS1. In one embodiment, the recombinant reassembled 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, compared to a corresponding virus having lysine at position 142 of PA. In one embodiment, the recombinant reassembled influenza virus has asparagine or glutamine at position 142 of PA, and has optionally selected amino acid residues at one or more specific positions of PB1, PB2, NP, M1, and / or NS1.In one embodiment, the recombinant reassembled influenza virus has an amino acid residue at position 247 of PB1 that results in enhanced growth in cells, including MDCK cells, Vero cells, or eggs, compared to a corresponding virus having glutamine at position 247 of PB1, that is, the residue at position 247 of PB1 in the PB1 gene segment of the recombinant influenza virus is not glutamine, but is a residue correlated with enhanced replication in MDCK cells, Vero cells, or eggs, and is an amino acid residue arbitrarily selected at one or more specific positions of PA, PB2, NP, M1, and / or NS1 as described herein. In one embodiment, the recombinant reassembled influenza virus has an amino acid residue at position 247 of PB1 that results in enhanced interaction with one or more host proteins in MDCK cells, Vero cells, or eggs, compared to a corresponding virus having glutamine at position 247 of PB1. In one embodiment, the recombinant reassembled influenza virus has histidine, arginine, or lysine at position 247 of PB1, and optionally selected amino acid residues at one or more specific positions of PA, PB2, NP, M1, and / or NS1 as described herein. In one embodiment, for example, the recombinant reassembled influenza virus has amino acid residues at positions 202 and / or 323 of PB2 that promote cell growth in cells, including MDCK cells, Vero cells, or eggs, compared to a 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 in the PB2 gene segment of the recombinant influenza virus are residues that are not methionine or phenylalanine, but correlate with enhanced replication in MDCK cells, Vero cells, or eggs, and optionally selected amino acid residues at one or more specific positions of PA, PB1, NP, M1, and / or NS as described herein. In one embodiment, the recombinant reassembled influenza virus has an amino acid residue at position 323 of PB2 that results in a modified cap-binding interaction compared to, for example, the corresponding virus which has phenylalanine at position 323 of PB2.In one embodiment, the recombinant reassembled influenza virus has leucine, alanine, threonine, valine, isoleucine, or glycine at positions 202 and / or 323 of PB2, and has optionally selected amino acid residues at one or more specific positions of PA, PB1, NP, M1, and / or NS as described herein. In one embodiment, the recombinant reassembled influenza virus has an amino acid residue at position 74 of NP that promotes cell growth, including in MDCK cells, Vero cells, or eggs, compared to a corresponding virus having arginine at position 74 of NP, for example; that is, 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 optionally selected amino acid residues at one or more specific positions of PA, PB1, PB2, M1, and / or NS as described herein. In one embodiment, a recombinant reassembled influenza virus has an amino acid residue at position 74 of the NP that can alter its folding, stability, and / or interaction with other viruses or host proteins compared to a corresponding virus having, for example, arginine at position 74 of the NP. In one embodiment, a recombinant reassembled influenza virus has lysine or histidine at position 74 of the NP and has optionally selected amino acid residues at one or more specific positions of PA, PB1, PB2, M1, and / or NS as described herein. In one embodiment, a recombinant reassembled influenza virus has amino acid residues at position 97 and / or 100 of M1 that result in enhanced intracellular growth, including in MDCK cells, Vero cells, or eggs, compared to a corresponding virus having, for example, valine at position 97 or tyrosine at position 100 of M1; that is, 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 amino acid residues selected at one or more specific positions of PA, PB1, PB2, NP, and / or NS1 as described herein.In one embodiment, the recombinant reassembled influenza virus has an amino acid residue at position 97 of M1 that can alter dimerization compared to, for example, a corresponding virus having valine at position 97 of M1. In one embodiment, the recombinant reassembled influenza virus has an amino acid residue at position 100 of M1 that can alter viral aggregation compared to, for example, a corresponding virus having tyrosine at position 100 of M1. In one embodiment, the recombinant reassembled influenza virus has leucine, threonine, isoleucine, alanine, or glycine at position 97 of M1 and / or lysine, arginine, or histidine at position 100, and the selected amino acid residue is located at one or more specific positions of PA, PB1, PB2, NP, and / or NS1 as described herein. In one embodiment, a recombinant reassembled influenza virus has an amino acid residue at position 55 of NS1 that promotes intracellular growth, including in MDCK cells, Vero cells, or eggs, compared to a corresponding virus having lysine at position 55 of NS1, for example, and the selected amino acid residue is located at one or more specific positions of PA, PB1, PB2, NP, and / or M1 as described herein. In one embodiment, a recombinant reassembled influenza virus has asparagine, aspartic acid, glutamic acid, or glutamine at position 55 of NS1, and the selected amino acid residue is located at one or more specific positions of PA, PB1, PB2, NP, and / or M1 as described herein. In one embodiment, the present invention provides an isolated recombinant reassembled influenza virus having six “internal” gene segments derived from a vaccine influenza virus having two or more selected amino acid residues at specific positions described herein, an NA gene segment selected from a first influenza virus isolate, and an HA gene segment derived 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 in 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 reassembled influenza virus has, for example, an amino acid residue at position 142 of PA that promotes growth in MDCK cells, Vero cells, or eggs compared to a corresponding virus having lysine at position 142 of PA; for example, an amino acid residue at position 247 of PB1 that promotes growth in MDCK cells, Vero cells, or eggs compared to a corresponding virus having glutamine at position 247 of PB1; for example, an amino acid residue at position 202 of PB2 that promotes growth in MDCK cells, Vero cells, or eggs compared to a corresponding virus having methionine at position 202 of PB2 or phenylalanine at position 323 of PB2 Having an amino acid residue at position 74 of NP that promotes growth in MDCK cells, Vero cells, or eggs, compared to a corresponding virus having arginine at position 74 of NP; having an amino acid residue at position 97 and / or 100 of M1 that promotes growth in MDCK cells, Vero cells, or eggs, compared to a corresponding virus having valine at position 97 of M1 or tyrosine at position 100 of M1; or having an amino acid residue at position 55 of NS1 that promotes growth in MDCK cells, Vero cells, or eggs, compared to a corresponding virus having lysine at position 55 of NS1; or having 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 in 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 reassembled influenza virus has two or more of the following: 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 isolated recombinant, for example, reassembled influenza viruses having selected amino acid residues, including one or more characteristic residues described herein, at one or more specific positions in one or more gene segments of PA, PB1, PB2, NP, M1, and / or NS1, for example, the selected amino acid residues at PB1, PB2, and NS; PB1, PB2, NP, and NS; PA, PB1, PB2, NP, and NS; PB1, PB2, NP, M, and NS; or specific positions of PA, PB1, PB2, NP, M, and NS. In one embodiment, the recombinant reassembled influenza virus has amino acid residues at positions 105 and / or 401 of PA that result in enhanced growth in cells, for example, MDCK cells, compared to a corresponding virus having, for example, phenylalanine or arginine at positions 105 and 401 of PA, respectively. In one embodiment, the recombinant reassembled influenza virus has, for example, methionine, arginine, threonine, glycine, alanine, asparagine, lysine, glutamic acid, aspartic acid, glutamic acid, proline, serine, glutamic acid, glycine, isoleucine, methionine, at positions 40, 54, 59, 62, 63, 66(F2), 73(F2), 75, 76, 78, 79, 80, 112, 180, 504, 507, 624, 644, 667, 694, 695, 697, 699, 700, 701, 702, 705, 713, or 714 of PB1, respectively. Compared to the corresponding virus having leucine, valine, isoleucine, asparagine, leucine, glutamic acid, phenylalanine, phenylalanine, proline, serine, tyrosine, serine, or methionine, PB1 has amino acid residues 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 that result in enhanced growth in cells, e.g., MDCK cells.In one embodiment, a recombinant reassembled 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 result in enhanced growth in cells, such as MDCK cells, compared to a corresponding virus having isoleucine, threonine, alanine, lysine, methionine, methionine, phenylalanine, arginine, glutamic acid, isoleucine, glutamine, glutamic acid, aspartic acid, or phenylalanine at positions 57, 58, 59, 61, 66, 202, 323, 368, 391, 504, 591, 677, 678, or 679 of PB2, respectively. In one embodiment, the recombinant reassembled 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 a corresponding virus having leucine, asparagine, arginine, methionine, aspartic acid, arginine, or threonine at positions 116, 224, 293, 371, 417, 422, or 442 of NP, respectively. In one embodiment, the recombinant reassembled influenza virus has amino acid residues at position 90 of M1 that promote growth in cells, compared to a corresponding virus having serine at position 90 of M1, for example. In one embodiment, the recombinant reassembled influenza virus has amino acid residues at positions 30, 49, 140, 161, or 223 of NS1 that promote growth in MDCK cells, compared to a corresponding virus that has proline, alanine, glutamine, threonine, or glutamic acid at positions 30, 49, 140, 161, or 223 of NS1, respectively. In one embodiment, the recombinant reassembled 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 is a recombinant influenza virus having an amino acid sequence having at least 80%, for example, an integer from 80 to 99, with respect to the corresponding polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15, such as a polypeptide having specific amino acid residues at 1, 2, 3 or more specific positions in PA, PB1, PB2, NP, M1, and / or NS1, and having at least 80%, for example, an integer from 80 to 99, with respect to 90%, 92%, 95%, 97%, 98%, or 99% amino acid sequence identity with respect to the corresponding polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15, such as a polypeptide having specific amino acid residues at 1, 2, 3 or more specific positions in PA, PB1, PB2, NP, M1, and / or NS1, and residues other than K142, S225, K356, or I550 in PA; E112, Q247, M507, or V644 in PB1; M202, F323, or I504 in PB2; R74, I112, I116, T442, or N417 in NP; V97, and / or Y100 in M1; and / or R140, or K55 in NS. Residues other than the specified residues may be conservative substitutions. A conservative amino acid substitution means the interchangeability of residues having similar side chains. For example, amino acid groups with aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; amino acid groups with aliphatic hydroxyl side chains are serine and threonine; amino acid groups with amide-containing side chains are asparagine and glutamine; amino acid groups with aromatic side chains are phenylalanine, tyrosine, and tryptophan; amino acid groups with basic side chains are lysine, arginine, and histidine; and amino acid groups with sulfur-containing side chains are cysteine ​​and methionine. In one embodiment, the conservative amino acid substituents 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 non-conservative amino acid substitutions, for example, two, three, or four, compared to the polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15.

