Improved influenza B virus replication for vaccine development

Abstract

The objective of this study is to provide a mutant influenza B virus that contains the "internal" segments of influenza B virus, but has enhanced viral titer, total protein yield, HA content, etc. in cultured cells and embryonated eggs, and is safe for humans. [Solution] The positions and types of favorable amino acid mutations in the "internal" segments of influenza B virus are selected and identified through screening, as well as through reverse genetics, and specific recombinant influenza B viruses obtained by combining these are constructed, and their functional effects are demonstrated through experiments.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. patent application Ser. No. 62 / 297,400, filed Feb. 19, 2016, the contents of which are incorporated herein by reference in their entirety.

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

[0003] Influenza B viruses are a major cause of human respiratory disease. The segmented nature of the influenza virus genome allows for segment reassortment during viral replication in cells infected with two or more influenza viruses. This segment reassortment, combined with genetic mutation and drift, can give rise to numerous divergent influenza virus lineages over time. New lineages exhibit antigenic variation in their hemagglutinin (HA) and / or neuraminidase (NA) proteins, and the gene encoding the HA protein is particularly prone to variability. The primary current treatment for influenza prevention is vaccination. Because 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 outbreaks are crucial for vaccine development. Based on epidemics and forecasts, vaccines are designed to stimulate protective immune responses against dominant and predicted influenza virus lineages (Park et al., 2004).

[0004] There are three general types of influenza viruses: A, B, and C, which are defined by the lack of serological cross-reactivity between their internal proteins. Influenza B viruses are further divided into two lineages based on antigenic and genetic differences in the glycoprotein HA.

[0005] The human burden of influenza A and B virus infections is significant, and the impact of influenza B virus infection may exceed that of influenza A virus infection in some seasons. Over the past several decades, viruses from two influenza B virus lineages (Victoria and Yamagata) have been circulating in humans, and both lineages are now represented by influenza viruses according to World Health Organization (WHO) recommendations. Although human influenza B virus vaccines have been available for over half a century, no organized effort has been made to develop high-yield candidates. Influenza viruses are classified into three types (influenza A, B, and C) based on their antigenicity; however, only influenza A and B viruses cause human health problems. Influenza A viruses are further divided into 18 hemagglutinin (HA, major viral antigen) and 11 neuramidanase (NA, secondary viral antigen) subtypes, designated H1-H18 and N1-N11, respectively. Influenza A viruses are responsible for annual epidemics (caused by antigenic escape mutants with point mutations in antigenic epitopes of the HA) and occasional pandemics. Pandemics are caused by avian or avian / human / swine reassortant influenza viruses that encode HA proteins to which humans lack a protective immune response. The epidemiology of influenza B viruses differs from that of influenza A viruses. Influenza B viruses circulate primarily in humans and do not cause pandemics. Nevertheless, the impact of influenza B virus infection on influenza-associated morbidity and mortality is substantial, exceeding that of influenza A viruses in some seasons (Paul-Glezen et al., 2013; Tafalla et al., 2016; van de Sandt et al., 2015). Until 1983, only one lineage of influenza B viruses circulated in humans.Since then, two lineages (Victoria, named after B / Victoria / 2 / 1987, and Yamagata, named after B / Yamagata / 16 / 1988) can be genetically and antigenically distinguished based on their HA. Until 2000, one of these two lineages tended to dominate the season; however, since 2001, both influenza B virus lineages have cocirculated annually in the human population (Belshe, 2010; Belshe et al., 2010).

[0006] Until recently, most influenza vaccines were trivalent: that is, they consisted of influenza A lineages of H1N1 and H3N2 subtypes and influenza B lineages. Two studies showed that the recommended influenza B vaccine lineage was the dominant lineage only half of the time in a given influenza season (Belshe, 2010; Ambrose et al., 2010). Based on these findings and the continued circulation of Yamagata-lineage and Victoria-lineage viruses, in 2012 the World Health Organization (WHO) recommended both lineages of influenza B viruses for human influenza vaccines. Thus, most seasonal influenza viruses are now quadrivalent.

[0007] Many influenza vaccines are produced by combining the HA and NA viral RNA (vRNA) segments of a WHO-recommended vaccine strain with the remaining six vRNA segments of a "backbone" strain. Viral backbones for live attenuated influenza A and B viruses were developed in the 1960s (Maassab, 1969). Most inactivated influenza A virus vaccines are based on the A / Puerto Rico / 8 / 34 (H1N1; PR8) viral backbone, which was selected for efficient replication in embryonated chicken eggs. For inactivated influenza B vaccines, the B / Lee / 40, B / Panama / 45 / 90, or wild-type strains are used as the backbone (www.who.int / influenza / vaccines / virus / recommendations / summary_b_vic_cvv_nh1516.pdf). Summary of the Invention

[0008] The present invention relates to several mutations in "internal" genes of influenza B viruses that can enhance virus titer and / or HA yield in cultured cells and in embryonated chicken eggs, as well as several mutations in the viral glycoproteins NA and NA of influenza B viruses. Exemplary reassortant or recombinant parental influenza B viruses represent two major influenza B virus lineages (i.e., the "B / Victoria" and "B / Yamagata" lineages). Viral libraries were generated for each lineage, and the libraries were then passaged in selected cells, and mutations that enhanced virus growth were identified. The use of one or more of these mutations in vaccine virus master strains (internal genes, "backbones," used with selected HA and NA, e.g., those of circulating or predicted circulating lineages) can result in high virus titers in vitro and / or in embryonated chicken eggs, enabling more efficient growth of influenza B viruses and more rapid and cost-effective vaccine production.

[0009] Several strategies have been developed to achieve enhanced properties (e.g., high titers (e.g., 10 to 100 in cultured cells and / or embryonated chicken eggs)). 8 PFU / mL or more, e.g., 5x10 8 , 10 9 , 5x10 9 or 10 10 Methods (including random mutagenesis and comprehensive testing of growth-enhancing mutations) can be used to develop influenza B viruses (e.g., based on reassortant or recombinant B / Yamagata and B / Victoria viruses) with replication rates of 100 PFU / mL. As described herein, numerous growth-enhancing mutations (both amino acid and non-coding nucleotide substitutions) that increase the yield of influenza B viruses have been identified. Individual growth-enhancing amino acid residues in influenza B virus polypeptides or non-coding nucleotide sequences in influenza B virus segments can be combined with one or more other growth-enhancing residues in the same influenza virus polypeptide or non-coding nucleotides in the same virus segment, or with one or more other growth-enhancing residues and / or nucleotide substitutions (e.g., growth-enhancing nucleotides in promoter sequences or nucleotides between the promoter sequence and the open reading frame) in other influenza virus polypeptides or virus segments, respectively. In particular, virus libraries with random mutations in the six "internal" influenza B virus RNA segments were screened for mutations that result in efficient replication. Candidate viruses that support high yields in cell culture were tested in the HA and NA genes of eight different viruses from the Victoria and Yamagata lineages. Mutation combinations that increased the titer of candidate vaccine viruses in mammalian systems used for human influenza virus propagation were identified and validated for use in embryonated chicken eggs, the most common propagation system for influenza viruses. These influenza B virus backbones can be used for improved vaccine virus production.

[0010] For example, one or more growth-enhancing residues in the NP protein, e.g., one, two, three, or four or more growth-enhancing residues in NP, one, two, three, or four or more growth-enhancing residues in the M protein (e.g., one, two, three, or four growth-enhancing factors in BM2, or one, two, three, or four or more growth-enhancing residues in M1), one, two, three, or four or more growth-enhancing residues in PA, or one, two, three, or four or more growth-enhancing residues in NS1, or growth-enhancing nucleotides in viral non-coding sequences of NP, PA, NS, or other viral segments, can be combined in preparing influenza B viruses (e.g., vaccines) to enhance viral titers. In one embodiment, growth-enhancing nucleotides in non-coding sequences can be introduced into viral segments, or, if present in viral segments, can be selected for inclusion in influenza B viruses. In one embodiment, one or more (e.g., 1, 2, 3, 4, or 5 or more) growth-enhancing residues in HA and / or NA can be introduced into the HA or NA viral segment of an influenza virus, or selected for inclusion when present in HA or NA, hi one embodiment, the one or more growth-enhancing residues can enhance viral growth by at least 1.2, 2, 2.8, 4, 3, 5, 6, 8, 10, 100, or 200 fold or more.

[0011] In one embodiment, the disclosure provides a method for preparing a virus comprising administering to a patient ... and optionally growth-enhancing non-coding nucleotide substitutions, and in one embodiment, provides isolated recombinant (e.g., reassortant) influenza B viruses comprising HA and NA genes of interest (e.g., HA and NA viral segments from annual and pandemic lineages or with selected amino acid residues as described herein), e.g., HA and NA genes / proteins of interest from annual and pandemic lineages, or HA and NA viral segments with selected amino acid residues as described herein, which viruses are more efficiently and cost-effectively produced in cell culture (in MDCK or Vero cells) or in embryonated chicken eggs.

[0012] In one embodiment, the reassortant or recombinant influenza B virus has amino acid residues at positions 28, 40, 51, 52, 57, 204, and / or 343 in NP, and / or a nucleotide other than c at position 500 in NP vRNA, or any combination thereof, that enhance growth in cells, including MDCK cells, Vero cells, and / or eggs, compared to the corresponding virus, e.g., having alanine, proline, proline, glutamic acid, serine, methionine, or proline at positions 28, 40, 51, 52, 57, 204, and 343 in NP, respectively, i.e., the residues at positions 28, 40, 51, 52, 57, 204, or 343 in the NP segment in the recombinant influenza B virus are not alanine, proline, proline, glutamic acid, serine, methionine, or proline, but are residues that correlate with enhanced replication in MDCK cells, Vero cells, or eggs. The recombinant virus may optionally include other selected amino acids at one or more specific positions in one or more of M1, BM2, PA, PB2, and / or NS1, and optionally in PB1, as described herein. In one embodiment, the recombinant influenza B virus has amino acid residues at positions 28, 40, 51, 52, 57, 204, and / or 343 in NP that result in enhanced interaction with one or more host proteins in MDCK cells, Vero cells, or eggs, compared to a corresponding virus having alanine, proline, proline, glutamic acid, serine, methionine, or proline at positions 28, 40, 51, 52, 57, 204, and / or 343 in NP, respectively. In one embodiment, the recombinant influenza B virus has growth-enhancing residues in NP, including, but not limited to, a residue other than alanine at position 28, a residue other than proline at position 40, a residue other than proline at position 51, a residue other than glutamic acid at position 52, a residue other than serine at position 57, a residue other than methionine at position 204, and / or a residue other than proline at position 343, and / or a nucleotide other than g at nucleotide position 1795 (italics indicate nucleotides; positions are relative to positive-sense cRNA), or any combination thereof.In one embodiment, the recombinant influenza B virus has in NP a threonine at position 28, a serine at position 40, a glutamine at position 51, a lysine at position 52, a glycine at position 57, a threonine at position 214, and / or a threonine at position 343, in NP, a nucleotide a at nucleotide position 1795 and / or a nucleotide t at nucleotide position 500, or any combination thereof, in NP vRNA, and an arbitrarily selected amino acid residue at one or more specific positions in the M, PA, PB1, PB2 and / or NS viral segments.

[0013] In one embodiment, the reassortant or recombinant influenza B virus has an amino acid residue at positions 34, 54, 77, 86, and / or 97 in M1 that enhances enhanced growth in cells, including MDCK cells, Vero cells, or eggs, compared to a corresponding virus, e.g., glycine, aspartic acid, arginine, methionine, or isoleucine, respectively, in M1, i.e., the residue at positions 34, 54, 77, 86, or 97 in M1 in the M segment is not glycine, aspartic acid, arginine, methionine, or isoleucine, but is a residue that correlates with enhanced replication in MDCK cells, Vero cells, or eggs. The recombinant virus may also optionally include selected amino acid residues at one or more specific positions in PA, BM2, PB2, NP, and / or NS1, and optionally PB1, as described herein. In one embodiment, the recombinant influenza B virus has amino acid residues at positions 34, 54, 77, 86, and / or 97 in M1 that enhance growth with one or more host proteins in MDCK cells, Vero cells, or eggs compared to a corresponding virus having, for example, glycine, aspartic acid, arginine, methionine, or isoleucine in M1 at positions 34, 54, 77, 86, or 97, respectively. In one embodiment, the recombinant influenza B virus has valine or asparagine, glycine, lysine, threonine, or asparagine in M1 at positions 34, 54, 77, 86, or 97, respectively, and an arbitrarily selected amino acid residue at one or more specific positions in NP, PA, PB1, PB2, and / or NS.

[0014] In one embodiment, the reassortant or recombinant influenza B virus has an amino acid residue at position 26, 27, 58, or 80 in BM2 that enhances growth in cells, including, by way of example, in MDCK cells, Vero cells, or eggs, compared to a corresponding virus having glycine, histidine, histidine, or arginine at residues 26, 27, 58, or 80, respectively, in BM2. The recombinant virus may also optionally include selected amino acid residues at one or more specific positions in NS1, PA, NP, PB2, and / or M1 described herein. In one embodiment, the residue at position 26 in BM2 is arginine, position 27 is arginine, position 58 is arginine, or position 80 is glycine.

[0015] In one embodiment, the reassortant or recombinant influenza B virus has an amino acid residue other than tyrosine at position 42, an amino acid residue other than methionine at position 117, an amino acid residue other than lysine at position 176, and / or an amino acid residue other than serine at position 252, a nucleotide other than a at position 39, an insertion of a nucleotide after position 38, or any combination thereof in NS1 that enhances growth in cells, including MDCK cells, Vero cells, or eggs, compared to a corresponding virus having, for example, a tyrosine at position 42, a methionine at position 117, a lysine at position 176, a serine at position 252, or an a at position 39. The recombinant virus can also include selected amino acid residues at one or more specific positions in PA, PB2, BM2, NP, and / or M1, and optionally PB1, as described herein. In one embodiment, the recombinant influenza virus has an asparagine at position 42 in NS1, a tyrosine at position 117, a glutamine at position 176, a threonine at position 252, a g at nucleotide position 39, an additional g after nucleotide position 38, or any combination thereof, and any selected amino acid at one or more specific positions in PA, PB2, BM2, NP, and / or M1 described herein.