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

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

[0013] In one embodiment, the influenza virus of the present invention is a recombinant influenza virus having specific amino acid residues at specific positions in PA, PB1, PB2, NP, M1, and / or NS1, and having an amino acid sequence identity of at least 80%, for example, 90%, 92%, 95%, 97%, 98%, or 99%, including any integer from 80 to 99, with respect 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 conserved 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 conserved substitutions of R74 and N417; an M gene segment having a conserved substitution of V97 and a non-conservative substitution of Y100; and an NS gene segment having a non-conservative substitution of K55.

[0014] In one embodiment, the influenza virus of the present invention is a recombinant influenza virus having specific amino acid residues at specific positions in PA, PB1, PB2, NP, M1, and / or NS1, and having an amino acid sequence having at least 80%, for example, an integer from 80 to 99, including 90%, 92%, 95%, 97%, 98%, or 99% amino acid sequence identity with respect 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.

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

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

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

[0018] In one embodiment, the parent strain has a gene segment having a sequence corresponding to sequence numbers 1-6 or 10-15. In one embodiment, the HA gene segment of the reassembled virus is a chimera of a different HA ectodomain sequence linked to a chimeric HA gene segment, for example, an HA signal peptide sequence and / or an HA transmembrane domain sequence derived from the HA gene segment of a parent isolate or strain, or its mutant. In one embodiment, the NA gene segment of the isolated recombinant virus is a chimeric NA gene segment, for example, a chimera of a different NA ectodomain sequence linked to an NA transmembrane domain sequence derived from the NA gene segment of a parent isolate or strain, or its mutant, and / or a stalk sequence derived from a parent isolate or strain, or its mutant. In one embodiment, the NA gene segment of the isolated recombinant virus is a chimeric NA gene, for example, a different NA ectodomain sequence linked to an NA transmembrane domain sequence derived from the NA gene segment of a parent isolate or strain, or its mutant, and / or a stalk sequence derived from a second isolate or strain, or its mutant. 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 used to prepare the vRNA may be one in which residues are introduced at specific positions by the recombinant method, or may be selected for 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 have been used to determine whether high-yield vaccine strain backbones can be prepared to meet the requirements of seasonal influenza and highly pathogenic pandemic viruses with respect to propagation in MDCK cells, chicken eggs, and Vero cells. 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, for example, the HA gene was optimized to enhance virus replication and HA content 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, for example, 10 to 12 times in MDCK cells, 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, the selection of sequences having one or more residues disclosed at specific positions of PA, PB1, PB2, NP, M1, and / or NS1, or substitution of such disclosed residues, which confer enhanced viral growth in cultured cells when used together with the HA and NA sequences of interest for vaccine viruses to be propagated or passaged in cultured cells, such as MDCK or Vero cells or eggs, may result in significantly higher viral titers. Thus, the present invention provides a method for selecting influenza viruses that exhibit enhanced replication in cell culture. The method comprises providing cells suitable for influenza vaccine production; serially culturing one or more influenza virus isolates in cells; and isolating the serially cultured viruses that exhibit enhanced growth compared to one or more isolates before serial 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 of PA, 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 useful for the preparation of reassembled viruses, for example, 6:1:1 reassembled viruses, 6:2 reassembled viruses, and 7:1 reassembled viruses. Within the scope of the present invention, a 6:1:1 reassembled virus is an influenza virus having six internal gene segments derived from a vaccine virus, an NA gene segment derived from a different (second) virus isolate, and an HA gene segment derived from a third isolate. Within the scope of the present invention, a 6:2 reassembled virus is an influenza virus having six internal gene segments derived from a vaccine virus, and an NA gene segment and an HA gene segment derived from a different (second) virus isolate; and within the scope of the present invention, a 7:1 reassembled virus is an influenza virus having six internal gene segments, an NA gene segment derived from a vaccine virus, and an HA gene segment derived from a different viral origin than the vaccine virus, or an influenza virus having six internal gene segments and an HA gene segment derived from a vaccine virus, wherein the NA gene segment is derived from a different viral origin than the vaccine virus.

[0024] One embodiment of the present invention includes a vector for vRNA production selected from a vector comprising a promoter operably linked to influenza virus PA DNA bound to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus PB1 DNA bound to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus PB2 DNA bound to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus HA DNA bound to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus NP DNA bound to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus NA DNA bound to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus M DNA bound to a transcription termination sequence, and a vector comprising a promoter operably linked to influenza virus NS DNA bound to a transcription termination sequence. In one embodiment, the DNA for vRNA production of PB1, PB2, PA, NP, M, and NS has sequences derived from cultured mammalian cells such as MDCK cells, Vero cells, or PER.C6® cells, or optionally from influenza viruses that replicate at high titers in hatched eggs, and / or, for example, vaccine viruses that do not cause severe illness in humans. The DNA for vRNA production of NA can be derived from any NA, e.g., any of N1 to N10, and the DNA for vRNA production of HA can be derived from any HA, e.g., 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 reassembly) or the same strain or the same isolate (6:2 reassembly), or NA can be derived from the same strain or the same isolate as the internal gene (7:1 reassembly).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. The 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 9 , , , 8 , , 50 ,

[0026] , 7 , 50 , 7 , 8 , 8 , 50 , , 6 , 7 , 50 , 10 ,

[0027] 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, for example, MDCK cells or Vero cells can be 1 log, 2 logs, 3 logs, or more of the titer of the corresponding virus lacking specific residues at specific positions.

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

[0028] In one embodiment, the DNA relating to the internal genes of PB1, PB2, PA, NP, M, and NS encodes a protein having substantially identical activity to the corresponding polypeptide encoded by one of sequence numbers 1-6 or 10-15. In this specification, “substantially identical activity” includes, for each activity or protein level, about 0.1%, 1%, 10%, 30%, 50%, 90%, e.g., 100% or more activity, or about 80%, 90% or more detectable protein levels of the corresponding full-length polypeptide. In one embodiment, the nucleic acid sequence is a sequence encoding a polypeptide that is substantially identical to the polypeptide encoded by one of sequence numbers 1-6 or 10-15 in having, for example, an integer percentage of 80-99, at least 80%, e.g., 90%, 92%, 95%, 97%, 98%, or 99% consecutive amino acid sequence identity. In one embodiment, the isolated and / or purified nucleic acid molecule contains, for example, one of sequence numbers 1-6 or 33-38 and a nucleotide sequence that is substantially identical to having a continuous nucleic acid sequence identity of any integer percentage from 50 to 100, at least 50%, for example, 60%, 70%, 80%, or 90%, or more.In one embodiment, the influenza virus polypeptide has one or more, for example, 2, 5, 10, 15, 20 or more, conserved amino acid substitutions, for example, a combination of conserved and non-conserved amino acid substitutions, with up to 10% or 20% of conserved substitutions, for example, up to 10% or 20% of residues compared to the polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15, and has two or more characteristic residues of PA, PB1, PB2, NP, M1, and / or NS1 compared to the polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15. , and also having two or more characteristic residues in the gene segments 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 of M1 and / or lysine, arginine, or histidine at position 100; 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, two, three, four, five, six, seven, or eight conserved and / or non-conserved amino acid substitutions, relative to the polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15, for example, that of the virus isolates 1, four, three, three, three, six, seven, or six of Table 4.

[0029] Accordingly, the present invention comprises the use of isolation and purification vectors or plasmids that express or encode influenza virus proteins, or influenza vRNA, both undenatured and recombinant vRNA. The vectors include influenza cDNA, e.g., influenza A (e.g., any influenza A gene including either the 16 HA or 9 NA subtype), B, or C DNA (see Fields Virology (Fields et al. (eds.), Lippincott, Williams and Wickens (2006)), which are specifically incorporated herein by reference). Any suitable promoter or transcription termination sequence can be used to express proteins or peptides, e.g., viral proteins or peptides, nonviral pathogen proteins or peptides, or therapeutic proteins or peptides.

[0030] The vector composition or a plurality of vectors of the present invention may also include heterologous genes or coding regions that can encode, for example, epitopes useful in cancer treatment or vaccines, or peptides or polypeptides useful in gene therapy, such as foreign genes encoding immunogenic peptides or proteins useful as vaccines or in gene substitution. When preparing a virus, the vector or plasmid containing the target gene or cDNA may be substituted for or added to a vector or plasmid relating to an influenza virus gene. Accordingly, other embodiments of the present invention include the above vector composition in which one of the vectors is substituted, or a plurality of such vectors, further comprising a 5' influenza virus sequence. Optionally, the 5' influenza virus sequence includes the 5' influenza virus coding sequence or a portion thereof, which is substituted for the target nucleic acid sequence, for example, a 3' influenza virus sequence that is substituted for the target cDNA, and optionally includes a 3' influenza virus coding sequence or a portion thereof. In one embodiment, the target nucleic acid sequence, such as cDNA, is oriented in the antisense (antigenome) direction. The introduction of these vectors, combined with the other vectors mentioned above, into host cells permissible for influenza virus replication produces recombinant viruses containing vRNAs corresponding to heterologous sequences of such vectors.

[0031] The promoter in the vector for vRNA production may be an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T7 promoter, or a T3 promoter, and optionally the vector may contain a transcription termination sequence such as an RNA polymerase I transcription termination sequence, an RNA polymerase II transcription termination sequence, an RNA polymerase III transcription termination sequence, or a ribozyme. Ribozymes within the scope of the present invention include, but are not limited to, tetrahymena ribozymes, ribonuclease P, hammerhead ribozymes, hairpin ribozymes, hepatitis ribozymes, 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 a vRNA or viral protein expression vector may be identical or different from the promoter or any other vector. In one embodiment, a vector or plasmid expressing influenza vRNA includes a promoter suitable for expression in at least one specific host cell, e.g., bird, or mammalian host cells such as primate cells including, e.g., dog, cat, horse, cattle, or human cells, or for expression in one or more hosts.

[0033] In one embodiment, at least one vector relating to vRNA includes an RNA polymerase II promoter, which is bound to a ribozyme sequence bound to a viral coding sequence, which is bound to another ribozyme sequence, and optionally to an RNA polymerase II transcription termination sequence. In one embodiment, at least two, e.g., 3, 4, 5, 6, 7, or 8 vectors for vRNA production include an RNA polymerase II promoter, a first ribozyme sequence, which is located 5' to the sequence corresponding to the viral coding sequence, 5' to the second ribozyme sequence, and 5' to the transcription termination sequence. Each RNA polymerase II promoter in each vRNA vector may be identical or different from the RNA polymerase II promoter in any other vRNA vector. Similarly, each ribozyme sequence in each vRNA vector may be identical or different from the ribozyme sequences 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 number of influenza virus vectors for reassembly, such vectors comprising a vector for vRNA production comprising a promoter operably linked to influenza virus PA DNA bound to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PB1 DNA bound to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PB2 DNA bound to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus HA DNA bound to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NP DNA bound to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NA DNA bound to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus M DNA bound to a transcription termination sequence, and influenza virus NSA vector for vRNA production containing a promoter operably linked to cDNA, wherein the DNA of PB1, PB2, PA, NP, NS, and M is derived from one or more influenza vaccine seed viruses and contains two or more characteristic residues at specific positions; including a vector for mRNA production containing a promoter operably linked to the DNA segment encoding influenza virus PA, a vector for mRNA production containing a promoter operably linked to the DNA segment encoding influenza virus PB1, a vector for mRNA production containing a promoter operably linked to the DNA segment encoding influenza virus PB2, and a vector for mRNA production containing a promoter operably linked to the DNA segment encoding influenza virus NP; optionally, a vector for mRNA production containing a promoter operably linked to the DNA segment encoding influenza virus HA, a vector for mRNA production containing a promoter operably linked to the DNA segment encoding influenza virus NA, a vector for mRNA production containing a promoter operably linked to the DNA segment encoding influenza virus M1, a vector for mRNA production containing a promoter operably linked to the DNA segment encoding influenza virus M2, or a vector for mRNA production containing a promoter operably linked to the DNA segment encoding influenza virus NS2. In one embodiment, at least one vector includes a sequence corresponding to a sequence encoding PB1, PB2, PA, NP, M, or NS, or a portion thereof, and having substantially the same activity as the corresponding polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15, for example, a sequence encoding a polypeptide having amino acid identity of any integer 80-100%, at least 80%, for example, 85%, 90%, 92%, 95%, 98%, 99%, or 100% with the polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15. Optionally, influenza virus M conjugated to a transcription termination sequence.Instead of a vector containing a promoter operably linked to cDNA, two vectors are available, for example, one containing a promoter operably linked to influenza virus M1 cDNA bound to a transcription termination sequence, and another containing a promoter operably linked to influenza virus M2 cDNA bound to a transcription termination sequence.

[0035] The numerous vectors of the present invention can be physically bound, or each vector can reside on an individual plasmid or on another, for example, a linear, nucleic acid delivery vehicle. In one embodiment, each vRNA-producing vector is on a separate plasmid. In another embodiment, each mRNA-producing vector is on a separate plasmid.

[0036] The present invention also provides a method for preparing influenza viruses. Such a method includes, for example, contacting cells with a number of vectors of the present invention in an amount effective to obtain infectious influenza viruses, either sequentially or simultaneously. The present invention also includes a step of isolating the virus from cells bound to the number of vectors. Accordingly, the present invention further provides isolated viruses and host cells bound to the number of vectors or viruses of the present invention. In other embodiments, the present invention includes a step of contacting cells with one or more vectors, vRNAs or protein-producing vectors prior to other vectors, vRNAs or protein-producing vectors. In one embodiment, the promoter for the vRNA vector used in the method is the RNA polymerase I promoter, the RNA polymerase II promoter, the RNA polymerase III promoter, the T3 promoter or the T7 promoter. In one embodiment, the RNA polymerase I promoter is the human RNA polymerase I promoter. In one embodiment, each vRNA vector used in the method resides on a separate plasmid. In one embodiment, the vRNA vector used in the method resides on one plasmid or on two or three different plasmids. In one embodiment, each mRNA vector used in the method resides on a separate plasmid. In one embodiment, the mRNA vectors for PA, PB1, PB2, and NP used in the method are located on one plasmid or on 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 the steps of: providing cells suitable for the production of an influenza vaccine; continuously culturing one or more influenza virus isolates in the cells; and isolating the continuously cultured virus having enhanced growth compared to the one or more isolates before continuous culture. In one embodiment, the cells are rodent or primate cells.

[0038] The method for producing viruses without the need for helper virus infection, as described herein, is useful in viral mutagenicity testing, the production of vaccines (e.g., for AIDS, influenza, hepatitis B, hepatitis C, rhinovirus, filovirus, malaria, herpes, and foot and oral diseases), and the production of gene therapy vectors (e.g., for cancer, AIDS, adenosine deaminase, muscular dystrophy, ornithine transcarbamylase deficiency, and central nervous system tumors). Therefore, it provides viruses for use in medical therapy (e.g., vaccines or gene therapy).

[0039] The present invention also provides isolated viral polypeptides and methods for preparing and using recombinant viruses of the present invention. Such methods include administering an effective amount of the influenza virus of the present invention, for example, an inactivated viral preparation, optionally in combination with an adjuvant and / or carrier, to a host organism, for example, a mammal, in an amount effective to prevent or alleviate infection of an animal, such as a mammal, by the virus or a virus closely related to its antigenicity. In one embodiment, the virus is administered intramuscularly, while in other embodiments, the virus is administered intranasally. In some administration protocols, the dose can be administered entirely intramuscularly or intranasally, and in other protocols, a combination of intramuscular and intranasal administration can be utilized. The vaccine may further include, for example, other isolates of influenza viruses, including recombinant influenza virus, other pathogens, further biological materials, or microbial components to form a polyvalent vaccine. In one embodiment, for example, intranasal vaccination including an inactivated influenza virus and a mucosal adjuvant can induce virus-specific IgA and neutralizing antibodies, as well as serum IgG, in the nasopharynx.

[0040] The influenza virus of the present invention can be used in combination with other antiviral agents, such as amantadine, rimantadine, and / or neuraminidase inhibitors, and can be administered separately, for example, before, during, and / or after administration, in combination with these antiviral agents. [Brief explanation of the drawing]