[0016] In one embodiment, the reassortant or recombinant influenza B virus has in PA an amino acid residue other than tyrosine at position 387, an amino acid residue other than valine at position 434, an amino acid residue other than aspartic acid at position 494, an amino acid residue other than threonine at position 524, and / or in PA vRNA a nucleotide other than a at position 2272, a nucleotide other than g at position 2213, a nucleotide other than a at position 1406, and / or a nucleotide other than c at position 1445, or any combination thereof, which enhances growth in cells, including MDCK cells, Vero cells, or eggs, compared to a corresponding virus having, for example, a tyrosine at position 387, a valine at position 434, an aspartic acid at position 494, a threonine at position 524, and / or a nucleotide other than a at position 2272, a g at position 2213, a nucleotide other than a at position 1406, and / or a nucleotide other than c at position 1445, or any combination thereof. The recombinant virus can also optionally include selected amino acid residues at one or more specific positions in NS1, BM2, NP, PB2, and / or M1, for example, as described herein, and optionally in PB1. In one embodiment, the recombinant influenza virus has a histidine at position 387, an alanine at position 434, or an asparagine at position 494, an alanine at position 534 in PA, and / or a t at position 2272, an a at position 2213, a g at position 1406, and / or a t at position 1445 in the PA vRNA, or any combination thereof.

[0017] In one embodiment, the reassortant or recombinant influenza B virus has an amino acid residue other than asparagine at position 16 in PB2 that confers enhanced growth in cells, including, by way of example, MDCK cells, Vero cells, or eggs, compared to a corresponding virus having asparagine at position 16. The recombinant virus may also optionally include selected amino acid residues at one or more specific positions in PA, NS1, BM2, NP, and / or M1, and optionally PB1, as described herein. In one embodiment, the recombinant influenza virus has serine at position 16 in PB2.

[0018] In one embodiment, the reassortant or recombinant influenza B virus exhibits enhanced growth in cells, including MDCK cells, Vero cells, or eggs, compared to a corresponding virus having, for example, threonine at position 34 in HA1, arginine at position 98, lysine at position 129, asparagine at position 168, asparagine at position 194, and / or threonine at position 196 in HA1, and / or lysine at position 39, serine at position 56, lysine at position 61, and aspartic acid at position 112 in HA2. an amino acid residue other than threonine, an amino acid residue other than arginine at position 98, an amino acid residue other than lysine at position 129, an amino acid residue other than asparagine at position 168, an amino acid residue other than asparagine at position 194, and / or an amino acid residue other than threonine at position 196, and / or in HA2, an amino acid residue other than lysine at position 39, an amino acid residue other than serine at position 56, an amino acid residue other than lysine at position 61, or an amino acid residue other than aspartic acid at position 112, or any combination thereof. The recombinant virus may also optionally comprise selected amino acid residues at one or more specific positions of PA, BM2, PB2, NS1, NP, and / or M1, and / or PB1 as described herein. In one embodiment, the recombinant influenza virus has an isoleucine at position 34, a glutamic acid at position 129, a glutamic acid or aspartic acid at position 168, a proline, alanine, isoleucine or asparagine at position 196, a lysine at position 98, an aspartic acid at position 194, a glycine at position 39 (in HA2), a glycine at position 56 (in HA2), an asparagine at position 51, or a glutamic acid at position 112 (in HA2), or any combination thereof.

[0019] In one embodiment, the reassortant or recombinant influenza B virus is grown in MDCK cells, Vero cells, or eggs, as compared to a corresponding virus having, for example, a T at position 76, an R at position 102, an E at position 105, a P at position 139, an N at position 169, a G at position 434, a T at position 436, and / or a D at position 457 in NA, which virus may optionally contain selected amino acid residues at one or more specific positions of PA, PB2, BM2, NP, NS1, and / or M1, and PB1 as described herein. and / or an amino acid residue other than aspartic acid (D) at position 457, or any combination thereof. In one embodiment, the recombinant influenza B virus has a methionine (M) at position 76, a lysine (K) at position 102, a lysine (K) at position 105, a serine (S) at position 139, a threonine (T) at position 169, a glutamic acid (E) at position 434, a methionine (M) at position 436, and / or an asparagine (N) at position 457, or any combination thereof.

[0020] In one embodiment, the invention provides an isolated recombinant reassortant influenza virus having six "internal" genes from a vaccine influenza virus with two or more selected amino acid residues at specific positions described herein, and an NA gene segment selected from a first influenza virus isolate, and an HA gene segment from the same isolate or a different isolate.

[0021] In one embodiment, the influenza virus of the present invention has specific amino acid residues at one, two, three or more specific positions in PA, PB1, PB2, NP, M1 and / or NS1, and has at least 80% (e.g., 90%, 92%, 95%, 97%, 98%, or 99%) contiguous amino acid sequence identity, including any integer between 80 and 99, with a corresponding polypeptide encoded by one of SEQ ID NOs: 1-6 (internal genes of B / Yamagata / 1 / 73), such as a polypeptide other than A at position 28, other than P at position 40, other than P at position 51, other than E at position 52, other than S at position 57, other than M at position 204, and / or other than P at position 343 in NP and / or a g at position 1795 or a t at position 500, or any combination thereof, in NP vRNA; in M1, a residue other than G at position 34, a residue other than D at position 54, a residue other than R at position 77, a residue other than M at position 86, a residue other than I at position 97, or any combination thereof; in BM2, a residue other than H at position 58, a residue other than R at position 80, a residue other than H at position 27, a residue other than G at position 26 (e.g., R at position 58, G at position 80, R at position 27), and / or in BM2, an R at position 26; in SN1, a residue other than Y at position 42, a residue other than M at position 117, a residue other than K at position 176, and / or a residue other than S at position 252, and / or in NS1 vRNA, a39g, an additional g after position 38, or any combination thereof; in PB2, a residue other than N at position 16; and / or in PA, a residue other than Y at position 387, a residue other than V at position 434, a residue other than D at position 494, a residue other than T at position 524, and / or PA The recombinant influenza B virus has, in its vRNA, a2272t, g2213a, a1406g, and / or c1445t, or any combination thereof. Residues other than those specified above may be conservatively substituted. Conservative amino acid substitution refers to the interchangeability of residues having similar side chains.For example, amino acids with aliphatic side chains are glycine, valine, leucine, and isoleucine; amino acids with aliphatic-hydroxyl side chains are serine and threonine; amino acids with amide-containing side chains are asparagine and glutamine; amino acids with aromatic side chains are phenylalanine, tyrosine, and tryptophan; amino acids with basic side chains are lysine, arginine, and histidine; and amino acids with sulfur-containing side chains are cysteine ​​and methionine. In one embodiment, conservative amino acid substitution groups are: threonine-valine-leucine-isoleucine-alanine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; glutamic acid-aspartic acid; and asparagine-glutamine. Non-conservative substitutions are also contemplated.

[0022] In one embodiment, the influenza B virus of the present invention is a recombinant influenza B virus having specific amino acid residues at one, two, three or more specific positions in PA, NS1, M, or NP, which is a polypeptide having an amino acid sequence with at least 80% (e.g., 90%, 92%, 95%, 97%, 98%, or 99%) contiguous amino acid sequence identity, including any integer between 80 and 99, to a corresponding polypeptide encoded by one of SEQ ID NOs: 1 to 6. In one embodiment, the influenza B virus of the present invention is a recombinant influenza B virus having specific amino acid residues at one or more specific positions in PA, PB1, PB2, NP, M1, and / or NS1, and which has an amino acid sequence with at least 80% (e.g., 90%, 92%, 95%, 97%, 98%, or 99%) contiguous amino acid sequence identity, including any integer between 80 and 99, to a corresponding polypeptide encoded by one of SEQ ID NOs: 1 to 6, such as a polypeptide having residues that are conservative substitutions.

[0023] Also included are any combinations of selected amino acid residues at the specific positions described herein.

[0024] PA, NP, M, and / or NS viral segments having residues at specific positions can be combined with PB1, PB2, HA, and NA viral segments to provide reassortant vaccine viruses of the invention. In one embodiment, the HA viral segment in the reassortant virus is heterologous to the PA, PB1, PB2, NP, M, and NS viral segments. In one embodiment, the NA gene segment in the reassortant is heterologous to the PA, PB1, PB2, NP, M, and NS viral segments. In one embodiment, the A viral segments in the reassortants comprise PA, PB1, PB2, NP, M, and NS viral segments from one influenza virus isolate or lineage ("parent"), or a variant thereof (e.g., comprising viral segments encoding influenza viral proteins with at least 95%, 96%, 97%, 98%, 99%, or 99.5% amino acid sequence identity, or comprising 1, 2, 5, 10, or 20 substitutions relative to the sequence of the parent influenza virus isolate or lineage). In one embodiment, the parent lineage comprises viral segments having a sequence corresponding to at least one of SEQ ID NOs: 1-6, and the recombinant virus comprises at least one of the viral segments with at least one of the PA, NS, NP, or M substitutions described herein, and at least one of the parent viral segments. In one embodiment, the HA gene segment in the reassortant virus is a chimeric HA gene segment (e.g., a chimera of a homologous HA ectodomain sequence linked to an HA signal peptide sequence and / or HA transmembrane domain sequence from the HA gene segment of the parent isolate or strain, or a variant thereof).In one embodiment, the NA gene segment in the isolated recombinant virus is a chimera of, for example, influenza B virus NA and influenza A virus NA, such as a chimeric NA gene segment (e.g., a homologous NA ectodomain sequence linked to an NA transmembrane domain sequence from the NA gene segment of the parental isolate or lineage, or a variant thereof, and / or a stalk sequence from the parental isolate or lineage, or a variant thereof). In one embodiment, the NA gene segment in the isolated recombinant virus is a chimeric NA gene segment, such as a chimeric NA gene segment (e.g., a chimera of a homologous NA ectodomain sequence linked to an NA transmembrane domain sequence from the NA gene segment of the parental isolate or lineage, or a variant thereof, and / or a stalk sequence from a second isolate or lineage, or a variant thereof). In one embodiment, the isolated recombinant virus has a homologous HA gene segment, a homologous NA gene segment, a chimeric HA gene segment, a chimeric NA gene segment, or any combination thereof. The nucleic acid sequence used to prepare vRNA or cRNA can be one that introduces the residue at a particular position by recombinant methodology, or can be selected to have the residue at the particular position.

[0025] As described herein, influenza virus isolates useful as vaccine viruses for carrying homologous NA and / or HA gene segments (e.g., B / Yamagata 1 / 73 reassortant carrying B / Yamagata lineage or B / Victoria lineage viruses) are serially passaged (e.g., about 10-12 times, although fewer passages are also possible) in MDCK cells to obtain viruses with enhanced replication in those cells. In one embodiment, the replication-enhanced virus obtained after serial passage has a titer at least 0.5 to 1 or 2 logs higher than a virus that has not been serially passaged. In one embodiment, the virus obtained after serial passage has substitutions in two or more internal genes relative to the parent virus.

[0026] Thus, for vaccine viruses grown or passaged in cells in culture (e.g., MDCK or Vero cells or eggs), the disclosed residues at one or more specific positions in PA, BM2, NP, M1, and / or NS1 that enhance viral growth in cultured cells, when used with the desired HA and NA sequences, can result in significantly higher viral titers. Accordingly, the present invention provides a method for selecting influenza viruses with enhanced replication in cell culture. The method includes providing cells suitable for influenza vaccine production; continuously culturing one or more influenza virus isolates in the cells; and isolating continuously cultured viruses that have enhanced growth relative to the one or more isolates prior to continuous culture. In one embodiment, the cells are canine or primate, e.g., human or simian, cells.

[0027] The present invention provides a plurality of influenza virus vectors of the invention, useful for preparing reassortants, including 6:1:1 reassortants, 6:2 reassortants, and 7:1 reassortants. A 6:1:1 reassortant within the scope of the present invention is an influenza virus having six internal gene segments from a vaccine virus, an NA gene segment from a different (second) virus isolate, and an HA gene segment from a third isolate; a 6:2 reassortant within the scope of the present invention is an influenza virus having six internal gene segments from a vaccine virus and NA and HA gene segments from a different (second) virus isolate; and a 7:1 reassortant within the scope of the present invention is an influenza virus having six internal gene segments and an NA gene segment from a vaccine virus and an HA gene segment from a viral source different from the vaccine virus, or an influenza virus having six internal gene segments and an HA gene segment from the vaccine virus, and the NA gene segment is from a viral source different from the vaccine virus.

[0028] In one embodiment of the invention, the plurality includes a vector for vRNA or cRNA production selected from vectors comprising a promoter operably linked to influenza virus PA DNA, e.g., cDNA, linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus PB1 DNA, e.g., cDNA, linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus PB2 DNA, e.g., cDNA, linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus HA DNA, e.g., cDNA, linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus NP DNA, e.g., cDNA, linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus NA DNA, e.g., cDNA, linked to a transcription termination sequence, a vector comprising a promoter operably linked to influenza virus M DNA, e.g., cDNA, linked to a transcription termination sequence, and a vector comprising a promoter operably linked to influenza virus NS DNA linked to a transcription termination sequence. In one embodiment, the DNA for producing vRNA or cRNA of PB1, PB2, PA, NP, M, and NS has a sequence derived from an influenza virus that replicates to high titers in cultured mammalian cells (e.g., MDCK cells, Vero cells, or PER.C6® cells, and optionally, embryonated chicken eggs) and / or derived from a vaccine virus (e.g., one that does not cause significant disease in humans). DNA, e.g., cDNA, for producing NA vRNA or cRNA can be derived from any NA, and DNA for producing HA vRNA or cRNA can be derived from any HA. In one embodiment, DNA for producing vRNA or cRNA can be for influenza A or C virus.DNA for vRNA or cRNA production of NA and HA can be from different lineages or isolates (6:1:1 reassortants) or the same lineage or isolate (6:2 reassortants), or NA can be from the same lineage or isolate as the internal gene (7:1 reassortants). The plurality also includes vectors for mRNA production selected from a vector encoding influenza virus PA, a vector encoding influenza virus PB1, a vector encoding influenza virus PB2, a vector encoding influenza virus NP, and optionally one or more vectors encoding NP, NS, M (e.g., M1 and BM2), HA, or NA. The vectors encoding the viral proteins can further include a transcription termination sequence.

[0029] Viruses capable of providing the internal genes of reassortant viruses within the scope of the present invention can be expressed in high titers in MDCK cells, e.g., at least about 10 5 Titer of PFU / mL (e.g., at least 10 6 PFU / mL, 10 7 PFU / mL or 10 8 PFU / mL); high titers in embryonated eggs, e.g., at least about 10 7 EID 50 / mL (e.g., at least 10 8 EID 50 / mL, 10 9 EID 50 / mL or 10 10 EID 50 / mL); high titer in cells such as MDCK cells, e.g., at least about 10 7 PFU / mL (e.g., at least 10 8 PFU / mL) or have high titers in two or more of these host cells.

[0030] In one embodiment, the titer of the reassortants of the invention in cells such as MDCK cells or Vero cells may be 1 log, 2 logs, 3 logs, or more than the titer of the corresponding virus that does not have a particular residue at a particular position.

[0031] In one embodiment, the DNA of the internal genes of PB1, PB2, PA, NP, M, and NS encodes a protein having substantially the same activity as the corresponding polypeptide encoded by one of SEQ ID NOs: 1-6. As used herein, "substantially the same activity" includes detectable protein having about 0.1%, 1%, 10%, 30%, 50%, 90%, e.g., up to 100% or more, or about 80%, 90% or more of the activity or protein level of the corresponding full-length polypeptide, respectively. In one embodiment, for example, the nucleic acid sequence encoding a polypeptide that is substantially identical to at least 80% (e.g., 90%, 92%, 95%, 97%, 98%, or 99% (including any integer between 80 and 99)) has continuous amino acid sequence identity to a polypeptide encoded by one of SEQ ID NOs: 1-6. In one embodiment, the isolated and / or purified nucleic acid molecule comprises a nucleic acid sequence that is substantially identical, e.g., at least 50% (e.g., 60%, 70%, 80%, or 90% (including any integer between 50 and 100)) or more contiguous amino acid sequence identity, to one of SEQ ID NOs: 1-6. In one embodiment, the isolated and / or purified nucleic acid molecule may also encode a polypeptide having at least 80% (e.g., 90%, 92%, 95%, 97%, 98%, or 99% (including any integer between 80 and 99)) contiguous amino acid sequence identity to a polypeptide encoded by one of SEQ ID NOs: 1-6. In one embodiment, the influenza virus polypeptide has a combination of conservative and non-conservative amino acid substitutions (conservative substitutions of up to 10% or 20% of the residues), or one or more (e.g., 2, 5, 10, 15, 20 or more) conservative amino acid substitutions ... up to 10% or 20% conservative substitutions) compared to a polypeptide encoded by one of SEQ ID NOs: 1-6, and has residues characteristic of one or more of PA, PB2, BM2, NP, M1 and / or NS1 compared to a polypeptide encoded by one of SEQ ID NOs: 1-6.In one embodiment, the influenza virus polypeptide has one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) conservative and / or non-conservative substitutions compared to a polypeptide encoded by one of SEQ ID NOs: 1-6.

[0032] Thus, the present invention encompasses the use of isolated and purified vectors or plasmids that express or encode influenza virus proteins or that express or encode influenza vRNA or cRNA, both natural and recombinant. The vectors can contain influenza cDNA (e.g., influenza A, B, or C DNA) (Fields et al. (eds.), Lippincott, Williams, and Wickens (2006), incorporated herein by reference in its entirety). Any suitable promoter or transcription termination sequence can drive the expression of a protein or peptide (e.g., a viral protein or peptide, a non-viral protein or peptide, or a therapeutic protein or peptide).

[0033] The compositions or vectors of the invention may contain a homologous gene or open reading frame of interest (e.g., a foreign gene encoding an immunogenic peptide or protein useful as a vaccine or in gene replacement), e.g., an epitope useful in cancer therapy or vaccines, or a peptide or polypeptide useful in gene therapy. When preparing virus, a vector or plasmid containing the gene or cDNA of interest may be used in place of a vector or plasmid for influenza virus genes, or may be added to a vector or plasmid for all influenza virus genes. Thus, other embodiments of the invention include the compositions or vectors described above, in which one of the vectors is linked to a desired nucleic acid sequence (e.g., a desired cDNA linked to a 3' influenza virus sequence, optionally including a 3' influenza coding sequence or portion thereof), replaced with, or further includes, a 5' influenza virus sequence, optionally including a 5' influenza virus coding sequence or portion thereof. In one embodiment, the desired nucleic acid sequence, such as a cDNA, is in the antisense (antigenome) orientation. Introduction of the conjugated vector into a host cell permissive for influenza virus replication results in recombinant viruses containing vRNA or cRNA corresponding to homologous sequences in the vector.

[0034] The promoter in a vector for vRNA or cRNA can be an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T7 promoter, or a T3 promoter, and optionally, the vector includes a transcription termination sequence such as an RNA polymerase I transcription termination sequence, an RNA polymerase II transcription termination sequence, an RNA polymerase III transcription termination sequence, or a ribozyme. Ribozymes within the scope of the present invention include, but are not limited to, tetrahymena ribozyme, RNase P, hammerhead ribozyme, hairpin ribozyme, hepatitis ribozyme, and synthetic ribozymes. In one embodiment, the RNA polymerase I promoter is a human RNA polymerase I promoter.

[0035] The promoter or transcription termination sequence in a vRNA, cRNA, or viral protein expression vector can be the same as or different from the promoter or any other vector. In one embodiment, the vector or plasmid expressing influenza vRNA or cRNA contains a promoter suitable for expression in at least one particular host cell, or in one or more hosts, such as avian or mammalian host cells, such as canine, feline, equine, bovine, ovine, or primate cells, including human cells.

[0036] In one embodiment, at least one vector for vRNA or cRNA contains an RNA polymerase II promoter linked to a viral coding sequence, linked to another ribozyme sequence, optionally linked 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 or cRNA production contain an RNA polymerase II promoter, a first ribozyme sequence 5' to a sequence corresponding to the viral sequence including the viral coding sequence, a second ribozyme sequence 5' to a transcription termination sequence. Each RNA polymerase II promoter in each vRNA or cRNA may be the same as or different from the RNA polymerase II promoter in any other vRNA or cRNA. Similarly, each ribozyme sequence in each vRNA or cRNA may be the same as or different from the ribozyme sequence in any other vRNA or cRNA. In one embodiment, the ribozyme sequences within a single vector are not identical.

[0037] In one embodiment, the invention provides an influenza virus PA DNA linked to a transcription termination sequence, e.g., a vector for producing vRNA or cRNA comprising a promoter operably linked to a cDNA; PB1 DNA linked to a transcription termination sequence, e.g., a vector for producing vRNA or cRNA comprising a promoter operably linked to a cDNA; PB2 DNA linked to a transcription termination sequence, e.g., a vector for producing vRNA or cRNA comprising a promoter operably linked to a cDNA; HA DNA linked to a transcription termination sequence, e.g., a vector for producing vRNA or cRNA comprising a promoter operably linked to a cDNA; NP DNA linked to a transcription termination sequence, e.g., a vector for producing vRNA or cRNA comprising a promoter operably linked to a cDNA; NA DNA linked to a transcription termination sequence, e.g., a vector for producing vRNA or cRNA comprising a promoter operably linked to a cDNA; M DNA linked to a transcription termination sequence, e.g., a vector for producing vRNA or cRNA comprising a promoter operably linked to a cDNA; and NS DNA linked to a transcription termination sequence. A number of influenza virus vectors for reassortant production are provided, including DNA, eg, vectors for vRNA or cRNA production comprising a promoter operably linked to a cDNA.wherein the DNA for PB1, PB2, PA, NP, NS, and M is derived from one or more influenza vaccine seed viruses and comprises two or more characteristic residues at the specified positions; and vectors 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, 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 BM2, or a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS2. In one embodiment, at least one vector comprises a corresponding sequence encoding PB1, PB2, PA, NP, M, or NS, or a portion thereof, having substantially the same activity as a corresponding polypeptide encoded by one of SEQ ID NOs: 1 to 6, for example, a sequence encoding a polypeptide having at least 80% (e.g., 85%, 90%, 92%, 95%, 98%, 99%, or 100% (including any integer between 80 and 100)) amino acid identity to a polypeptide encoded by one of SEQ ID NOs: 1 to 6.If desired, two vectors can be used in place of the vector containing a promoter operably linked to influenza virus M cDNA linked to a transcription termination sequence (e.g., a vector containing a promoter operably linked to influenza virus M1 cDNA linked to a transcription termination sequence and a vector containing a promoter operably linked to influenza virus BM2 cDNA linked to a transcription termination sequence).

[0038] The vectors of the present invention may be physically linked, or each vector may be present on an individual plasmid or other, e.g., linear, nucleic acid delivery vehicle. In one embodiment, each vRNA or cRNA production vector is present on a separate plasmid. In one embodiment, each mRNA production vector is present on a separate plasmid.

[0039] The present invention also provides a method for preparing influenza virus. The method comprises contacting cells with a plurality of the vectors of the present invention, e.g., sequentially or simultaneously, in an amount effective to produce infectious influenza virus. The present invention also comprises isolating virus from cells contacted with a plurality of the vectors. Thus, the present invention further provides isolated virus, as well as host cells contacted with a plurality of the vectors or viruses of the present invention. In another embodiment, the present invention comprises contacting the cells with one or more vectors, vRNA or cRNA or protein production vectors, before any other vector, vRNA or protein production vector. In one embodiment, the promoter of the vRNA or cRNA vector used in the method is an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T3 promoter, or a T7 promoter. In one embodiment, the RNA polymerase I promoter is a human RNA polymerase I promoter. In one embodiment, each vRNA or cRNA vector used in the method is on a separate plasmid. In one embodiment, the vRNA or cRNA vectors used in the method are on one plasmid or on two or three different plasmids. In one embodiment, each mRNA vector used in the method is on a separate plasmid. In one embodiment, the mRNA vectors for PA, PB1, PB2, and NP used in the method are on one plasmid or on two or three different plasmids.

[0040] In one embodiment, the present invention provides a method for selecting influenza viruses with enhanced replication in cell culture. The method includes providing cells suitable for influenza vaccine production; continuously culturing one or more influenza virus isolates in the cells; and isolating continuously cultured viruses that have enhanced growth compared to the one or more isolates prior to continuous culture. In one embodiment, the cells are rodent or primate cells.

[0041] The methods of producing the viruses described herein, which do not require helper virus infection, are useful in viral mutation studies and in the production of vaccines (e.g., AIDS, influenza, hepatitis B, hepatitis C, rhinovirus, filoviruses, malaria, herpes, and foot and mouth disease) and gene therapy vectors (cancer, AIDS, adenosine deaminase, muscular dystrophy, ornithine transcarbamylase deficiency, and central nervous system tumors). Thus, viruses for use in medical therapy (e.g., for vaccines or gene therapy) are provided.

[0042] The present invention also provides isolated viral polypeptides and methods for preparing and using the recombinant viruses of the invention. The methods include administering to a host organism (e.g., a mammal) an effective amount of an influenza virus of the invention (in combination with an inactivated virus preparation, optionally with an adjuvant and / or a carrier), e.g., in an amount effective to prevent or ameliorate infection of an animal, such as a mammal, with the virus or an antigenically closely related virus. In one embodiment, the virus is administered intramuscularly, while in other embodiments, the virus is administered intranasally. In some administration protocols, the entire dose is administered intramuscularly or intranasally, while in others a combination of intramuscular and intranasal administration is used. The vaccine may further include other isolates of influenza virus, including recombinant influenza viruses, other pathogens, additional biological agents, or microbial components (e.g., forming a multivalent vaccine). In one embodiment, for example, intranasal vaccination with an inactivated influenza virus and a mucosal adjuvant can induce virus-specific IgA and neutralizing antibodies in the nasopharynx and serum IgG.

[0043] The influenza viruses of the present invention may be used in conjunction with other antiviral agents (e.g., amantadine, rimantadine, and / or neuraminidase inhibitors), e.g., the viruses may be administered separately, e.g., before and / or after, or in combination with, those antiviral agents. [Prior art documents] [Non-patent literature]

[0044] [Non-Patent Document 1] Ambrose and Levin, Hum. Vaccin. Immunother., 8:81 (2012). [Non-patent document 2] Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, Ltd., Williams and Wilkins, Baltimore, MD (1987). [Non-licensed document 3] Aymard-Henry et al., Virology: A Practical Approach, Oxford IRL Press, Oxford, 119-150 (1985).