[0041] [Figure 1A] Nucleotide sequences of the PR8 (Cambridge) gene (SEQ ID NOs: 10-15). [Figure 1B] Nucleotide sequences of the PR8 (Cambridge) gene (SEQ ID NOs: 10-15). [Figure 1C] Nucleotide sequences of the PR8 (Cambridge) gene (SEQ ID NOs: 10-15). [Figure 2] Overview of library passaging and identification of high-yield candidates. [Figure 3] The number of clones with random mutations possessing a specific HA titer. [Figure 4] The titer of a clone with a selective mutation. [Figure 5A] Growth curves of UW-PR8 viruses with previously identified mutations in PB2(A), PB1(B), PA(C), NP, M, or NS1(D). [Figure 5B] Growth curves of UW-PR8 viruses with previously identified mutations in PB2(A), PB1(B), PA(C), NP, M, or NS1(D). [Figure 5C] Growth curves of UW-PR8 viruses with previously identified mutations in PB2(A), PB1(B), PA(C), NP, M, or NS1(D). [Figure 5D] Growth curves of UW-PR8 viruses with previously identified mutations in PB2(A), PB1(B), PA(C), NP, M, or NS1(D). [Figure 6] An overview of mutations that confer high replication characteristics in MDCK cells. [Figure 7A] A) Virus strains were tested for HA titer (2n units) and viral titer (PFU / mL units). B) Growth curves in MDCK cells. [Figure 7B] A) Virus strains were tested for HA titer (2n units) and viral titer (PFU / mL units). B) Growth curves in MDCK cells. [Figure 8A-B]A) HA titers of wild-type (UW-PR8) and clone #4. B) Viral proteins of wild-type (UW-PR8) and #4. C) SDS-PAGE analysis of viral proteins of wild-type and #4. [Figure 8C] A) HA titers of wild-type (UW-PR8) and clone #4. B) Viral proteins of wild-type (UW-PR8) and #4. C) SDS-PAGE analysis of viral proteins of wild-type and #4. [Figure 9A] A) Comparison of titers between wild-type virus (UW-PR8) and high-replicating virus with M1 mutation. B) Growth rate of wild-type virus (UW-PR8) and high-replicating virus with M1 mutation. [Figure 9B] A) Comparison of titers between wild-type virus (UW-PR8) and high-replicating virus with M1 mutation. B) Growth rate of wild-type virus (UW-PR8) and high-replicating virus with M1 mutation. [Figure 10A] A) Canine codon utilization table. B) Relative adaptability between wild-type (UW-PR8) and "rare" codon-optimized PB2 virus. C) Relative adaptability between wild-type (UW-PR8) and "all" codon-optimized PB2 virus. D) Growth rate of PB2 codon-optimized virus. E) Growth rate of viruses having codon-optimized PB2, PB1, PA, or NP gene segments or combinations of segments. F) Sequences of PB2, PB1, PA, and NP gene segments of UW-PR8 and sequences of canine codon-optimized PB2, PB1, PA, and NP gene segments of UW-PR8. [Figure 10B] A) Canine codon utilization table. B) Relative adaptability between wild-type (UW-PR8) and "rare" codon-optimized PB2 virus. C) Relative adaptability between wild-type (UW-PR8) and "all" codon-optimized PB2 virus. D) Growth rate of PB2 codon-optimized virus. E) Growth rate of viruses having codon-optimized PB2, PB1, PA, or NP gene segments or combinations of segments. F) Sequences of PB2, PB1, PA, and NP gene segments of UW-PR8 and sequences of canine codon-optimized PB2, PB1, PA, and NP gene segments of UW-PR8. [Figure 10C]A) Canine codon utilization table. B) Relative adaptability between wild-type (UW-PR8) and "rare" codon-optimized PB2 virus. C) Relative adaptability between wild-type (UW-PR8) and "all" codon-optimized PB2 virus. D) Growth rate of PB2 codon-optimized virus. E) Growth rate of viruses having codon-optimized PB2, PB1, PA, or NP gene segments or combinations of segments. F) Sequences of PB2, PB1, PA, and NP gene segments of UW-PR8 and sequences of canine codon-optimized PB2, PB1, PA, and NP gene segments of UW-PR8. [Figure 10D] A) Canine codon utilization table. B) Relative adaptability between wild-type (UW-PR8) and "rare" codon-optimized PB2 virus. C) Relative adaptability between wild-type (UW-PR8) and "all" codon-optimized PB2 virus. D) Growth rate of PB2 codon-optimized virus. E) Growth rate of viruses having codon-optimized PB2, PB1, PA, or NP gene segments or combinations of segments. F) Sequences of PB2, PB1, PA, and NP gene segments of UW-PR8 and sequences of canine codon-optimized PB2, PB1, PA, and NP gene segments of UW-PR8. [Figure 10E] A) Canine codon utilization table. B) Relative adaptability between wild-type (UW-PR8) and "rare" codon-optimized PB2 virus. C) Relative adaptability between wild-type (UW-PR8) and "all" codon-optimized PB2 virus. D) Growth rate of PB2 codon-optimized virus. E) Growth rate of viruses having codon-optimized PB2, PB1, PA, or NP gene segments or combinations of segments. F) Sequences of PB2, PB1, PA, and NP gene segments of UW-PR8 and sequences of canine codon-optimized PB2, PB1, PA, and NP gene segments of UW-PR8. [Figure 10F]A) Canine codon utilization table. B) Relative adaptability between wild-type (UW-PR8) and "rare" codon-optimized PB2 virus. C) Relative adaptability between wild-type (UW-PR8) and "all" codon-optimized PB2 virus. D) Growth rate of PB2 codon-optimized virus. E) Growth rate of viruses having codon-optimized PB2, PB1, PA, or NP gene segments or combinations of segments. F) Sequences of PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 3, 2, 1, and 4, respectively) and sequences of canine codon-utilization optimized PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 25, 26, 27, and 28, respectively). [Figure 10G] A) Canine codon utilization table. B) Relative adaptability between wild-type (UW-PR8) and "rare" codon-optimized PB2 virus. C) Relative adaptability between wild-type (UW-PR8) and "all" codon-optimized PB2 virus. D) Growth rate of PB2 codon-optimized virus. E) Growth rate of viruses having codon-optimized PB2, PB1, PA, or NP gene segments or combinations of segments. F) Sequences of PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 3, 2, 1, and 4, respectively) and sequences of canine codon-utilization optimized PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 25, 26, 27, and 28, respectively). [Figure 10H] A) Canine codon utilization table. B) Relative adaptability between wild-type (UW-PR8) and "rare" codon-optimized PB2 virus. C) Relative adaptability between wild-type (UW-PR8) and "all" codon-optimized PB2 virus. D) Growth rate of PB2 codon-optimized virus. E) Growth rate of viruses having codon-optimized PB2, PB1, PA, or NP gene segments or combinations of segments. F) Sequences of PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 3, 2, 1, and 4, respectively) and sequences of canine codon-utilization optimized PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 25, 26, 27, and 28, respectively). [Figure 10I]A) Canine codon utilization table. B) Relative adaptability between wild-type (UW-PR8) and "rare" codon-optimized PB2 virus. C) Relative adaptability between wild-type (UW-PR8) and "all" codon-optimized PB2 virus. D) Growth rate of PB2 codon-optimized virus. E) Growth rate of viruses having codon-optimized PB2, PB1, PA, or NP gene segments or combinations of segments. F) Sequences of PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 3, 2, 1, and 4, respectively) and sequences of canine codon-utilization optimized PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 25, 26, 27, and 28, respectively). [Figure 10J] A) Canine codon utilization table. B) Relative adaptability between wild-type (UW-PR8) and "rare" codon-optimized PB2 virus. C) Relative adaptability between wild-type (UW-PR8) and "all" codon-optimized PB2 virus. D) Growth rate of PB2 codon-optimized virus. E) Growth rate of viruses having codon-optimized PB2, PB1, PA, or NP gene segments or combinations of segments. F) Sequences of PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 3, 2, 1, and 4, respectively) and sequences of canine codon-utilization optimized PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 25, 26, 27, and 28, respectively). [Figure 10K] A) Canine codon utilization table. B) Relative adaptability between wild-type (UW-PR8) and "rare" codon-optimized PB2 virus. C) Relative adaptability between wild-type (UW-PR8) and "all" codon-optimized PB2 virus. D) Growth rate of PB2 codon-optimized virus. E) Growth rate of viruses having codon-optimized PB2, PB1, PA, or NP gene segments or combinations of segments. F) Sequences of PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 3, 2, 1, and 4, respectively) and sequences of canine codon-utilization optimized PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 25, 26, 27, and 28, respectively). [Figure 10L]A) Canine codon utilization table. B) Relative adaptability between wild-type (UW-PR8) and "rare" codon-optimized PB2 virus. C) Relative adaptability between wild-type (UW-PR8) and "all" codon-optimized PB2 virus. D) Growth rate of PB2 codon-optimized virus. E) Growth rate of viruses having codon-optimized PB2, PB1, PA, or NP gene segments or combinations of segments. F) Sequences of PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 3, 2, 1, and 4, respectively) and sequences of canine codon-utilization optimized PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 25, 26, 27, and 28, respectively). [Figure 10M] A) Canine codon utilization table. B) Relative adaptability between wild-type (UW-PR8) and "rare" codon-optimized PB2 virus. C) Relative adaptability between wild-type (UW-PR8) and "all" codon-optimized PB2 virus. D) Growth rate of PB2 codon-optimized virus. E) Growth rate of viruses having codon-optimized PB2, PB1, PA, or NP gene segments or combinations of segments. F) Sequences of PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 3, 2, 1, and 4, respectively) and sequences of canine codon-utilization optimized PB2, PB1, PA, and NP gene segments of UW-PR8 (sequence numbers 25, 26, 27, and 28, respectively). [Figure 11A] A) Nucleotide position 4 of the PR8 and Indo / NC / 09 genes. B) All 3'C4U mutant strains. C) Growth rate of recombinant UW-PR8 virus (black) encoding "C" at position 4 of the PB2, PB1, and PA genes, and mutant strains (red) encoding "U" at position 4 of all eight segments. [Figure 11B] A) Nucleotide position 4 of the PR8 and Indo / NC / 09 genes. B) All 3'C4U mutant strains. C) Growth rate of recombinant UW-PR8 virus (black) encoding "C" at position 4 of the PB2, PB1, and PA genes, and mutant strains (red) encoding "U" at position 4 of all eight segments. [Figure 11C]A) Nucleotide position 4 of the PR8 and Indo / NC / 09 genes. B) All 3'C4U mutant strains. C) Growth rate of recombinant UW-PR8 virus (black) encoding "C" at position 4 of the PB2, PB1, and PA genes, and mutant strains (red) encoding "U" at position 4 of all eight segments. [Figure 12A] Representative H7 and N9 nucleotide and amino acid sequences for use with the internal gene segment sequences disclosed herein, which are effective in providing high-titer influenza viruses for vaccines. [Figure 12B] Representative H7 and N9 nucleotide and amino acid sequences for use with the internal gene segment sequences disclosed herein, which are effective in providing high-titer influenza viruses for vaccines. [Figure 12C] Representative H7 and N9 nucleotide and amino acid sequences for use with the internal gene segment sequences disclosed herein, which are effective in providing high-titer influenza viruses for vaccines. [Figure 13A] A) Diagram of chimeric HA and NA genes that enhance viral titer. B) Growth rate of chimeric viruses. [Figure 13B] A) Diagram of chimeric HA and NA genes that enhance viral titer. B) Growth rate of chimeric viruses. [Figure 14A] A) Growth rate of the virus with the mutation combination. B) PFU and HA titer of the virus with the mutation combination. [Figure 14B] A) Growth rate of the virus with the mutation combination. B) PFU and HA titer of the virus with the mutation combination. [Figure 15] Screening of eggs. [Figure 16] HA titers of 216 clones isolated from Vero cells. [Figure 17] Recombinant viruses created using different PR8 backbone mutations. [Figure 18A] An overview of the creation of a virus that promotes growth in MDCK cells and Vero cells. [Figure 18B] An overview of the creation of a virus that promotes growth in MDCK cells and Vero cells. [Modes for carrying out the invention]

[0042] Detailed description of the present invention definition In this specification, the term “isolation” means the in vitro preparation and / or isolation of nucleic acid molecules of the present invention, such as vectors or plasmids, peptides or polypeptides (proteins), or viruses, and thus unrelated to in vivo substances or substantially purified from in vitro substances. The preparation of isolated viruses is usually obtained by in vitro culture and growth and / or through passage in eggs, and is substantially free of other infectious pathogens.