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Brief Description of the Drawings

[0045] [Figure 1A] Nucleic acid sequence of B / Yamagata 1 / 1973 (SEQ ID NOs: 1-6, 9 and 10) and amino acid sequence of HA (SEQ ID NO: 7) and NA (SEQ ID NO: 8). [Figure 1B] Nucleic acid sequence of B / Yamagata 1 / 1973 (SEQ ID NOs: 1-6, 9 and 10) and amino acid sequence of HA (SEQ ID NO: 7) and NA (SEQ ID NO: 8). [Figure 1C] Nucleic acid sequence of B / Yamagata 1 / 1973 (SEQ ID NOs: 1-6, 9 and 10) and amino acid sequence of HA (SEQ ID NO: 7) and NA (SEQ ID NO: 8). [Figure 1D] Nucleic acid sequence of B / Yamagata 1 / 1973 (SEQ ID NOs: 1-6, 9 and 10) and amino acid sequence of HA (SEQ ID NO: 7) and NA (SEQ ID NO: 8). [Figure 1E] Nucleic acid sequence of B / Yamagata 1 / 1973 (SEQ ID NOs: 1-6, 9 and 10) and amino acid sequence of HA (SEQ ID NO: 7) and NA (SEQ ID NO: 8). [Figure 1F] Nucleic acid sequence of B / Yamagata 1 / 1973 (SEQ ID NOs: 1-6, 9 and 10) and amino acid sequence of HA (SEQ ID NO: 7) and NA (SEQ ID NO: 8). [Figure 1G] Nucleic acid sequence of B / Yamagata 1 / 1973 (SEQ ID NOs: 1-6, 9 and 10) and amino acid sequence of HA (SEQ ID NO: 7) and NA (SEQ ID NO: 8). [Figure 2A] Overview of library passage in MDCK cells and identification of high-yield (HY) candidates (HY(Yam) and HY(Vic)). Viral libraries from the Yamagata and Victoria lineages, carrying random mutations in the intended vRNAs, were cultured in MDCK cells for 12 passages or passaged twice, mixed, and passaged an additional 10 times. More than 700 viral plaques were selected for each viral lineage. Based on hemagglutination and viral titer, the top eight candidates for each lineage were identified through a stepwise selection process. Testing combination mutations resulted in the selection of RG(Yam)#8 and RG(Vic)#2. Introduction of additional mutations (identified by viral library screening in Vero cells) resulted in the high-yield vaccine backbones HY(Yam) and HY(ViC). HA titer, hemagglutination titer. [Figure 2B] Overview of library passage in MDCK cells and identification of high-yield (HY) candidates (HY(Yam) and HY(Vic)). Viral libraries from the Yamagata and Victoria lineages, carrying random mutations in the intended vRNAs, were cultured in MDCK cells for 12 passages or passaged twice, mixed, and passaged an additional 10 times. More than 700 viral plaques were selected for each viral lineage. Based on hemagglutination and viral titer, the top eight candidates for each lineage were identified through a stepwise selection process. Testing combination mutations resulted in the selection of RG(Yam)#8 and RG(Vic)#2. Introduction of additional mutations (identified by viral library screening in Vero cells) resulted in the high-yield vaccine backbones HY(Yam) and HY(ViC). HA titer, hemagglutination titer. [Figure 3A] Viral titers (growth kinetics) and hemagglutination (HA) titers for a selection of high-yield candidates from the B / Yamagata lineage (A) and B / Victoria lineage (B). Viruses carrying the high-yield vaccine backbone of the Yamagata lineage (A) and Victoria lineage (B). Mutations detected in the candidate viruses are shown in Tables 1 and 2, respectively. Data are from three independent experiments; mean titers ± SD are shown. Statistical significance was determined using a linear mixed model (*p<0.05; **p<0.01); where titers of high-yield vaccine candidates were lower than those of wild-type virus, P values ​​are not shown. The asterisk color indicates a comparison of each virus with the WT virus. [Figure 3B]Viral titers (growth kinetics) and hemagglutination (HA) titers for a selection of high-yield candidates from the B / Yamagata lineage (A) and B / Victoria lineage (B). Viruses carrying the high-yield vaccine backbone of the Yamagata lineage (A) and Victoria lineage (B). Mutations detected in the candidate viruses are shown in Tables 1 and 2, respectively. Data are from three independent experiments; mean titers ± SD are shown. Statistical significance was determined using a linear mixed model (*p<0.05; **p<0.01); where titers of high-yield vaccine candidates were lower than those of wild-type virus, P values ​​are not shown. The asterisk color indicates a comparison of each virus with the WT virus. [Figure 4A] Viral titers and HA titers of regenerated high-yield candidates of the B / Yamagata lineage (A) and B / Victoria lineage (B). Mutations introduced into the B / Yamagata lineage (A) and B / Victoria lineage (B) viruses are shown in Tables 3 and 4, respectively. Data are from three independent experiments; mean titers ± SD are shown. P values ​​were calculated using a linear mixed model (*p<0.05; **p<0.01); where the titers of high-yield vaccine candidates were lower than those of the wild-type virus, P values ​​are not shown. The color of the asterisk indicates a comparison of the respective viruses with the WT virus. [Figure 4B] Viral titers and HA titers of regenerated high-yield candidates of the B / Yamagata lineage (A) and B / Victoria lineage (B). Mutations introduced into the B / Yamagata lineage (A) and B / Victoria lineage (B) viruses are shown in Tables 3 and 4, respectively. Data are from three independent experiments; mean titers ± SD are shown. P values ​​were calculated using a linear mixed model (*p<0.05; **p<0.01); where the titers of high-yield vaccine candidates were lower than those of the wild-type virus, P values ​​are not shown. The color of the asterisk indicates a comparison of the respective viruses with the WT virus. [Figure 5]Chimeric HA and NA constructs. The ectodomains of influenza B virus HA (green) and NA (blue) proteins were inserted between the remaining sequences of influenza A virus PR8 HA (purple) and NA (dark orange) vRNAs. Wide bars indicate coding regions. Small bars indicate non-coding regions (NCRs). SP, signal peptide; TM, transmembrane domain; CT, cytoplasmic tail. [Figure 6A] Growth kinetics and HA titers of high-yielding Yamagata lineage and Victoria lineage viruses. (A) The indicated wild-type Yamagata lineage viruses were compared with the Yamagata lineage high-yielding candidate RG(Yam)#8 (Table 3) and RG(Yam)#8 with the PA-a2272t mutation; the latter virus was selected as lead candidate H(Yam). (B) The indicated Victoria lineage wild-type viruses were compared with the Victoria lineage high-yielding candidate RG(Vic)#2 (Table 4) and RG(Vic) with the PA-a2272t mutation; the latter virus was selected as lead candidate HY(Vic). In both sets of experiments, MDCK cells were infected in triplicate with the indicated viruses at 0.001 molar ratio and incubated at 35°C. At the indicated time points, virus and hemagglutination titers were determined by performing plaque assays or hemagglutination assays, respectively. Values ​​shown are the mean ± SD of three independent experiments. P values ​​were calculated using a linear mixed model (*P < 0.05; **P < 0.01). Red and blue asterisks indicate a comparison of the respective virus with the WT virus; beige asterisks indicate a comparison of the viruses depicted in red and blue. [Figure 6B]Growth kinetics and HA titers of high-yielding Yamagata lineage and Victoria lineage viruses. (A) The indicated wild-type Yamagata lineage viruses were compared with the Yamagata lineage high-yielding candidate RG(Yam)#8 (Table 3) and RG(Yam)#8 with the PA-a2272t mutation; the latter virus was selected as lead candidate H(Yam). (B) The indicated Victoria lineage wild-type viruses were compared with the Victoria lineage high-yielding candidate RG(Vic)#2 (Table 4) and RG(Vic) with the PA-a2272t mutation; the latter virus was selected as lead candidate HY(Vic). In both sets of experiments, MDCK cells were infected in triplicate with the indicated viruses at 0.001 molar ratio and incubated at 35°C. At the indicated time points, virus and hemagglutination titers were determined by performing plaque assays or hemagglutination assays, respectively. Values ​​shown are the mean ± SD of three independent experiments. P values ​​were calculated using a linear mixed model (*P < 0.05; **P < 0.01). Red and blue asterisks indicate a comparison of the respective virus with the WT virus; beige asterisks indicate a comparison of the viruses depicted in red and blue. [Figure 7A] Comparison of wild-type and high-yield viruses with different HA and NA vRNAs. Viruses with the indicated viral HA and NA vRNAs combined with internal vRNA segments from the respective natural wild-type isolate (WT) or from the HA(Yam) (A-C) or HY(Vic) (D-F) isolates (viruses indicated by black bars have the eight wild-type vRNA segments of a human influenza B virus isolate). Values ​​shown are the mean ± SD of three independent experiments. P values ​​were calculated using a linear mixed model (*P < 0.05; **P < 0.01). Red asterisks indicate a comparison of the respective virus to the WT virus. [Figure 7B]Comparison of wild-type and high-yield viruses with different HA and NA vRNAs. Viruses with the indicated viral HA and NA vRNAs combined with internal vRNA segments from the respective natural wild-type isolate (WT) or from the HA(Yam) (A-C) or HY(Vic) (D-F) isolates (viruses indicated by black bars have the eight wild-type vRNA segments of a human influenza B virus isolate). Values ​​shown are the mean ± SD of three independent experiments. P values ​​were calculated using a linear mixed model (*P < 0.05; **P < 0.01). Red asterisks indicate a comparison of the respective virus to the WT virus. [Figure 7C] Comparison of wild-type and high-yield viruses with different HA and NA vRNAs. Viruses with the indicated viral HA and NA vRNAs combined with internal vRNA segments from the respective natural wild-type isolate (WT) or from the HA(Yam) (A-C) or HY(Vic) (D-F) isolates (viruses indicated by black bars have the eight wild-type vRNA segments of a human influenza B virus isolate). Values ​​shown are the mean ± SD of three independent experiments. P values ​​were calculated using a linear mixed model (*P < 0.05; **P < 0.01). Red asterisks indicate a comparison of the respective virus to the WT virus. [Figure 7D] Comparison of wild-type and high-yield viruses with different HA and NA vRNAs. Viruses with the indicated viral HA and NA vRNAs combined with internal vRNA segments from the respective natural wild-type isolate (WT) or from the HA(Yam) (A-C) or HY(Vic) (D-F) isolates (viruses indicated by black bars have the eight wild-type vRNA segments of a human influenza B virus isolate). Values ​​shown are the mean ± SD of three independent experiments. P values ​​were calculated using a linear mixed model (*P < 0.05; **P < 0.01). Red asterisks indicate a comparison of the respective virus to the WT virus. [Figure 7E]Comparison of wild-type and high-yield viruses with different HA and NA vRNAs. Viruses with the indicated viral HA and NA vRNAs combined with internal vRNA segments from the respective natural wild-type isolate (WT) or from the HA(Yam) (A-C) or HY(Vic) (D-F) isolates (viruses indicated by black bars have the eight wild-type vRNA segments of a human influenza B virus isolate). Values ​​shown are the mean ± SD of three independent experiments. P values ​​were calculated using a linear mixed model (*P < 0.05; **P < 0.01). Red asterisks indicate a comparison of the respective virus to the WT virus. [Figure 7F] Comparison of wild-type and high-yield viruses with different HA and NA vRNAs. Viruses with the indicated viral HA and NA vRNAs combined with internal vRNA segments from the respective natural wild-type isolate (WT) or from the HA(Yam) (A-C) or HY(Vic) (D-F) isolates (viruses indicated by black bars have the eight wild-type vRNA segments of a human influenza B virus isolate). Values ​​shown are the mean ± SD of three independent experiments. P values ​​were calculated using a linear mixed model (*P < 0.05; **P < 0.01). Red asterisks indicate a comparison of the respective virus to the WT virus. [Figure 8A]HY(Yam) and HY(Vic) backbone exchange. (A and B) Comparison of the virus and hemagglutination titers of two wild-type Yamagata lineage viruses with viruses carrying the same HA and NA vRNAs combined with the internal genes of HY(Yam) or HY(Vic). (C and D) Comparison of the virus and hemagglutination titers of two wild-type Yamagata lineage viruses with viruses carrying the same HA and NA vRNAs combined with the internal genes of HY(Yam) or HY(Vic). Values ​​shown are the mean ± SD of three independent experiments. P values ​​were calculated using a linear mixed model (*P < 0.05; **P < 0.01). Red and blue asterisks indicate a comparison between the respective viruses and the WT virus; beige asterisks indicate a comparison between the viruses depicted in red and blue. [Figure 8B] HY(Yam) and HY(Vic) backbone exchange. (A and B) Comparison of the virus and hemagglutination titers of two wild-type Yamagata lineage viruses with viruses carrying the same HA and NA vRNAs combined with the internal genes of HY(Yam) or HY(Vic). (C and D) Comparison of the virus and hemagglutination titers of two wild-type Yamagata lineage viruses with viruses carrying the same HA and NA vRNAs combined with the internal genes of HY(Yam) or HY(Vic). Values ​​shown are the mean ± SD of three independent experiments. P values ​​were calculated using a linear mixed model (*P < 0.05; **P < 0.01). Red and blue asterisks indicate a comparison between the respective viruses and the WT virus; beige asterisks indicate a comparison between the viruses depicted in red and blue. [Figure 8C]HY(Yam) and HY(Vic) backbone exchange. (A and B) Comparison of the virus and hemagglutination titers of two wild-type Yamagata lineage viruses with viruses carrying the same HA and NA vRNAs combined with the internal genes of HY(Yam) or HY(Vic). (C and D) Comparison of the virus and hemagglutination titers of two wild-type Yamagata lineage viruses with viruses carrying the same HA and NA vRNAs combined with the internal genes of HY(Yam) or HY(Vic). Values ​​shown are the mean ± SD of three independent experiments. P values ​​were calculated using a linear mixed model (*P < 0.05; **P < 0.01). Red and blue asterisks indicate a comparison between the respective viruses and the WT virus; beige asterisks indicate a comparison between the viruses depicted in red and blue. [Figure 8D] HY(Yam) and HY(Vic) backbone exchange. (A and B) Comparison of the virus and hemagglutination titers of two wild-type Yamagata lineage viruses with viruses carrying the same HA and NA vRNAs combined with the internal genes of HY(Yam) or HY(Vic). (C and D) Comparison of the virus and hemagglutination titers of two wild-type Yamagata lineage viruses with viruses carrying the same HA and NA vRNAs combined with the internal genes of HY(Yam) or HY(Vic). Values ​​shown are the mean ± SD of three independent experiments. P values ​​were calculated using a linear mixed model (*P < 0.05; **P < 0.01). Red and blue asterisks indicate a comparison between the respective viruses and the WT virus; beige asterisks indicate a comparison between the viruses depicted in red and blue. [Figure 9A]Comparison of high-yield influenza A and B vaccine virus backbones. (A and B) Virus yields and