[0043] In this specification, “substantially purified” means that the species of interest is the dominant species in the composition, for example, in abundance on a molar basis compared to any other individual species, preferably at least about 80% of the species present in the composition, optionally 90% or more, for example 95%, 98%, 99% or more of the species present.

[0044] In this specification, “substantially absent” means below the detection level for a particular infectious pathogen, using a standard detection method 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 its viral genome. Reassembled viruses can be prepared using recombinant or non-recombinant technologies.

[0046] In this specification, the terms “recombinant nucleic acid” or “recombinant DNA sequence or segment” mean nucleic acids, such as DNA, that originate from or are isolated from a certain origin and can subsequently be chemically modified in vitro, whose sequence is not of natural origin or corresponds to a sequence of natural origin that is not present in an unmodified genome. An example of DNA “originating” from a certain origin may be a DNA sequence that is recognized as a useful fragment and subsequently chemically synthesized in an essentially pure form. An example of DNA “isolated” from such an origin may be a useful DNA sequence that has been excised or removed from such origin by chemical means, such as the use of restriction enzymes, and can be further manipulated, for example, amplified, by genetic engineering methods for use in the present invention.

[0047] In this specification, “heterogeneous” influenza virus gene or gene segment means a recombinant, for example, reassembled influenza virus gene or gene segment in an influenza virus that originates from an influenza virus source different from that of most other influenza viruses.

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

[0049] As used herein, the terms “recombinant protein” or “recombinant polypeptide” refer to protein molecules expressed from recombinant DNA molecules. In contrast, as used herein, the term “undenatured protein” is used to refer to proteins isolated from natural sources (i.e., non-recombinant sources). Molecular biological techniques can be used to produce recombinant forms of proteins that have the same properties as the undenatured form of the protein.

[0050] Methods for comparing sequences are well known in the art. Therefore, measuring the percentage identity between any two sequences can be achieved using mathematical algorithms.

[0051] The computer execution of these mathematical algorithms can be used for sequence comparison to measure sequence identity. Sequence 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 determines whether the word matches or satisfies a positive threshold T when aligned with a word of equal length in the database sequence. T is called the neighbor word score threshold. These initial neighbor word hits serve as a seed to initiate a search for longer HSPs containing them. Subsequently, word hits are extended bidirectionally along each sequence as long as the cumulative sequence comparison score can be increased. The cumulative score is calculated with respect to the nucleotide sequence using parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, the score matrix is ​​used to calculate the cumulative score. Word hit extension in each direction is stopped when the cumulative sequence comparison score decreases from its maximum achieved value to amount X, when the cumulative score becomes zero or less due to the accumulation of 1 or more negative score residue sequence comparisons, or when one of the sequence ends is reached.

[0052] Furthermore, for the calculation of 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 sum probability (P(N)) that indicates the likelihood that the match between two nucleotide or amino acid sequences may occur by chance. For example, a test nucleic acid sequence is considered similar to a reference nucleic acid sequence if the minimum sum probability of the comparison of the test nucleic acid sequence with the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0053] The BLASTN program (for nucleotide sequences) can use a word length (W) of 11, an expected value (E) of 10, a cutoff value of 100, M=5, N=4, and a comparison of both strands as defaults. For amino acid sequences, the BLASTP program can use a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 score matrix as defaults. See http: / / www.ncbi.n1m.nih.gov. Sequence comparison can also be performed manually by observation.

[0054] In sequence comparison, typically one sequence serves as the reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence compared to the reference sequence based on the specified program parameters.

[0055] Structure and replication of influenza virus The influenza A virus has a genome of eight single-strand 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. Low pH in late endosomes induces a conformational change in the HA, resulting in the exposure of the N-terminus of the HA2 subunit (the so-called fusion peptide). The fusion peptide induces fusion of the virus and the endosomal membrane, releasing the matrix protein (M1) and the RNP complex into the cytoplasm. The RNP consists of a nucleoprotein (NP) that capsids the vRNA, and a viral polymerase complex formed by the PA, PB1, and PB2 proteins. The RNP is transported into the nucleus where transcription and replication take place. The RNA polymerase complex catalyzes three distinct reactions: the synthesis of mRNA with a 5' cap and 3' poly-A structure, the synthesis of full-length complementary RNA (cRNA), and the synthesis of genomic vRNA using cRNA as a template. Newly synthesized vRNA, NP, and polymerase proteins are subsequently assembled in RNPs, expelled from the nucleus, and transported to the cell membrane, where progeny viral particles budding occurs. Neuraminidase (NA) proteins play a crucial role in the late stages of infection by removing sialic acid from sialyl oligosaccharides, thereby releasing newly assembled viral particles from the cell surface and preventing self-aggregation of the viral particles. Viral 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 viruses, but some differences exist. For example, influenza B virus lacks the M2 protein, which has ion channel activity, but it does have BM2 and a gene segment containing 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 cell, including mutant cells such as any bird cell, or, for example, human cells such as 293T or PER.C6® cells, or canine cells such as MDCK, cattle, horses, cats, pigs, sheep, rodent cells such as mink cells such as MvLu1 cells, or hamster cells such as CHO cells, or non-human primate cells such as Vero cells, and other mammalian cells that assist in the efficient replication of the influenza virus, can be used to isolate and / or propagate the influenza virus. The isolated virus can be used to prepare a reassembled virus. In one embodiment, the host cells for vaccine production are maintained mammalian or bird strain cells or cell lines. Complete characterization of the cells used can be carried out so as to enable appropriate testing for the purity of the final product. Data that can be used to determine the properties of cells include (a) information on their origin, derivation, and passage history; (b) information on their growth and morphological characteristics; (c) test results for adventitious agents; (d) distinctive features that can be clearly recognized among other cell lines, such as biochemical, immunological, and cytogenetic patterns; and (e) test results for tumorigenic potential. In one embodiment, the passage level or population doubling of the host cells used should be as low as possible.

[0058] In one embodiment, the cells are serial cell lines approved or potentially approved by the WHO. Requirements for certification of such cell lines include characterization of at least one of the following: lineage, growth characteristics, immunomarkers, virus susceptibility to tumorigenicity, and storage conditions, and testing in animals, eggs, and cell cultures. Such characterization is used to confirm that the cells are free of detectable exogenous pathogens. In some countries, nuclear testing may also be required. Furthermore, tumorigenicity can be tested in cells at the same passage level as those used in vaccine production. The virus can be purified prior to vaccine production by a process that has been shown to produce consistent results (see, e.g., World Health Organization, 1982).

[0059] Viruses produced by host cells can be highly purified before being formulated as vaccines or gene therapies. Generally, purification methods result in the extensive removal of cellular DNA and other cellular components, as well as foreign pathogens. Methods that extensively degrade or denature DNA can also be used.

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

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

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

[0063] The split vaccine contains viral particles that have been subjected to treatment with a lipid-soluble agent. The split vaccine can be prepared as follows: The aqueous suspension of the obtained purified virus, whether inactivated or uninactivated, is treated with a lipid solvent such as ethyl ether or chloroform, in combination with a surfactant, under stirring. Dissolution of the viral coat lipids results in fragmentation of the viral particles. The removed aqueous phase contains the split vaccine, mainly consisting of hemagglutinin and neuraminidase, along with the removed viral lipid environment and the core or its degradation products. Subsequently, the residual infectious particles are inactivated if they have not yet been inactivated. The split vaccine can be combined with the attenuated virus of the present invention in a polyvalent vaccine.

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

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

[0066] Attenuated live virus vaccine For example, attenuated live influenza virus vaccines, such as the recombinant virus vaccine 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 transferring attenuating genes from an attenuated donor virus to a replicated isolate or a reassorted virus. Since resistance to influenza A virus is first mediated by the development of an immune response to HA and / or NA glycoproteins, the genes encoding these surface antibodies are derived from the reassorted virus or clinical isolate. The attenuating genes are derived from the attenuated parent. In this approach, the genes conferring attenuation do not typically encode HA and NA glycoproteins.

[0067] A virus (donor influenza virus) capable of reproducibly attenuating influenza viruses can be used; for example, a cold-adapted (ca) donor virus can be used in the production of attenuated vaccines. Live, attenuated reassembled virus vaccines can be produced by conjugating a ca donor virus with a pathogenic replicating virus. The progeny of the reassembled virus are then selected at 25°C (limited to the replication of pathogenic viruses) in the presence of a suitable antiserum, which inhibits the replication of viruses that have surface antibodies to the attenuated ca donor virus. Useful reassembled viruses are (a) infectious, (b) attenuated for seronegative non-adult mammals and immunologically first stimulated adult mammals, (c) immunogenic, and (d) genetically stable. The immunogenicity of the ca reassembled viruses is comparable to their replication levels. Thus, the acquisition of six transmissible genes of ca donor viruses by novel wild-type viruses reproducibly attenuates these viruses for use in the 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 containing these mutant genes can be rescued. Attenuating mutations can be introduced into the uncoding and coding regions of the genome. These attenuating mutations can also be introduced into genes other than HA or NA, such as the PB2 polymerase gene. Therefore, novel donor viruses containing attenuating mutations introduced by site-directed mutagenesis can also be produced, and such potentially novel donor viruses can be used in the production of attenuated bioreassembled vaccines in a manner similar to the above method for ca donor viruses. Similarly, other well-known and suitable attenuated donor strains can be reassembled with the influenza virus to obtain attenuated vaccines suitable for use in mammalian vaccination.

[0069] In one embodiment, such an attenuated virus retains viral genes encoding antigenic determinants substantially similar to those of the original clinical isolate. This is because the aim is for the attenuated vaccine to exhibit substantially the same antigenicity as the original clinical isolate of the virus, while simultaneously lacking pathogenicity to the extent that it is unlikely to induce a severe disease state in vaccinated mammals.