hemagglutination titers of (i) the indicated viruses; (ii) viruses with the indicated HA and NA vRNAs combined with the internal genes of HY (Yam); and (iii) viruses with the indicated chimeric A / B HA and NA vRNAs combined with the internal genes of a high-yield influenza A virus. (C and D) Similar experiments were performed with viruses of the Victoria lineage. (E and F) Comparison of the indicated wild-type and hybrid viruses in embryonated chicken eggs. Values ​​shown are the mean ± SD of three independent experiments. Statistical significance was determined by using a linear mixed model (A–D) or by two-way analysis of variance followed by Tukey's post hoc test (E and F) (*P < 0.05; **P < 0.01); where the titer of the high-yield vaccine candidate was lower than that of the wild-type virus, no P value is shown. Red and blue asterisks indicate a comparison of the respective virus to the WT virus, and beige asterisks indicate a comparison of the viruses depicted in red and blue. [Figure 9B]Comparison of high-yield influenza A and B vaccine virus backbones. (A and B) Virus yields and hemagglutination titers of (i) the indicated viruses; (ii) viruses with the indicated HA and NA vRNAs combined with the internal genes of HY (Yam); and (iii) viruses with the indicated chimeric A / B HA and NA vRNAs combined with the internal genes of a high-yield influenza A virus. (C and D) Similar experiments were performed with viruses of the Victoria lineage. (E and F) Comparison of the indicated wild-type and hybrid viruses in embryonated chicken eggs. Values ​​shown are the mean ± SD of three independent experiments. Statistical significance was determined by using a linear mixed model (A–D) or by two-way analysis of variance followed by Tukey's post hoc test (E and F) (*P < 0.05; **P < 0.01); where the titer of the high-yield vaccine candidate was lower than that of the wild-type virus, no P value is shown. Red and blue asterisks indicate a comparison of the respective virus to the WT virus, and beige asterisks indicate a comparison of the viruses depicted in red and blue. [Figure 9C]Comparison of high-yield influenza A and B vaccine virus backbones. (A and B) Virus yields and hemagglutination titers of (i) the indicated viruses; (ii) viruses with the indicated HA and NA vRNAs combined with the internal genes of HY (Yam); and (iii) viruses with the indicated chimeric A / B HA and NA vRNAs combined with the internal genes of a high-yield influenza A virus. (C and D) Similar experiments were performed with viruses of the Victoria lineage. (E and F) Comparison of the indicated wild-type and hybrid viruses in embryonated chicken eggs. Values ​​shown are the mean ± SD of three independent experiments. Statistical significance was determined by using a linear mixed model (A–D) or by two-way analysis of variance followed by Tukey's post hoc test (E and F) (*P < 0.05; **P < 0.01); where the titer of the high-yield vaccine candidate was lower than that of the wild-type virus, no P value is shown. Red and blue asterisks indicate a comparison of the respective virus to the WT virus, and beige asterisks indicate a comparison of the viruses depicted in red and blue. [Figure 9D]Comparison of high-yield influenza A and B vaccine virus backbones. (A and B) Virus yields and hemagglutination titers of (i) the indicated viruses; (ii) viruses with the indicated HA and NA vRNAs combined with the internal genes of HY (Yam); and (iii) viruses with the indicated chimeric A / B HA and NA vRNAs combined with the internal genes of a high-yield influenza A virus. (C and D) Similar experiments were performed with viruses of the Victoria lineage. (E and F) Comparison of the indicated wild-type and hybrid viruses in embryonated chicken eggs. Values ​​shown are the mean ± SD of three independent experiments. Statistical significance was determined by using a linear mixed model (A–D) or by two-way analysis of variance followed by Tukey's post hoc test (E and F) (*P < 0.05; **P < 0.01); where the titer of the high-yield vaccine candidate was lower than that of the wild-type virus, no P value is shown. Red and blue asterisks indicate a comparison of the respective virus to the WT virus, and beige asterisks indicate a comparison of the viruses depicted in red and blue. [Figure 9E]Comparison of high-yield influenza A and B vaccine virus backbones. (A and B) Virus yields and hemagglutination titers of (i) the indicated viruses; (ii) viruses with the indicated HA and NA vRNAs combined with the internal genes of HY (Yam); and (iii) viruses with the indicated chimeric A / B HA and NA vRNAs combined with the internal genes of a high-yield influenza A virus. (C and D) Similar experiments were performed with viruses of the Victoria lineage. (E and F) Comparison of the indicated wild-type and hybrid viruses in embryonated chicken eggs. Values ​​shown are the mean ± SD of three independent experiments. Statistical significance was determined by using a linear mixed model (A–D) or by two-way analysis of variance followed by Tukey's post hoc test (E and F) (*P < 0.05; **P < 0.01); where the titer of the high-yield vaccine candidate was lower than that of the wild-type virus, no P value is shown. Red and blue asterisks indicate a comparison of the respective virus to the WT virus, and beige asterisks indicate a comparison of the viruses depicted in red and blue. [Figure 9F]Comparison of high-yield influenza A and B vaccine virus backbones. (A and B) Virus yield and hemagglutination titers of (i) the indicated viruses; (ii) viruses with the indicated HA and NA vRNAs combined with the internal genes of HY (Yam); and (iii) viruses with the indicated chimeric A / B HA and NA vRNAs combined with the internal genes of a high-yield influenza A virus. (C and D) Similar experiments were performed with viruses of the Victoria lineage. (E and F) Comparison of the indicated wild-type and hybrid viruses in embryonated chicken eggs. Values ​​shown are the mean ± SD of three independent experiments. Statistical significance was determined by using a linear mixed model (A–D) or by two-way analysis of variance followed by Tukey's post hoc test (E and F) (*P < 0.05; **P < 0.01); where the titer of the high-yield vaccine candidate was lower than that of the wild-type virus, no P value is shown. Red and blue asterisks indicate a comparison of the respective virus to the WT virus, and beige asterisks indicate a comparison of the viruses depicted in red and blue. [Figure 10A]Assessment of total viral protein yield and HA content of HY(Yam) and HY(Vic) viruses. A) Comparison of viruses harboring the indicated Yamagata lineage virus HA and NA vRNAs in combination with the same native wild-type virus (WT) or HY(Yam) internal vRNA. B) Comparison of viruses containing the indicated Victoria lineage virus HA and NA vRNAs in combination with the same wild-type (WT) or HY (Vic9) internal vRNAs. Total viral protein yields of sucrose gradient-purified virus samples grown in MDCK cells are shown (left and center). PNGaseF treatment deglycosylates HA1 and HA2; this treatment was performed because glycosylated HA2 migrates at a similar molecular weight to M1. HA content (right) was calculated based on the total viral protein amount and the relative amount of HA. Values ​​shown are the mean ± SD of three independent experiments. Statistical significance was assessed using one-way analysis of variance followed by Dunnett's test to compare the total viral protein yield and HA content of wild-type virus with that of recombinant high-yield vaccine viruses (*P < 0.05; **P < 0.01). [Figure 10B]Assessment of total viral protein yield and HA content of HY(Yam) and HY(Vic) viruses. A) Comparison of viruses harboring the indicated Yamagata lineage virus HA and NA vRNAs in combination with the same native wild-type virus (WT) or HY(Yam) internal vRNA. B) Comparison of viruses containing the indicated Victoria lineage virus HA and NA vRNAs in combination with the same wild-type (WT) or HY (Vic9) internal vRNAs. Total viral protein yields of sucrose gradient-purified virus samples grown in MDCK cells are shown (left and center). PNGaseF treatment deglycosylates HA1 and HA2; this treatment was performed because glycosylated HA2 migrates at a similar molecular weight to M1. HA content (right) was calculated based on the total viral protein amount and the relative amount of HA. Values ​​shown are the mean ± SD of three independent experiments. Statistical significance was assessed using one-way analysis of variance followed by Dunnett's test to compare the total viral protein yield and HA content of wild-type virus with that of recombinant high-yield vaccine viruses (*P < 0.05; **P < 0.01). [Figure 11A]Pathogenicity of HY(Yam) and HY(Vic) viruses in mice. A-C) Comparison of wild-type Yamagata lineage virus (B / Massachusetts / 2 / 2012), a virus with B / Massachusetts / 2 / 2012 HA and NA vRNAs, combined with residual vRNAs from B / Yamagata / 1 / 73 (used to generate the viral library), and a virus with B / Massachusetts / 2 / 2012 HA and RA in combination with residual vRNAs from HY(Yam). D-F) Comparison of wild-type Victoria lineage virus (B / Brisbane / 60 / 2008), a virus with B / Brisbane / 60 / 2008 HA and NA vRNAs combined with residual vRNAs from B / Yamagata / 1 / 73 (used to generate the viral library), and a virus with B / Brisbane / 60 / 2008 HA and NA vRNAs in combination with residual vRNAs from HY(Vic). BALB / c mice (5 per group) were inoculated intranasally with 10 pfu of the indicated viruses and monitored daily for weight change (A and D) and survival (B and E). To assess viral replication in mice, 10 additional mice were infected with 10 pfu of the indicated viruses. On days 3 and 6 postinfection, mice from each group were sacrificed, and lung virus titers were measured using a plaque assay in MDCK cells (C and F). Statistical significance was assessed using one-way analysis of variance followed by Dunnett's test (*P < 0.05; **P < 0.01). [Figure 11B]Pathogenicity of HY(Yam) and HY(Vic) viruses in mice. A-C) Comparison of wild-type Yamagata lineage virus (B / Massachusetts / 2 / 2012), a virus with B / Massachusetts / 2 / 2012 HA and NA vRNAs, combined with residual vRNAs from B / Yamagata / 1 / 73 (used to generate the viral library), and a virus with B / Massachusetts / 2 / 2012 HA and RA in combination with residual vRNAs from HY(Yam). D-F) Comparison of wild-type Victoria lineage virus (B / Brisbane / 60 / 2008), a virus with B / Brisbane / 60 / 2008 HA and NA vRNAs combined with residual vRNAs from B / Yamagata / 1 / 73 (used to generate the viral library), and a virus with B / Brisbane / 60 / 2008 HA and NA vRNAs in combination with residual vRNAs from HY(Vic). BALB / c mice (5 per group) were inoculated intranasally with 10 pfu of the indicated viruses and monitored daily for weight change (A and D) and survival (B and E). To assess viral replication in mice, 10 additional mice were infected with 10 pfu of the indicated viruses. On days 3 and 6 postinfection, mice from each group were sacrificed, and lung virus titers were measured using a plaque assay in MDCK cells (C and F). Statistical significance was assessed using one-way analysis of variance followed by Dunnett's test (*P < 0.05; **P < 0.01). [Figure 11C]Pathogenicity of HY(Yam) and HY(Vic) viruses in mice. A-C) Comparison of wild-type Yamagata lineage virus (B / Massachusetts / 2 / 2012), a virus with B / Massachusetts / 2 / 2012 HA and NA vRNAs, combined with residual vRNAs from B / Yamagata / 1 / 73 (used to generate the viral library), and a virus with B / Massachusetts / 2 / 2012 HA and RA in combination with residual vRNAs from HY(Yam). D-F) Comparison of wild-type Victoria lineage virus (B / Brisbane / 60 / 2008), a virus with B / Brisbane / 60 / 2008 HA and NA vRNAs combined with residual vRNAs from B / Yamagata / 1 / 73 (used to generate the viral library), and a virus with B / Brisbane / 60 / 2008 HA and NA vRNAs in combination with residual vRNAs from HY(Vic). BALB / c mice (5 per group) were inoculated intranasally with 10 pfu of the indicated viruses and monitored daily for weight change (A and D) and survival (B and E). To assess viral replication in mice, 10 additional mice were infected with 10 pfu of the indicated viruses. On days 3 and 6 postinfection, mice from each group were sacrificed, and lung virus titers were measured using a plaque assay in MDCK cells (C and F). Statistical significance was assessed using one-way analysis of variance followed by Dunnett's test (*P < 0.05; **P < 0.01). [Figure 11D]Pathogenicity of HY(Yam) and HY(Vic) viruses in mice. A-C) Comparison of wild-type Yamagata lineage virus (B / Massachusetts / 2 / 2012), a virus with B / Massachusetts / 2 / 2012 HA and NA vRNAs, combined with residual vRNAs from B / Yamagata / 1 / 73 (used to generate the viral library), and a virus with B / Massachusetts / 2 / 2012 HA and RA in combination with residual vRNAs from HY(Yam). D-F) Comparison of wild-type Victoria lineage virus (B / Brisbane / 60 / 2008), a virus with B / Brisbane / 60 / 2008 HA and NA vRNAs combined with residual vRNAs from B / Yamagata / 1 / 73 (used to generate the viral library), and a virus with B / Brisbane / 60 / 2008 HA and NA vRNAs in combination with residual vRNAs from HY(Vic). BALB / c mice (5 per group) were inoculated intranasally with 10 pfu of the indicated viruses and monitored daily for weight change (A and D) and survival (B and E). To assess viral replication in mice, 10 additional mice were infected with 10 pfu of the indicated viruses. On days 3 and 6 postinfection, mice from each group were sacrificed, and lung virus titers were measured using a plaque assay in MDCK cells (C and F). Statistical significance was assessed using one-way analysis of variance followed by Dunnett's test (*P < 0.05; **P < 0.01). [Figure 11E]Pathogenicity of HY(Yam) and HY(Vic) viruses in mice. A-C) Comparison of wild-type Yamagata lineage virus (B / Massachusetts / 2 / 2012), a virus with B / Massachusetts / 2 / 2012 HA and NA vRNAs, combined with residual vRNAs from B / Yamagata / 1 / 73 (used to generate the viral library), and a virus with B / Massachusetts / 2 / 2012 HA and RA in combination with residual vRNAs from HY(Yam). D-F) Comparison of wild-type Victoria lineage virus (B / Brisbane / 60 / 2008), a virus with B / Brisbane / 60 / 2008 HA and NA vRNAs combined with residual vRNAs from B / Yamagata / 1 / 73 (used to generate the viral library), and a virus with B / Brisbane / 60 / 2008 HA and NA vRNAs in combination with residual vRNAs from HY(Vic). BALB / c mice (5 per group) were inoculated intranasally with 10 pfu of the indicated viruses and monitored daily for weight change (A and D) and survival (B and E). To assess viral replication in mice, 10 additional mice were infected with 10 pfu of the indicated viruses. On days 3 and 6 postinfection, mice from each group were sacrificed, and lung virus titers were measured using a plaque assay in MDCK cells (C and F). Statistical significance was assessed using one-way analysis of variance followed by Dunnett's test (*P < 0.05; **P < 0.01). [Figure 11F]Pathogenicity of HY(Yam) and HY(Vic) viruses in mice. A-C) Comparison of wild-type Yamagata lineage virus (B / Massachusetts / 2 / 2012), a virus with B / Massachusetts / 2 / 2012 HA and NA vRNAs, combined with residual vRNAs from B / Yamagata / 1 / 73 (used to generate the viral library), and a virus with B / Massachusetts / 2 / 2012 HA and RA in combination with residual vRNAs from HY(Yam). D-F) Comparison of wild-type Victoria lineage virus (B / Brisbane / 60 / 2008), a virus with B / Brisbane / 60 / 2008 HA and NA vRNAs combined with residual vRNAs from B / Yamagata / 1 / 73 (used to generate the viral library), and a virus with B / Brisbane / 60 / 2008 HA and NA vRNAs in combination with residual vRNAs from HY(Vic). BALB / c mice (5 per group) were inoculated intranasally with 10 pfu of the indicated viruses and monitored daily for weight change (A and D) and survival (B and E). To assess viral replication in mice, 10 additional mice were infected with 10 pfu of the indicated viruses. On days 3 and 6 postinfection, mice from each group were sacrificed, and lung virus titers were measured using a plaque assay in MDCK cells (C and F). Statistical significance was assessed using one-way analysis of variance followed by Dunnett's test (*P < 0.05; **P < 0.01). [Figure 12A]Growth kinetics and hemagglutination titers of single reassortant viruses. A) Comparison of the parental virus used to generate the Yamagata lineage virus library (i.e., B / Yamagata / 1 / 73 with HA and NA vRNAs from B / Yokohama / UT-K31 / 2012) with a virus also carrying individual vRNAs from HY (Yam). B) Comparison of the parental virus used to generate the Victoria lineage virus library (i.e., B / Yamagata / 1 / 73 with HA and NA vRNAs from B / Yokohama / UT-K1A / 2011) with a virus also carrying individual vRNAs from HY (Vic). Data are from three independent experiments; mean titers ± SD are shown. Values ​​shown are the mean ± SD of three independent experiments. Statistical significance was determined using a linear mixed model (*P < 0.05; **P < 0.01). The color of the asterisk indicates the comparison of each virus with the comparator virus (depicted in black). [Figure 12B] Growth kinetics and hemagglutination titers of single reassortant viruses. A) Comparison of the parental virus used to generate the Yamagata lineage virus library (i.e., B / Yamagata / 1 / 73 with HA and NA vRNAs from B / Yokohama / UT-K31 / 2012) with a virus also carrying individual vRNAs from HY (Yam). B) Comparison of the parental virus used to generate the Victoria lineage virus library (i.e., B / Yamagata / 1 / 73 with HA and NA vRNAs from B / Yokohama / UT-K1A / 2011) with a virus also carrying individual vRNAs from HY (Vic). Data are from three independent experiments; mean titers ± SD are shown. Values ​​shown are the mean ± SD of three independent experiments. Statistical significance was determined using a linear mixed model (*P < 0.05; **P < 0.01). The color of the asterisk indicates the comparison of each virus with the comparator virus (depicted in black). [Figure 13A]Luciferase activity in a minireplicon assay at 35°C. Effect of mutations in the NP protein or PA and NS vRNAs on viral polymerase activity. 293T (A) or MDCK (B) cells were transfected with plasmids expressing the polymerase protein and wild-type or mutant NP proteins, and with a plasmid transcribing a viral-like RNA encoding luciferase. Luciferase activity was measured 48 hours later. In parallel, MDCK cells were transfected with the above protein expression plasmids and wild-type or mutant viral-like RNA encoding luciferase and carrying mutations in the noncoding regions of the PA vRNA (C) or NS vRNA (D). Luciferase activity was measured 48 hours later. Data are from three independent experiments; mean titers ± SD are shown. Statistical significance was assessed using one-way analysis of variance followed by Dunnett's test (*P < 0.05; **P < 0.01). [Figure 13B] Luciferase activity in a minireplicon assay at 35°C. Effect of mutations in the NP protein or PA and NS vRNAs on viral polymerase activity. 293T (A) or MDCK (B) cells were transfected with plasmids expressing the polymerase protein and wild-type or mutant NP proteins, and with a plasmid transcribing a viral-like RNA encoding luciferase. Luciferase activity was measured 48 hours later. In parallel, MDCK cells were transfected with the above protein expression plasmids and wild-type or mutant viral-like RNA encoding luciferase and carrying mutations in the noncoding regions of the PA vRNA (C) or NS vRNA (D). Luciferase activity was measured 48 hours later. Data are from three independent experiments; mean titers ± SD are shown. Statistical significance was assessed using one-way analysis of variance followed by Dunnett's test (*P < 0.05; **P < 0.01). [Figure 13C]Luciferase activity in a minireplicon assay at 35°C. Effect of mutations in the NP protein or PA and NS vRNAs on viral polymerase activity. 293T (A) or MDCK (B) cells were transfected with plasmids expressing the polymerase protein and wild-type or mutant NP proteins, and with a plasmid transcribing a viral-like RNA encoding luciferase. Luciferase activity was measured 48 hours later. In parallel, MDCK cells were transfected with the above protein expression plasmids and wild-type or mutant viral-like RNA encoding luciferase and carrying mutations in the noncoding regions of the PA vRNA (C) or NS vRNA (D). Luciferase activity was measured 48 hours later. Data are from three independent experiments; mean titers ± SD are shown. Statistical significance was assessed using one-way analysis of variance followed by Dunnett's test (*P < 0.05; **P < 0.01). [Figure 13D] Luciferase activity in a minireplicon assay at 35°C. Effect of mutations in the NP protein or PA and NS vRNAs on viral polymerase activity. 293T (A) or MDCK (B) cells were transfected with plasmids expressing the polymerase protein and wild-type or mutant NP proteins, and with a plasmid transcribing a viral-like RNA encoding luciferase. Luciferase activity was measured 48 hours later. In parallel, MDCK cells were transfected with the above protein expression plasmids and wild-type or mutant viral-like RNA encoding luciferase and carrying mutations in the noncoding regions of the PA vRNA (C) or NS vRNA (D). Luciferase activity was measured 48 hours later. Data are from three independent experiments; mean titers ± SD are shown. Statistical significance was assessed using one-way analysis of variance followed by Dunnett's test (*P < 0.05; **P < 0.01). [Figure 14A]Contribution of the M1 mutation in the HY backbone to the integration and composition of HA, NP, and M1 VLPs. 293T cells were transfected with protein expression plasmids for HA, NA, NP, BM2, NS2, and wild-type or mutant M1. Forty-eight hours after transfection, cell lysates and VLPs in cell culture supernatants were Western blotted with anti-HA, anti-NP, and anti-M1 monoclonal antibodies. The band intensities of HA, NP, and M1 were quantified using ImageJ software (NIH), and the relative percentages of HA, NP, and M1 in VLPs are shown in (B). Data are from three independent experiments. Mean titers ± SD are shown. Statistical significance was determined using one-way analysis of variance followed by Tukey's post-hoc test (*p<0.05; **p<0.01). [Figure 14B] Contribution of the M1 mutation in the HY backbone to the integration and composition of HA, NP, and M1 VLPs. 293T cells were transfected with protein expression plasmids for HA, NA, NP, BM2, NS2, and wild-type or mutant M1. Forty-eight hours after transfection, cell lysates and VLPs in cell culture supernatants were Western blotted with anti-HA, anti-NP, and anti-M1 monoclonal antibodies. The band intensities of HA, NP, and M1 were quantified using ImageJ software (NIH), and the relative percentages of HA, NP, and M1 in VLPs are shown in (B). Data are from three independent experiments. Mean titers ± SD are shown. Statistical significance was determined using one-way analysis of variance followed by Tukey's post-hoc test (*p<0.05; **p<0.01). [Figure 15A]Effect of NS1 mutations on IFN activity. A) To compare the ability of wild-type and mutant NS1 to inhibit IFN-β synthesis, 293T cells were transfected with wild-type or mutant NS1 protein expression plasmids and the reporter plasmid pGL-IFN-β, encoding firefly luciferase under the control of the IFN-β promoter. Cells were incubated for 24 hours, infected with 5 ml of Sendai virus, and incubated again for 24 hours, then lysed and firefly luciferase assayed. B) To determine the ability of wild-type and mutant NS1 to inhibit IFN-β-stimulated gene synthesis, 293T cells were transfected with wild-type or mutant NS1 protein expression plasmids and the reporter plasmid plSRE-Luc, encoding firefly luciferase under the control of an interferon-regulated promoter. After 24 hours, cells were stimulated with human IFN-β. Luciferase activity was measured 48 hours after transfection. Data are from three independent experiments. Mean titers ± SD are shown. Statistical significance was determined using one-way analysis of variance followed by Tukey's post-hoc test (*p<0.05; **p<0.01). [Figure 15B]Effect of NS1 mutations on IFN activity. A) To compare the ability of wild-type and mutant NS1 to inhibit IFN-β synthesis, 293T cells were transfected with wild-type or mutant NS1 protein expression plasmids and the reporter plasmid pGL-IFN-β, encoding firefly luciferase under the control of the IFN-β promoter. Cells were incubated for 24 hours, infected with 5 ml of Sendai virus, and incubated again for 24 hours, then lysed and firefly luciferase assayed. B) To determine the ability of wild-type and mutant NS1 to inhibit IFN-β-stimulated gene synthesis, 293T cells were transfected with wild-type or mutant NS1 protein expression plasmids and the reporter plasmid plSRE-Luc, encoding firefly luciferase under the control of an interferon-regulated promoter. After 24 hours, cells were stimulated with human IFN-β. Luciferase activity was measured 48 hours after transfection. Data are from three independent experiments. Mean titers ± SD are shown. Statistical significance was determined using one-way analysis of variance followed by Tukey's post-hoc test (*p<0.05; **p<0.01). [Figure 16A] Nucleotide sequences of viral segments in selected HY clones (PB2 and PB1 from Yamagata 1 / 73; SEQ ID NOs: 11 and 12); PA from Yamagata 1 / 73 with a1406g / c1445t / a2272t (SEQ ID NO: 13); NP from Yamagata 1 / 73 with P40S, c500t (SEQ ID NO: 14) or NP from Yamagata 1 / 73 with P40S / M204T (SEQ ID NO: 15); M from Yamagata 1 / 73 with R77K (SEQ ID NO: 16) or M from Yamagata 1 / 73 with M86T (SEQ ID NO: 17); and NS from Yamagata 1 / 73 with a39g K176Q (SEQ ID NO: 18) or NS from Yamagata 1 / 73 with 38(+1)g (SEQ ID NO: 19). [Figure 16B]Nucleotide sequences of viral segments in selected HY clones (PB2 and PB1 from Yamagata 1 / 73; SEQ ID NOs: 11 and 12); PA from Yamagata 1 / 73 with a1406g / c1445t / a2272t (SEQ ID NO: 13); NP from Yamagata 1 / 73 with P40S, c500t (SEQ ID NO: 14) or NP from Yamagata 1 / 73 with P40S / M204T (SEQ ID NO: 15); M from Yamagata 1 / 73 with R77K (SEQ ID NO: 16) or M from Yamagata 1 / 73 with M86T (SEQ ID NO: 17); and NS from Yamagata 1 / 73 with a39g K176Q (SEQ ID NO: 18) or NS from Yamagata 1 / 73 with 38(+1)g (SEQ ID NO: 19). [Figure 16C] Nucleotide sequences of viral segments in selected HY clones (PB2 and PB1 from Yamagata 1 / 73; SEQ ID NOs: 11 and 12); PA from Yamagata 1 / 73 with a1406g / c1445t / a2272t (SEQ ID NO: 13); NP from Yamagata 1 / 73 with P40S, c500t (SEQ ID NO: 14) or NP from Yamagata 1 / 73 with P40S / M204T (SEQ ID NO: 15); M from Yamagata 1 / 73 with R77K (SEQ ID NO: 16) or M from Yamagata 1 / 73 with M86T (SEQ ID NO: 17); and NS from Yamagata 1 / 73 with a39g K176Q (SEQ ID NO: 18) or NS from Yamagata 1 / 73 with 38(+1)g (SEQ ID NO: 19). [Figure 16D]Nucleotide sequences of viral segments in selected HY clones (PB2 and PB1 from Yamagata 1 / 73; SEQ ID NOs: 11 and 12); PA from Yamagata 1 / 73 with a1406g / c1445t / a2272t (SEQ ID NO: 13); NP from Yamagata 1 / 73 with P40S, c500t (SEQ ID NO: 14) or NP from Yamagata 1 / 73 with P40S / M204T (SEQ ID NO: 15); M from Yamagata 1 / 73 with R77K (SEQ ID NO: 16) or M from Yamagata 1 / 73 with M86T (SEQ ID NO: 17); and NS from Yamagata 1 / 73 with a39g K176Q (SEQ ID NO: 18) or NS from Yamagata 1 / 73 with 38(+1)g (SEQ ID NO: 19). [Figure 16E] Nucleotide sequences of viral segments in selected HY clones (PB2 and PB1 from Yamagata 1 / 73; SEQ ID NOs: 11 and 12); PA from Yamagata 1 / 73 with a1406g / c1445t / a2272t (SEQ ID NO: 13); NP from Yamagata 1 / 73 with P40S, c500t (SEQ ID NO: 14) or NP from Yamagata 1 / 73 with P40S / M204T (SEQ ID NO: 15); M from Yamagata 1 / 73 with R77K (SEQ ID NO: 16) or M from Yamagata 1 / 73 with M86T (SEQ ID NO: 17); and NS from Yamagata 1 / 73 with a39g K176Q (SEQ ID NO: 18) or NS from Yamagata 1 / 73 with 38(+1)g (SEQ ID NO: 19). [Figure 16F]Nucleotide sequences of viral segments in selected HY clones (PB2 and PB1 from Yamagata 1 / 73; SEQ ID NOs: 11 and 12); PA from Yamagata 1 / 73 with a1406g / c1445t / a2272t (SEQ ID NO: 13); NP from Yamagata 1 / 73 with P40S, c500t (SEQ ID NO: 14) or NP from Yamagata 1 / 73 with P40S / M204T (SEQ ID NO: 15); M from Yamagata 1 / 73 with R77K (SEQ ID NO: 16) or M from Yamagata 1 / 73 with M86T (SEQ ID NO: 17); and NS from Yamagata 1 / 73 with a39g K176Q (SEQ ID NO: 18) or NS from Yamagata 1 / 73 with 38(+1)g (SEQ ID NO: 19). [Figure 16G] Nucleotide sequences of viral segments in selected HY clones (PB2 and PB1 from Yamagata 1 / 73; SEQ ID NOs: 11 and 12); PA from Yamagata 1 / 73 with a1406g / c1445t / a2272t (SEQ ID NO: 13); NP from Yamagata 1 / 73 with P40S, c500t (SEQ ID NO: 14) or NP from Yamagata 1 / 73 with P40S / M204T (SEQ ID NO: 15); M from Yamagata 1 / 73 with R77K (SEQ ID NO: 16) or M from Yamagata 1 / 73 with M86T (SEQ ID NO: 17); and NS from Yamagata 1 / 73 with a39g K176Q (SEQ ID NO: 18) or NS from Yamagata 1 / 73 with 38(+1)g (SEQ ID NO: 19). [Figure 16H]Nucleotide sequences of viral segments in selected HY clones (PB2 and PB1 from Yamagata 1 / 73; SEQ ID NOs: 11 and 12); PA from Yamagata 1 / 73 with a1406g / c1445t / a2272t (SEQ ID NO: 13); NP from Yamagata 1 / 73 with P40S, c500t (SEQ ID NO: 14) or NP from Yamagata 1 / 73 with P40S / M204T (SEQ ID NO: 15); M from Yamagata 1 / 73 with R77K (SEQ ID NO: 16) or M from Yamagata 1 / 73 with M86T (SEQ ID NO: 17); and NS from Yamagata 1 / 73 with a39g K176Q (SEQ ID NO: 18) or NS from Yamagata 1 / 73 with 38(+1)g (SEQ ID NO: 19). [Figure 16I] Nucleotide sequences of viral segments in selected HY clones (PB2 and PB1 from Yamagata 1 / 73; SEQ ID NOs: 11 and 12); PA from Yamagata 1 / 73 with a1406g / c1445t / a2272t (SEQ ID NO: 13); NP from Yamagata 1 / 73 with P40S, c500t (SEQ ID NO: 14) or NP from Yamagata 1 / 73 with P40S / M204T (SEQ ID NO: 15); M from Yamagata 1 / 73 with R77K (SEQ ID NO: 16) or M from Yamagata 1 / 73 with M86T (SEQ ID NO: 17); and NS from Yamagata 1 / 73 with a39g K176Q (SEQ ID NO: 18) or NS from Yamagata 1 / 73 with 38(+1)g (SEQ ID NO: 19). DETAILED DESCRIPTION OF THE INVENTION