[0070] Therefore, the viruses in a polyvalent vaccine can be attenuated or inactivated to induce an immune response in an animal, such as a mammal, and then formulated and administered as a vaccine according to well-known methods. Methods for determining whether such an attenuated or inactivated vaccine retains similar antigenicity to a clinical isolate or a high-growth strain derived therefrom are well-known in the art. Such well-known methods include the use of antiserum or antibodies to remove viruses expressing the antigenic determinants of the donor virus; chemical selection (e.g., amantadine or rimantadine); HA and NA activity and suppression; and nucleic acid screening (e.g., probe hybridization or PCR, etc.) to confirm that the donor gene encoding the antigenic determinant (e.g., HA or NA gene) is not present in the attenuated virus.

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

[0072] Conventional vaccines generally contain about 0.1 to 200 μg, for example, 30 to 100 μg, of HA derived from each strain added to their composition. The vaccine forming the main component of the vaccine composition of the present invention may include a combination of influenza viruses, such as one influenza virus or one or more reassembled influenza viruses, for example, at least two or three influenza viruses.

[0073] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and / or emulsions, and may 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 injectable organic esters such as ethyl oleate. Carriers or occlusive dressings may be used to increase skin penetration and enhance antigen absorption. Liquid dosage forms for oral administration generally include liposome solutions containing liquid dosage forms. Suitable forms of liposome suspension 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 may also contain adjuvants, wetting agents, emulsifiers and suspending agents, or sweeteners, flavorings, or fragrances.

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

[0075] The heterogeneity of the vaccine can be provided by mixing at least two influenza virus strains, for example 2 to 20 strains, or any range or value of replicated influenza viruses. The vaccine can be provided for variations within a single strain of influenza virus using techniques well known in the art.

[0076] The pharmaceutical compositions of the present invention may further or additionally comprise at least one chemotherapeutic agent, for example, a chemotherapeutic compound for gene therapy, an immunosuppressant, an anti-inflammatory agent or an immunoenhancing agent, and a chemotherapeutic agent for vaccines, which may include, but are 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, phosphonoacetate, acyclovir, dideoxynucleoside, protease inhibitor, or ganciclovir.

[0077] Such compositions may also contain small, variable amounts of endotoxin-free formaldehyde and preservatives, which are known to be safe and not contribute to undesirable effects in organisms to which the composition is administered.

[0078] For medical purposes The administration of such a composition (or the antiserum it induces) may be for “preventive” or “therapeutic” purposes. When used for prevention, the vaccine composition of the present invention is provided before any symptom or clinical sign of a pathogen infection becomes apparent. Prophylactic administration of such a composition helps prevent or reduce subsequent infections. When used for prevention, the gene therapy composition of the present invention is provided before any symptom or clinical sign of a disease becomes apparent. Prophylactic administration of such a composition helps prevent or reduce one or more symptoms or clinical signs associated with the disease.

[0079] When provided therapeutically, viral vaccines are used to detect the symptoms or clinical signs of actual infection. Therapeutic administration of the compound helps to attenuate the actual infection. When provided therapeutically, gene therapy compositions are used to detect the symptoms or clinical signs of disease. Therapeutic administration of the compound helps to attenuate the symptoms or clinical signs of that disease.

[0080] Accordingly, the vaccine composition of the present invention can be administered before the onset of infection (to prevent or reduce the predicted infection) or after the actual onset of infection. Similarly, such composition can be administered for gene therapy before any symptom or clinical sign of a disease or disorder manifests or after one or more symptoms are detected.

[0081] A composition may be considered "pharmaceutically acceptable" if the recipient mammal can tolerate its administration. Such a drug is said to be administered in a "therapeutably effective dose" if the amount administered is physiologically significant. The compositions of the present invention are physiologically effective if their presence causes 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 to at least one strain of infectious influenza virus.

[0082] The “prevention” provided does not need to be absolute; that is, it is sufficient if there is a statistically significant improvement compared to a mammalian control group or population, and influenza infection does not need to be completely prevented or eradicated. Prevention may be limited to reducing the severity or rate of onset of symptoms or clinical signs of influenza virus infection.

[0083] Medication The compositions of the present invention can confer resistance to one or more pathogens, such as one or more influenza virus strains, by passive or active immunity. In active immunity, the attenuated live 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 immunity, the induced antiserum can be collected and administered to a recipient suspected of being infected with an infection caused by at least one influenza virus strain. The gene therapy compositions of the present invention can yield the desired gene product at a prophylactic or therapeutic level by active immunity.

[0084] In one embodiment, the vaccine is administered to a female mammal (during or before pregnancy or childbirth) under conditions of sufficient time and quantity to produce an immune response, thereby helping to protect both the female and the fetus or newborn (through passive uptake of antibodies via the placenta or in breast milk).

[0085] Accordingly, the present invention includes methods for preventing or mitigating a disorder or disease, for example, infection by at least one strain of a pathogen. In this specification, a vaccine is said to prevent or mitigate a disease if its administration results in whole or partial reduction (i.e., suppression) of the clinical signs or condition of the disease, or whole or partial immunity of an individual to the disease. In this specification, a gene therapy composition is said to prevent or mitigate a disease if its administration results in whole or partial reduction (i.e., suppression) of the clinical signs or condition of the disease, or whole or partial immunity of an individual to the disease.

[0086] A composition comprising at least one influenza virus of the present invention, comprising a weakened virus and one or more other isolated viruses, one or more of its isolated viral proteins, one or more isolated nucleic acid molecules encoding one or more of its viral proteins, or a combination thereof, can be administered by any means to achieve the objective.

[0087] For example, these compositions can be administered via various parenteral routes, such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral, or transdermal routes. Parenteral administration can be achieved by rapid intravenous injection or slow perfusion.

[0088] A typical dosing regimen for preventing, suppressing, or treating symptoms associated with the influenza virus involves administering an effective amount of the vaccine composition herein, either as a single treatment or repeated as an augmentation or bolus dose, for a period ranging from one week to approximately 24 months, or any range or value within that range.

[0089] According to the present invention, the “effective amount” of the composition is an amount sufficient to achieve the desired effect. It is understood that the effective dose may depend on the recipient’s species, age, sex, health, and weight, the type of concomitant treatment if used in combination, the frequency of treatment, and the nature of the desired effect. The effective dose range provided below is not intended to limit the dose range of the present invention.

[0090] For example, the dosage of attenuated live or dead virus vaccines for animals such as adult mammals is approximately 10 2 ~10 15 For example, 10 3 ~10 12 The plaque-forming units (PFUs) / kg may be any of the above ranges. The dose of the inactivated vaccine may be approximately 0.1 to 1000, for example, 30 to 100 μg of HA protein. However, the dose should be a safe and effective amount determined by standard methods, starting with an existing vaccine.

[0091] The amount of immunoreactive HA in each dose of replicated viral vaccine can be standardized to a suitable dose, e.g., 30–100 μg or any range or value within this range, or to an amount recommended by a government agency or authorized professional institution. The amount of NA can also be standardized, although this glycoprotein may be unstable during purification and storage.

[0092] The dose of immune response HA in each dose of replicated viral vaccine can be standardized to contain an appropriate amount, e.g., 1 to 50 μg or any range or value within this range, as recommended by the US Public Heath Service (PHS), typically 15 μg per component for older children (3 years and older) and 7.5 μg for children under 3 years of age. The amount of NA can also be standardized, although this glycoprotein can be unstable during processor purification and storage (Kendal et al., 1980; Kerr et al., 1975). Each 0.5-ml dose of vaccine may contain approximately 1 to 50 billion viral particles, preferably 10 billion particles.

[0093] The present invention will be explained by the following non-limiting embodiments. [Examples]

[0094] Example 1 method Cells and viruses 293T human embryonic kidney cells are maintained in Dulbecco's modified Eagle minimal basal medium (DMEM) with 10% fetal calf serum and antibiotics. Maidin' Derby canine kidney (MDCK) cells are grown in MEM with 5% neonatal calf serum and antibiotics. African green monkey cub (WCB) cells, established after biosafety testing for use in human vaccine production (Sugawara et al., 2002), are maintained in serum-free VP-SFM medium (GIBCO-BRL) containing antibiotics. Cells are maintained at 37°C in 5% CO2. The WHO-recommended vaccine seed virus is NIBRG-14.

[0095] Plasmid creation and reverse genetics Reverse genetics based on plasmids (Neumann et al., 1999) was used to generate a reassembly of influenza A virus. Full-length cDNA was cloned into plasmids under the control of a human polymerase I promoter and a mouse RNA polymerase I terminator (PolI plasmid).

[0096] A pre-generated series of PolI constructs derived from A / WSN / 33(H5N1;WSN) or PR8 strains are used in 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] For all reverse genetics experiments, use chicken triactin, e.g., beta-actin, or plasmids expressing WSN or PR8 NP, PA, PB1, or PB2 under promoter control (Horimoto et al., 2006; Neumann et al., 1999). Specifically, the PolI plasmid and protein expression plasmid are mixed with the translocation reagent, Trans-IT 293T (Panvera), incubated at room temperature for 15 minutes, and then added to 293T cells. Translocation cells are incubated in Opti-MEMI (GIBCO-BRL) for 48 hours. For reverse genetics in Vero WCB cells, the plasmid mixture is translocated using an electroporator (Amaxa) according to the manufacturer's instructions. Sixteen hours after translocation, freshly prepared Vero WCB cells are added to the translocation cells, and TPCK-trypsin (1 g / mL) is added to the culture six hours later. Transplanted cells are incubated in serum-free VP-SFM for a total of 4 days. The supernatant containing infectious virus is collected and can be bebiologically cloned by limiting dilution.

[0098] A recombinant virus was prepared containing the HA and NA genes from A / Hong Kong / 213 / 2003 (H5N1) and the remainder from the UW-PR8 influenza A virus. The titer of the recombinant virus was 1010.67 EID. 50 The concentration was / mL, and the HA titer was 1:1600.