[0046] definition As used herein, the term "isolated" refers to a nucleic acid molecule (e.g., a vector or plasmid), peptide or polypeptide (protein), or virus of the invention, such that it is free from association with in vivo materials or is substantially purified from in vitro materials. Isolated virus preparations are generally obtained by in vitro culture and propagation, and / or through passaging in eggs, and are substantially free of other infectious agents.

[0047] As used herein, "substantially purified" means that the species of interest is the predominant species, e.g., on a molar basis, over any other individual species in the composition, preferably greater than at least about 80% of the species present, and optionally 90% or more (e.g., 95%, 98%, 99% or more) of the species present in said composition.

[0048] As used herein, "substantially free" means below the detectable level of a particular infectious agent using standard detection methods for that agent.

[0049] A "recombinant" virus is a virus that has been manipulated in vitro, e.g., using recombinant DNA techniques, to introduce an alteration into the genome of the virus. Reassortant viruses can be prepared by recombinant or non-recombinant techniques.

[0050] As used herein, the term "recombinant nucleic acid" or "recombinant DNA sequence or segment" refers to a nucleic acid, e.g., DNA, derived from or isolated from a source, that can subsequently be modified chemically in vitro, such that the sequence does not exist in nature or corresponds to a naturally occurring sequence that is not positioned as it exists in a natural genome. An example of DNA "derived" from a source would be a sequence that is identified as a useful fragment and then chemically synthesized in essentially pure form. An example of such DNA "isolated" from a source is a useful DNA sequence that is excised or removed from the source by chemical means, for example, by use of restriction enzymes, so that it can be further manipulated (e.g., amplified) for the present invention by genetic engineering methodologies.

[0051] As used herein, a "heterologous" influenza virus gene or gene segment is derived from an influenza virus source that is different from the majority of other influenza virus genes or gene segments in the recombinant (e.g., reassortant) influenza virus.

[0052] The terms "isolated polypeptide," "isolated peptide," or "isolated protein" include polypeptides, peptides, or proteins encoded by cDNA or recombinant RNA, including those of synthetic origin, or some combination thereof.

[0053] As used herein, the term "recombinant protein" or "recombinant polypeptide" refers to a protein molecule that is expressed from a recombinant DNA molecule. In contrast, the term "native protein" is used herein to refer to a protein that has been isolated from a naturally occurring (e.g., non-recombinant) source. Molecular biology techniques can be used to produce recombinant forms that have identical properties compared to the native form of the protein.

[0054] Methods for alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm.

[0055] Computer implementations of these mathematical algorithms can be used to compare sequences to determine sequence identity. Alignments using these programs can be performed using default parameters. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by recognizing short words of length W in the query sequence that, when aligned to words of the same length in a database sequence, either match or satisfy a certain positive threshold score T. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as possible to increase the cumulative alignment score. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score, and extension of the word hits in each direction is stopped when the cumulative alignment score falls off by an amount X from its maximum achieved value, due to the accumulation of one or more negative-scoring residue alignments, causing the cumulative score to fall below zero, or the end of either sequence is reached.

[0056] In addition to calculating the 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 may be the smallest sum probability (P(N)), which provides an indication of the likelihood that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered to be similar to a reference sequence if the smallest sum probability in a 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.

[0057] The BLAST program (for nucleotide sequences) can use as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program can use as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See http: / / www.ncbi.nlm.nih.gov. Alignments can also be performed manually by inspection.

[0058] For sequence comparison, typically, one sequence serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent identity of the test sequence relative to the reference sequence based on the designated program parameters.

[0059] Structure and propagation of influenza B viruses Influenza B viruses possess a genome of eight single-stranded, negative-sense viral RNAs (vRNAs) encoding at least 10 proteins. The influenza virus life cycle begins with the binding of hemagglutinin (HA) to sialic acid-containing receptors on the surface of the host cell, followed by receptor-mediated endocytosis. The low pH in late endosomes induces a conformational shift in HA, thereby exposing the N-terminus of the HA2 subunit (called the fusion peptide). The fusion peptide initiates fusion of the viral and endosomal membranes, releasing matrix protein (M1) and the RNP complex into the cytoplasm. RNPs consist of a nucleoprotein (NP) that encapsulates vRNA and a viral polymerase complex formed by PA, PB1, and PB2 proteins. RNPs are transported into the nucleus, where transcription and replication occur. The RNA polymerase complex catalyzes three distinct reactions: the synthesis of mRNA with a 5' cap and 3' poly(A) structure, full-length complementary RNA (cRNA), and genomic vRNA using the cRNA as a template. The newly synthesized vRNA, NP, and polymerase protein are then assembled into RNPs, exported from the nucleus, and transported to the plasma membrane where budding of progeny virus particles occurs. The neuraminidase (NA) protein plays a key role in the late stages of infection by removing sialic acid from sialyloligosaccharides, releasing newly assembled virions from the cell surface and preventing self-aggregation of virus particles. Viral assembly involves protein-protein and protein-vRNA interactions, although the nature of these interactions is largely unknown.

[0060] Influenza B virus of the present invention Mutations that increase the replication capacity of viruses in cell culture and / or embryonated eggs are effective in amplifying influenza viruses and establishing a potent influenza vaccine platform. Currently, most influenza B vaccines are produced in embryonated chicken eggs. Influenza vaccines produced in MDCK cells are currently approved for human use in the United States and Europe, and Vero cell-derived influenza vaccines are approved for human use in Europe. As described herein, a viral library carrying random mutations in the "internal" viral genes (viral genes excluding those encoding the viral surface glycoproteins HA and NA) of a vaccine virus isolate, e.g., the NA and HA genes from B / Yokohama / UT-K31 / 2012 (representing the Yamagata lineage) or the internal genes of B / Yamagata 1 / 73 with the NA and HA genes from B / Yokohama / UT-K1A / 2011 (representing the Victoria lineage), is generated and passaged in cells (e.g., MDCK or Vero cells). The identified mutations result in higher viral titers in cells (and may also increase viral titers in heterologous cells and / or embryonated chicken eggs), enabling more efficient influenza B virus growth and more cost-effective vaccine production. In addition to mutations in the coding regions of the internal viral segments and viral glycoproteins, mutations in non-coding regions have been observed to increase viral titers, e.g., g1795a in the NP segment, a39g in the NS segment, an additional g after position 38 in the NS segment, or g2213a or a2272t in the PA segment. The resulting coding sequences that result in enhanced growth can also have codon usage optimized for expression in mammalian cells, such as canine or primate cells, or avian cells, such as chicken embryos. The mutations can be used in various combinations, with the results dependent on the cell line (or egg) being used and the desired level of improvement in viral replication.One or more selected mutations may be introduced into one or more internal genes of a vaccine virus isolate, and the one or more internal genes with one or more mutations may be selected for inclusion in a reassortant useful as a vaccine virus, which may then be combined with other viruses, such as one or more influenza A viruses and / or one or more influenza B viruses, to form a multivalent vaccine.

[0061] Cell lines that can be used in the present invention Any cell, e.g., any avian or mammalian cell, such as human (293T or PER.C6® cells), or canine (MDCK), bovine, equine, feline, porcine, ovine, rodent (e.g., mink (e.g., MvLu1 cells)), or hamster (e.g., CHO cells), or non-human primate (e.g., Vero cells, including mutant cells), can support efficient replication of influenza virus and can be used to isolate and / or propagate influenza virus. Isolated virus can be used to prepare reassortant virus. In one embodiment, the host cell for vaccine production is a continuous mammalian or avian cell line or cell line. Full characterization of the cells used can be performed, including appropriate testing for purity of the final product. Data that can be used to characterize a cell include (a) its origin, derivation, and subculture history; (b) information about its growth and morphological characteristics; (c) test results for exogenous agents; (d) distinguishing characteristics such as biochemical, immunological, and cytogenetic patterns that make the cell distinctive among other cell lines; and (e) test results for tumorigenicity. In one embodiment, the host cells used have as low a subculture level or population doubling as possible.

[0062] In one embodiment, the cells are a WHO-certified or verifiable continuous cell line. Requirements for certifying such cell lines include characterization of at least one of pedigree, growth characteristics, immunological markers, viral susceptibility to tumorigenicity, and storage conditions, as well as testing in animals, eggs, and cell cultures. Such characterization is used to confirm that the cells are free of detectable adventitious agents. In some countries, cytokaryochemistry may also be required. Additionally, tumorigenicity may be tested in cells at the same passage level as those used in vaccine production. The virus may be purified by a process shown to provide consistent results prior to vaccine production (see, e.g., World Health Organization, 1982).

[0063] The virus produced by the host cells can be highly purified prior to vaccine or gene therapy formulation. Generally, the purification procedure results in extensive removal of cellular DNA and other cellular components, as well as foreign agents. Procedures that significantly degrade or denature DNA can also be used.

[0064] Influenza vaccine Vaccines of the present invention comprise an isolated recombinant influenza virus of the present invention and, optionally, one or more other isolated viruses, including other isolated influenza viruses, one or more immunogenic proteins or glycoproteins of one or more isolated influenza viruses or one or more other pathogens (e.g., one or more immunogenic proteins from bacteria, non-influenza viruses, yeast, or fungi), or an isolated nucleic acid encoding one or more viral proteins (e.g., a DNA vaccine) comprising one or more immunogenic proteins of an isolated influenza virus of the present invention. In one embodiment, the influenza virus of the present invention can be a vaccine vector for influenza virus or other pathogens.

[0065] Intact virion vaccines can be concentrated by ultrafiltration and then purified by zonal centrifugation or chromatography. Viruses other than those of the invention, such as those included in multivalent vaccines, can be inactivated before or after purification, for example, with formalin or β-propiolactone.

[0066] Subunit vaccines contain purified glycoproteins. Such vaccines can be prepared as follows: surface antigens are purified, for example, by ultracentrifugation, using a virus suspension fragmented by treatment with a detergent. Subunit vaccines therefore contain primarily HA and NA proteins. The detergent used can be, for example, a cationic detergent such as hexadecyltrimethylammonium bromide (Bachmeyer, 1975), an anionic detergent such as ammonium deoxycholate (Laver & Webster, 1976), or a nonionic detergent such as that sold under the name TRITON X 100. The hemagglutinin can also be purified after treating the virions with a protease such as bromelain. The subunit vaccines can be combined with the attenuated viruses of the present invention in multivalent vaccines.

[0067] Split vaccines contain virions that have been treated with a lipid-dissolving agent. Split vaccines can be prepared as follows: an aqueous suspension of purified virus obtained as described above, with or without inactivation, is treated with a lipid solvent, such as ethyl ether or chloroform associated with a surfactant, under stirring. Dissolution of the viral envelope lipids results in fragmentation of the viral particles. The aqueous phase containing the split vaccine, primarily composed of hemagglutinin and neuraminidase, and core or its degradation products, from which the original lipid environment has been removed, is recovered. Residual infectious particles are then inactivated, if not already complete. The split vaccine can be combined with the attenuated virus of the present invention in a multivalent vaccine.

[0068] Inactivated vaccines Inactivated influenza virus vaccines are provided by activating replicating virus using known methods, such as, but not limited to, formalin or β-propiolactone treatment. Inactivated vaccine types that can be used in the present invention can include whole virus (WV) vaccines or subvirion (SV) (split) vaccines. The WV vaccines include intact inactivated vaccines, while the SV vaccines include purified virus that has been disrupted with a detergent to solubilize the lipid-containing viral envelope, followed by chemical inactivation of residual virus.

[0069] In addition, vaccines that can be used include vaccines that contain isolated HA and NA surface proteins, referred to as surface antigen or subunit vaccines.

[0070] Live attenuated virus vaccines Live attenuated influenza virus vaccines, such as vaccines comprising the recombinant viruses of the invention, can be used to prevent or treat influenza virus infection. Attenuation can be achieved in a single step by transferring attenuating genes from an attenuated donor virus to a cloned isolate or reassortant virus by known methods. Because resistance to influenza A viruses is primarily mediated by the generation of an immune response against the HA and / or NA glycoproteins, the genes encoding these surface antigens are derived from the reassortant virus or clinical isolate. The attenuating genes are derived from the attenuated parent. In this approach, the genes conferring attenuation generally do not encode the HA and NA glycoproteins.