[0099] [Table 1]

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

[0101] [ka]

[0102] [ka]

[0103] [ka]

[0104] [ka]

[0105] [ka]

[0106] [ka]

[0107] [ka]

[0108] The high-titer A / PR / 8 / 34 (H1N1, UW-PR8) virus grows 10 times 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 will produce a vaccine strain that can be safely generated, confirming the use of UW-PR8 as a master vaccine strain.

[0109] Genes contributing to the differing growth characteristics between UW-PR8 and PR8(Cambridge) were measured using non-HA and non-NA genes (Figure 1) of the NIBRG-14 vaccine strain. High titers were obtained in eggs when the majority of internal genes were derived from UW-PR8. The highest titers were found in strains possessing 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). Polymerase subunits (PA, PB1, and PB2) and the NP gene of UW-PR8 enhanced the growth of H5N1 vaccine seed virus in chicken hatchling eggs, while the NS gene of PR8(Cambridge) enhanced the growth of H5N1 vaccine seed virus in chicken hatchling eggs. The tyrosine (Y) at position 360 in PB2 of UW-PR8 likely contributes to the high growth rate of this virus in MDCK cells.

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

[0111] [Table 2]

[0112] The viral library was passaged 12 times in MDCK cells, or after 2 passages, the library was mixed and then passaged another 10 times (Figure 2). After 10 to approximately 12 consecutive passages in MDCK cells, plaque assays were performed, and over 1400 individual plaques were selected. Figure 3 shows the number of clones with varying 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 produced from selective mutations. The sequences of the 36 viruses with the highest HA titers from the random mutation library were determined (Table 2).

[0113] [Table 3] [Table 4]

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

[0115] [Table 5]

[0116] In the third approach, candidate strains from approaches 1 and 2 were combined, and HA titer 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 were identified, including #1, #4, #36, #38, P17, P16, and P61. To identify the growth characteristics of these viruses, their growth rates in MDCK cells were 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 shows the SDS-PAGE analysis of the viral proteins for the wild type (UW-PR8) and #4. Further analysis demonstrated that viruses with the V97A / Y100H mutation in M1 produced higher HA titers than the parent virus, but lower viral titers overall (see Figures 9A-B). The V97A / Y100H mutation in M1 could result in particles with a larger surface area, allowing for the incorporation of more HA protein. Since inactivated influenza viruses are administered based on their HA content, mutant strains with high HA content are attractive vaccine candidates.

[0119] To identify mutations in the influenza promoter region that lead to enhanced replication, viruses with a "U" at position 4 of the 3' end of all eight vRNA segments were prepared using the internal genes of UW-PR8 PA, PB1, and PB2 (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 containing combinations of promoter mutations and amino acid changes were prepared, and their titers were measured (Table 5).

[0120] [Table 8]

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

[0122] [Table 9]

[0123] Optimization of rare codons in PB2 resulted in increased titer compared to the wild-type virus (UW-PR8) (see Figure 10D). Codon optimization of other gene segments was performed, and the titers of viruses with those segments or combinations of optimized segments were measured (Figure 10E). Another approach to enhance viral titers in MDCK cells involved preparing chimeric HA and NA genes (Figure 13A) and measuring the titers of viruses containing these genes (Figure 13B).

[0124] Viruses possessing the above-mentioned combinations of mutations (high-growth backbone mutations, promoter mutations, chimeric HA and NA genes, and inucodon optimization) were prepared, and the growth rate, PFU, and HA titer of these viruses were measured (see Figure 14). Representative backbone mutations include inucodon opti-PB2+C4U+M202L, F323L; PB1:C4U+Q247H; PA:C4U+K142N; NP:inucodon opti-NP+R74K; M:V97A, Y100H; and NS:K55E. Any mutations or any combination thereof described herein may be combined with, for example, seasonal H1N1 and H3N2, H3N2 mutants, PdmH1N1, H5N1, H7N9 or H9N2, or other branch groups 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 viral libraries were prepared and passaged 10 times in eggs. High-growth mutants were screened using three limiting dilutions (Figure 15). In one embodiment, a mutant strain exhibiting high growth characteristics in MDCK cells has a PB2 gene segment having a promoter mutation (C4U) and a mutation resulting in I504V (relative to the parent virus); a PB1 gene segment having a promoter mutation (C4U) and a mutation resulting in E112G; a PA gene segment having a promoter mutation (C4U) and a mutation resulting in S225C; an NP gene segment having mutations resulting in R74K and N417D; an M gene segment having mutations resulting in V97A and Y100H; and an NS gene segment having a mutation resulting in K55E, wherein optionally, the sequences of one or more gene segments, for example, the NP gene segment, are modified to include optimized inucodons. In one embodiment, a mutant strain exhibiting high growth characteristics in MDCK cells has an inucodon-optimized PB2 gene segment having a promoter mutation (C4U) and mutations resulting in M202L and F323L; a PB1 gene segment having a promoter mutation (C4U) and a mutation resulting in Q247H; a PA gene segment having a promoter mutation (C4U) and a mutation resulting in K142N; an inucodon-optimized NP gene segment having a mutation resulting in R74K; an M gene segment having mutations resulting in V97A and Y100H; and an NS gene segment having a mutation resulting in K55E.

[0125] Similar experiments were performed in Vero cells using clones with high replication characteristics in MDCK cells, for example, after approximately 3-5 passages in Vero cells (see Figure 16). Figure 17 shows five viruses that may have high replication characteristics in Vero cells. In one embodiment, a PR8(UW) mutant strain with high growth characteristics in Vero cells has the following mutations, which can be used in various combinations to enhance the replication capacity 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 of NS1), or R118K (amino acid change of NS1).

[0126] 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 incorporated herein by reference. In the foregoing specification, the present invention is described in relation to certain preferred embodiments, and many things are described in detail for illustrative purposes, but it will be apparent to those skilled in the art that the invention can be adapted to further embodiments, and some of the details described herein can be considerably modified without departing from the fundamental principles of the invention. [1] An isolated recombinant influenza virus having PA, PB1, PB2, NP, NS, and M gene segments, heterologous or chimeric influenza virus NA gene segment, and heterologous or chimeric HA gene segment derived from a first influenza vaccine virus isolate, wherein two or more of the 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; and positions 40, 54, 59, 62, 63, 75, 76 of PB1. , 78, 79, 80, 112, 180, 247, 327, 507, 624, 644, 667, 694, 695, 697, 699, 700, 701, 702, 705, 713, and / or 714th positions of PB2; 57, 58, 59, 61, 66, 202, 323, 368, 391, 504, 591, 677, 678, and / or 679th positions of PB2; 74, 112, 116, 224, 293, 371, 377, 417, 422, or 442nd positions of NP; 90, 97, and / or 100th positions of M1; or 30, 49, 55, 118, 140, 161, and / or 223rd positions of NS1 Isolated recombinant influenza virus. [2] The isolated virus according to claim 1, wherein PA has 142N, 225C, 356R, or 550L; PB1 has one or more 112G, 247H, 507V, or 644A; PB2 has one or more 202L, 323L, or 504V; NP has one or more 74K, 112L, 116L, 417D, or 442A; M1 has 97A and / or 100H; and / or NS1 has 55E and / or 140Q, or a combination thereof. [3] The isolated virus according to claim 1 or 2, having at least one of PB2's 202L and / or 323L, PB1's 247H, or NP's 74K, and optionally having at least one of PA1's 142N, NS1's 55K, or M1's 97A and / or 100H. [4] The isolated virus according to claim 1 or 2, having at least one of PB2's 202L and / or 323L, PB1's 247H, or NP's 74K, and having at least one of PA1's 142N, NS1's 55K, or M1's 97A and / or 100H. [5] The isolated virus according to claim 1, wherein PB2 has 202L and / or 323L. [6] The isolated virus according to claim 1 or 5, wherein PB1 has 247H. [7] The isolated virus according to claim 1, 5, or 6, wherein the NP has 74K. [8] The isolated virus according to any one of claims 1 to 7, wherein the gene segment of PB1, PB2, or PA has U at position 4. [9] The isolated virus according to claim 1 or 2, wherein PB1 has 40I, 40L, 112G, 180W, 247H, 507V, or 644A.

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

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

[12] An isolated virus according to any one of claims 1 to 2 or 9 to 11, wherein NS1 has 30P, 118K, 161T, or 140Q.

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

[14] The isolated virus according to claim 1, wherein selected amino acid residues at specific positions of PA are located at positions 97, 105, 142, 149, 225, 356, 357, 401, 404, and / or 421 (one or more).

[15] The isolated virus according to claim 1 or 14, wherein the selected amino acid residues at specific positions of PB1 are located at positions 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 (one or more).

[16] The isolated virus according to any one of claims 1 or 14 to 15, wherein selected amino acid residues at specific positions of PB2 are located at positions 57, 58, 59, 61, 66, 202, 243, 323, 504, 677, 678, and / or 679 (one or more).

[17] The isolated virus according to claim 1 or any one of claims 14 to 16, wherein selected amino acid residues at specific positions of the NP are located at positions 74, 112, 116, 224, 293, 417, and / or 442 (one or more).

[18] The isolated virus according to any one of claims 1 to 17, wherein the selected amino acid residues at specific positions of M1 are located at positions 90, 97, and / or 100 (one or more).

[19] The isolated virus according to any one of claims 1 to 18, wherein the selected amino acid residues at specific positions of NS1 are located at positions 49, 30, 55, 161, and / or 223 (one or more).

[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 are: PB1 having the amino acid sequence encoded by SEQ ID NO: 2 or PB1 having at least 95% amino acid sequence identity to 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% amino acid sequence identity to 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% amino acid sequence identity to PA encoded by SEQ ID NO: 1; NP having the amino acid sequence encoded by SEQ ID NO: 4 or NP having at least 95% amino acid sequence identity to 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% amino acid sequence identity to 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% amino acid sequence identity to NS encoded by SEQ ID NO: 6 It contains at least one sequence of NS having 95% amino acid sequence identity; or the PA, PB1, PB2, NP, NS, and M gene segments include: PB1 having the amino acid sequence encoded by SEQ ID NO: 10 or PB1 having at least 95% amino acid sequence identity to 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% amino acid sequence identity to 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% amino acid sequence identity to 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% amino acid sequence identity to 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% amino acid sequence identity to M encoded by SEQ ID NO: 14;Alternatively, an isolated virus according to any one of claims 1 to 21, comprising at least one sequence of an NS having the amino acid sequence encoded by SEQ ID NO: 15 or an NS having at least 95% amino acid sequence identity with respect to the NS encoded by SEQ ID NO: 15.

[23] An 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 having an isolated recombinant virus according to any one of claims 1 to 23.

[25] Multiple influenza virus vectors for preparing reassemblies, 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 vRNA production vector are derived from one or more influenza vaccine virus isolates, and the NA in the vRNA production vector is derived from NA The DNA has sequences relating to 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, 71 3, or 714, and / or 247; PB2's 57, 58, 59, 61, 66, 202, 323, 368, 391, 504, 591, 677, 678, 679, 202, and / or 323; NP's 74, 112, 116, 224, 293, 371, 377, 417, 422, and / or 442; M1's 90, 97, and / or 100;Or a vector having 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 containing this.

[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 polypeptides having at least 95% amino acid sequence identity with the corresponding polypeptides 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 the RNA polymerase I promoter, the RNA polymerase II promoter, the RNA polymerase III promoter, the T3 promoter, or the 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 mutations for promoter C.

[31] A method for preparing an influenza virus, A sufficient amount to obtain 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 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 sequences related to heterologous NA, and the HA DNA in the vector for HA vRNA production is derived from PA 30, 31, 105, 142, 149, 225, 356, 357, 401, and / or 550; PB1 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; PB2 57, 5 Vectors having heterologous HA sequences of 8, 59, 61, 66, 202, 323, 368, 391, 504, 591, 677, 678, 679, 202, and / or 323; NP 74, 112, 116, 224, 293, 371, 377, 417, 422, and / or 442; M1 90, 97, and / or 100; or NS 30, 49, 55, 118, 140, 161 or 223, and A vector for mRNA production containing a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for mRNA production containing a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector for mRNA production containing a promoter operably linked to a DNA segment encoding influenza virus PB2, a vector for mRNA production containing a promoter operably linked to a DNA segment encoding influenza virus NP, optionally a vector for mRNA production containing a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for mRNA production containing a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for mRNA production containing a promoter operably linked to a DNA segment encoding influenza virus M1, a vector for mRNA production containing a promoter operably linked to a DNA segment encoding influenza virus M2, or a vector for mRNA production containing a promoter operably linked to a DNA segment encoding influenza virus NS2. A method for preparing an influenza virus, including contacting it with cells.

[32] The method according to claim 31, wherein the cells are chicken cells.

[33] The method according to claim 31, wherein the cells are mammalian cells.

[34] The method according to claim 31, wherein the cells are Vero cells, human cells or MDCK cells.

[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 have sequences corresponding to DNA encoding polypeptides having at least 95% amino acid sequence identity with the corresponding polypeptides 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 described in any one of claims 31 to 37.

[39] A vector for the expression of influenza virus PA vRNA or mRNA 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 the expression of influenza virus PB1 vRNA or mRNA 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 the expression of PB2 vRNA or mRNA 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 the expression of influenza virus NP vRNA or mRNA 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 the expression of influenza virus NS1 vRNA or mRNA 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.

[44] A vector for the expression of influenza virus M1 vRNA or mRNA, having at least 95% amino acid sequence identity to the M1 polypeptide encoded by SEQ ID NO: 5 and having serine at position 90.

Claims

1. A composition comprising a dose of isolated recombinant influenza A virus having the viral segments PA, PB1, PB2, NP, NS, and M, a heterologous influenza virus NA viral segment, and a heterologous HA viral segment, wherein two or more of the PA, PB1, or PB2 viral segments are Code a PA having 401K, Code PB1 having 40L and 180W, 112G, or 247H, Code PB2 having 202L or 323L; or 1) The PB2 has 202L and 323L, and the PB1 has 247H, 2) The PB2 has 202L and 323L, and the PA has 401K, 3) The PB1 has 247H, and the PA has 401K, 4) The PB2 has 202L and 323L, and the PB1 has 112G, 5) The PA has 225C and the PB1 has 112G, 6) The PB2 has 202L and 323L, and the PB1 has 40L and 180W, or 7) The PB2 has 202L and 323L, and the PB1 has 247H, And the recombinant virus is 3 x 10 7 The composition having a potency exceeding PFU / ml.

2. The composition according to claim 1, further comprising 644A in PB1, 74K and 417D or 116L in NP, or 97A and / or 100H in M1.

3. The composition according to claim 1 or 2, wherein one of the viral segments PB1, PB2, or PA has U at position 4.

4. The composition according to claim 1, wherein PB1 further comprises 40I, 40L, or 644A, or NP further comprises 74K and 417D, or 116L, or NS1 further comprises 30P, or 161T.

5. The composition according to claim 1, wherein PA contains 401K, PB1 contains 40L and / or 180W, PB2 contains 202L and 323L, or NP contains 116L.

6. The composition according to any one of claims 1 to 5, wherein at least one of the viral segments PA, PB1, PB2, NP, NS, and M has a promoter mutation from C to U.

7. The composition 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.

8. The viral segments PA, PB1, PB2, NP, NS, and M in recombinant viruses 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 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% amino acid sequence identity with PB2 encoded by SEQ ID NO: 3; PA having the amino acid sequence encoded by Sequence ID No. 1, or PA having at least 95% amino acid sequence identity with PA encoded by Sequence ID No. 1; NP having the amino acid sequence encoded by SEQ ID NO: 4, or NP having at least 95% amino acid sequence identity with 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% amino acid sequence identity with 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% amino acid sequence identity with NS encoded by SEQ ID NO: 6, It contains at least one of the following sequences, or The aforementioned virus segments PA, PB1, PB2, NP, NS, and M are as follows: PB1 having the amino acid sequence encoded by SEQ ID NO: 10, or PB1 having at least 95% amino acid sequence identity with 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% amino acid sequence identity with 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% amino acid sequence identity with 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% amino acid sequence identity with 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% amino acid sequence identity with M encoded by SEQ ID NO: 14, NS having the amino acid sequence encoded by SEQ ID NO: 15, or NS having at least 95% amino acid sequence identity with NS encoded by SEQ ID NO: 15 A composition according to any one of claims 1 to 7, comprising at least one of the sequences.

9. The isolated virus, 10 8 A composition according to any one of claims 1 to 8, having a potency exceeding PFU / ml.

10. A vaccine manufacturing method provides a dosage of isolated recombinant influenza A virus having the viral segments PA, PB1, PB2, NP, NS, and M, the viral segment of heterologous influenza virus NA, and the viral segment of heterologous HA, wherein two or more of the PA, PB1, or PB2 viral segments are Code a PA having 401K, Code PB1 having 40L and 180W, 112G, or 247H, Code PB2 having 202L or 323L, or 1) Here, PB2 has 202L and 323L, and PB1 has 247H, 2) Here, if PB2 has 202L and 323L, and PA has 401K, 3) Here, PB1 has 247H and PA has 401K, 4) Here, PB2 has 202L and 323L, and PB1 has 112G, 5) Here, PA has 225C and PB1 has 112G, 6) Here, PB2 has 202L and 323L, and PB1 has 40L and 180W, or 7) Here, PB2 has 202L and 323L, and PB1 has 247H, The aforementioned recombinant virus 3 × 10 7 A method for producing the vaccine having a potency exceeding PFU / ml.

11. Multiple vectors for preparing influenza A virus, 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 containing a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence. A vRNA production vector containing a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence. A vRNA production vector containing a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence. A vRNA production vector containing a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence. A vRNA production vector comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence, and A vRNA production vector containing a promoter operably linked to influenza virus NS DNA linked to a transcription termination sequence. Includes, Here, the DNA of PB1, PB2, PA, NP, NS, and M in the vRNA production vector is derived from one or more influenza virus isolates. The NA DNA in the NA vRNA production vector has a heterologous NA sequence. The HA DNA in the HA vRNA production vector has the sequence of a heterologous HA. Here, two or more of the PA, PB1, or PB2 viral segments Code a PA having 401K, Code PB1 having 40L and 180W, 112G, or 247H, Code PB2 having 202L or 323L, or 1) Here, PB2 has 202L and 323L, and PB1 has 247H, 2) Here, PB2 has 202L and 323L, and PA has 401K, 3) Here, PB1 has 247H and PA has 401K, 4) Here, PB2 has 202L and 323L, and PB1 has 112G, 5) Here, PA has 225C and PB1 has 112G, 6) Here, PB2 has 202L and 323L, and PB1 has 40L and 180W, or 7) The plurality of vectors wherein PB2 has 202L and 323L, and PB1 has 247H.

12. The vector according to claim 11, wherein the DNA of PB1, PB2, PA, NP, NS, and M in the vector for vRNA production has sequences corresponding to sequences that encode polypeptides having at least 95% amino acid sequence identity with respect to the corresponding polypeptides encoded by SEQ ID NOs. 1-6 or 10-15.

13. The vector according to claim 11 or 12, wherein at least one of the PA, PB1, or PB2 viral segments has a promoter mutation from C to U.

14. The composition according to claim 1, wherein PB2 comprises C4U and 504V, PB1 comprises C4U, 40L and 180W, PA comprises C4U and 401K, NP comprises 116L, and / or NS1 comprises 30P or 118K.

15. The composition according to claim 1, wherein PB2 has 504V and PB1 has 112G, or wherein PB2 has 504V, PB1 has 40L and 180W, PA has 401K, and NS1 has 30P or R118K; or wherein PB2 has 202L and 323L, and PB1 has 247H.

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