[0071] Viruses (donor influenza viruses) capable of reproducibly attenuating influenza viruses are available; for example, cold-adapted (ca) donor viruses can be used to produce attenuated vaccines. Live, attenuated reassortant virus vaccines can be produced by mating the ca donor virus with replicated viruses of the pathogen. Reassortant progeny are then selected at 25°C (restrictive for virulent virus replication) in the presence of appropriate antisera that inhibit replication of viruses bearing the surface antigens of the attenuated ca donor virus. Useful reassortant viruses are (a) infectious, (b) attenuated for seronegative non-adult mammals and immunologically primed adult mammals, (c) immunogenic, and (d) genetically stable. The immunogenicity of the ca reassortant viruses is comparable to their replication levels. Thus, acquisition of the six transferable genes of the ca donor virus by new wild-type viruses reproducibly attenuated these viruses for use in vaccinating susceptible mammals, both adults and non-adults.

[0072] To rescue viruses harboring these mutant genes, other attenuating mutations can be introduced into influenza virus genes by site-directed mutagenesis. Attenuating mutations can be introduced into non-coding as well as coding regions of the genome. Such attenuating mutations can also be introduced into genes other than HA or NA, such as the PB2 polymerase gene. Thus, novel donor viruses can be generated that have attenuating mutations introduced by site-directed mutagenesis, and such novel donor viruses can be used in the generation of live, attenuated reassortant vaccine candidates in a manner similar to that described above for the ca donor virus. Similarly, other known and suitable attenuated donor strains can be reassorted with influenza virus to obtain attenuated vaccines suitable for use in the vaccination of mammals.

[0073] In one embodiment, such an attenuated virus maintains genes from the virus that encode antigenic determinants substantially similar to those of the original clinical isolate, since the goal of the attenuated vaccine is to provide substantially the same antigenicity as the original clinical isolate of the virus, but lacks pathogenicity to the extent that it minimizes the likelihood of the vaccine inducing a significant disease state in a vaccinated mammal.

[0074] Thus, the viruses in a multivalent vaccine can be attenuated or inactivated, formulated, and administered according to known methods as a vaccine to induce an immune response in an animal (e.g., a mammal). Methods for determining whether such attenuated or inactivated vaccines maintain similar antigenicity to clinical isolates or high-growth strains derived therefrom are well known in the art. Such known methods include the use of serum or antibodies to eliminate viruses expressing antigenic determinants of the donor virus; chemical selection (e.g., amantadine or rimantidine); HA and NA activity and inhibition; and nucleic acid screening (e.g., probe hybridization or PCR) to confirm that the donor genes encoding the antigenic determinants (e.g., HA or NA genes) are absent from the attenuated virus.

[0075] Pharmaceutical Composition Pharmaceutical compositions of the present invention suitable for inoculation (e.g., nasal, parenteral, or oral administration) comprise one or more influenza isolates, e.g., one or more attenuated or inactivated influenza viruses, subunits thereof, and / or isolated nucleic acids encoding one or more proteins thereof, and optionally further include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The compositions can further comprise adjuvants or excipients known in the art. Compositions of the present invention are generally presented in the form of individual doses (unit doses).

[0076] Conventional vaccines generally contain about 0.1-200 μg, for example 30-100 μg, of HA from each strain present in the composition. The vaccines that form the basis of the vaccine compositions of the invention can contain a single influenza virus or a combination of influenza viruses, such as at least two or three influenza viruses, including one or more reassortant viruses.

[0077] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and / or emulsions, which may contain auxiliary substances or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Carriers or occlusive dressings may be used to increase skin penetration and enhance antigen absorption. Liquid dosage forms for oral administration generally contain liposome solutions containing the liquid dosage forms. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Such compositions may also contain adjuvants, wetting agents, emulsifying and suspending agents, or sweeteners, flavoring agents, or perfuming agents in addition to inert diluents.

[0078] When the compositions of the present invention are used for individual administration, they can further contain salts, buffers, adjuvants, or other substances as desired to improve the effectiveness of the compositions. For vaccines, adjuvants, substances that can enhance a specific immune response, can be used. Typically, the adjuvant and the composition are mixed before presentation to the immune system, or are separate but present at the same site in the organism being immunized.

[0079] Heterogeneity in a vaccine can be provided by mixing influenza viruses replicated from at least two strains of influenza virus, e.g., 2-20 strains, or any range or value therein. Using techniques known in the art, a vaccine can be provided for variation within a single strain of influenza virus.

[0080] The pharmaceutical compositions according to the present invention may comprise at least one chemotherapeutic compound, such as, but not limited to, gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon-α, interferon-β, interferon-γ, tumor necrosis factor-alpha, thiosemicarbazones, methiazone, rifampin, ribavirin, pyrimidine analogs, purine analogs, foscarnet, phosphonoacetic acid, or the like, for example, immunosuppressants, anti-inflammatory agents, or immunostimulants for gene therapy, and vaccines. The compositions may further or additionally include chemotherapy drugs including benzodiazepines, ...

[0081] The compositions may also contain variable but small amounts of endotoxin-free formaldehyde, and preservatives that have been found to be safe and have no undesirable effects in organisms to which the compositions are administered.

[0082] Purpose of the drug Administration of the composition (or the antisera it elicits) may be for "prophylactic" or "therapeutic" purposes. When provided prophylactically, the compositions of the invention, which are vaccines, are provided before the onset of symptoms or clinical signs of pathogen infection. Prophylactic administration of the composition serves to prevent or reduce any subsequent infection. When provided prophylactically, the gene therapy compositions of the invention are provided before the onset of any symptoms or clinical signs of disease. Prophylactic administration of the composition serves to prevent or reduce one or more symptoms or clinical signs associated with the disease.

[0083] When provided therapeutically, a viral vaccine is provided upon detection of a symptom or clinical sign of an actual infection. The therapeutic administration of the compound serves to attenuate any actual infection. When provided therapeutically, a gene therapy composition is provided upon detection of a symptom or clinical sign of the disease. The therapeutic administration of the compound serves to attenuate the symptom or clinical sign of the disease.

[0084] Thus, the vaccine compositions of the invention can be provided either before the onset of symptoms of infection (to prevent or attenuate anticipated infection) or after the onset of actual infection. Similarly, for gene therapy, the compositions can be provided before any symptoms or clinical signs of a disorder or disease appear, or after one or more symptoms are detected.

[0085] A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient mammal. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. A composition of the invention is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, for example, if it enhances at least one primary or secondary humoral or cellular immune response to at least one strain of infectious influenza virus.

[0086] The "protection" provided need not be absolute, i.e., it need not completely prevent or eradicate said influenza infection, provided there is a statistically significant improvement compared to a control population or set of mammals. Protection may be limited to reducing the severity or rapidity of symptomatic expression or clinical signs of said influenza infection.

[0087] Medicines Management The compositions of the invention can confer resistance to one or more pathogens, e.g., one or more influenza virus strains, through either passive or active immunization. In active immunization, the live-attenuated vaccine composition is administered prophylactically to a host (e.g., a mammal), and the host's immune response to the administration protects against infection and / or disease. For passive immunization, the induced antisera can be collected and administered to a recipient suspected of having an infection caused by at least one influenza virus strain. The gene therapy compositions of the invention can deliver prophylactic or therapeutic levels of a desired gene product through active immunization.

[0088] In one embodiment, the vaccine is provided to a mammalian female (at or before pregnancy or parturition) under conditions of time and in an amount sufficient to cause the production of an immune response that serves to protect both the female and her fetus or newborn (through passive uptake of antibodies in the placenta or breast milk).

[0089] Thus, the present invention includes methods for preventing or attenuating a disorder or disease, such as infection by at least one strain of a pathogen. As used herein, a vaccine is said to prevent or attenuate a disease if its administration results in either a full or partial attenuation (i.e., suppression) of clinical signs or symptoms of the disease, or total or partial immunity of an individual to the disease. As used herein, a gene therapy composition is said to prevent or attenuate a disease if its administration results in either a full or partial attenuation (i.e., suppression) of clinical signs or symptoms of the disease.

[0090] Compositions comprising one or more influenza viruses of the present invention, including attenuated influenza viruses and one or more other isolated viruses, one or more isolated viral proteins thereof, one or more isolated nucleic acid molecules encoding one or more viral proteins thereof, or combinations thereof, can be administered by any means that achieves the intended purpose.

[0091] For example, administration of such compositions may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral or transdermal routes, etc. Parenteral administration may be accomplished by bolus injection or gradual perfusion over time.

[0092] A typical regimen for preventing, suppressing, or treating a condition associated with influenza virus is administered as a single treatment, or repeated as augmentation or booster doses over a period of from 1 week to about 24 months, or any range or value therein.

[0093] According to the present invention, an "effective amount" of a composition is an amount sufficient to achieve the desired effect. It is understood that the effective dosage may depend on the species, age, sex, health, and weight of the recipient, type of concurrent treatment, if any, frequency of treatment, and the nature of the desired effect. The ranges of effective doses provided below are not intended to limit the invention and represent dosage ranges.

[0094] The dosage of a live attenuated or killed virus vaccine for an animal, such as an adult mammal, is about 10 2 -10 15 , e.g., 10 3 -10 12 The dose may be in units of plaque-forming units (PFU), or any range or value therein. The dose of an inactivated vaccine may range from about 0.1 to 1000, e.g., 30 to 100 μg of HA protein. However, using an existing vaccine as a starting point, the dose must be a safe and effective amount as determined by conventional methods.

[0095] The amount of immunoreactive HA in each dose of the replicated viral vaccine can be standardized to contain an appropriate amount, e.g., 30-100 μg, or any range or value therein, or an amount recommended by a government agency or certified professional. The amount of NA can also be standardized, although this glycoprotein can be unstable during purification and storage.

[0096] The amount of immunoreactive HA in each dose of replicated viral vaccine can be standardized to contain an appropriate amount, e.g., 1-50 μg, or any range or value therein, or the amount recommended by the U.S. Public Health Service (PHS), which is typically 15 μg per component for older children (ages 3 and older) and 7.5 μg per component 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 volume of vaccine can contain approximately 1-50 billion viral particles, preferably 10 billion particles.

[0097] Illustrative Embodiments In one embodiment, the recombinant or reassortant influenza B virus comprises: For example, but not limited to, residues in HA including a residue other than T at position 34, a residue other than K at position 129, a residue other than N at position 168, a residue other than T at position 196, or any combination thereof; residues in NA including, but not limited to, a residue other than N at position 169 and / or a residue other than G at position 434; residues in NP including, but not limited to, a residue other than alanine (A) at position 28, a residue other than P at position 40, a residue other than P at position 51, a residue other than E at position 52, a residue other than S at position 57, a residue other than M at position 204, a nucleotide other than g at nucleotide position 1795, or combinations thereof; residues in M1 including, but not limited to, a residue other than G at position 34, a residue other than aspartic acid (D) at position 54, a residue other than R at position 77, and a residue other than M at position 86; residues in BM2 including, but not limited to, a residue other than H at position 58 and / or a residue other than R at position 80; It has amino acids that enhance replication in MDCK cells, such as, but not limited to, a residue other than M at position 117, a residue other than K at position 176, and / or a residue other than S at position 252, a nucleotide other than a at position 39, or any combination thereof. In one embodiment, the recombinant influenza B virus comprises, for example, in HA1, a residue I at position 34, a residue E at position 129, an E, D at position 168, a P, isoleucine (I), A or N at position 196, or any combination thereof; in NA, a residue T at position 169 and / or a residue E at position 434; in NP, a residue T at position 28, a residue S at position 40, a residue Q at position 51, a residue K at position 52, a residue G at position 57, a residue T at position 214, a g at nucleotide position 1795, or any combination thereof; in M1, a residue valine (V) or N at position 34, a residue G at position 54, a residue K at position 77, a residue T at position 86, a residue N at position 97, or any combination thereof; in BM2, a residue R at position 58 and / or a residue G at position 80; NS1 has amino acids that result in enhanced replication in MDCK cells, such as a tyrosine (Y) residue at position 117, a glutamine (Q) residue at position 176, a T residue at position 252, a g at nucleotide position 39, an additional g after nucleotide position 38, or any combination thereof.

[0098] In one embodiment, the recombinant or reassortant influenza B virus comprises residues in HA1 including, for example, but not limited to, a residue other than R at position 98, a residue other than N at position 194, and / or a residue other than T at position 196, and / or residues in HA2 including, but not limited to, a residue other than K at position 39, a residue other than S at position 56, a residue other than K at position 61, or a residue other than D at position 112, or any combination thereof; residues in Na including, but not limited to, a residue other than T at position 76, a residue other than R at position 102, a residue other than E at position 105, a residue other than P at position 139, a residue other than T at position 436, a residue other than D at position 457, or any combination thereof; residues in NP including, but not limited to, a residue other than P at position 343; residues in M1 including, but not limited to, a residue other than G at position 34, a residue other than I at position 97, or any combination thereof; Residues in BM2 that include a residue other than H at position 58, a residue other than R at position 80, a residue other than H at position 27, a residue other than G at position 26, or any combination thereof; residues in NS1 that include a residue other than Y at position 42; residues in PA that include a residue other than Y at position 387, a residue other than V at position 434, a residue other than D at position 494, a residue other than T at position 524, a nucleotide other than a at nucleotide position 2272, and a nucleotide other than g at nucleotide position 2213, and residues in PB2 that include a residue other than N at position 16; or any combination thereof.In one embodiment, the recombinant influenza B virus comprises, in HA, a P, I, A or N (in HA1) at position 196, a K (in HA1) at position 98, a D (in HA1) at position 194, a G (in HA2) at position 39, a G (in HA2) at position 56, a N (in HA2) at position 51, or an E (in HA2) at position 112, or any combination thereof; in NA, a M at position 76, a K at position 102, a K at position 105, a S at position 139, a M at position 436, and / or an N at position 457, or any combination thereof; in NP, a T at position 343; in M1, a V or N at position 34 and / or an N at position 97; In BM2, residue R at position 58, residue G at position 80, residue R at position 27, residue R at position 26, or any combination thereof; in NS1, residue N; in PA, residue H at position 387, residue A at position 434, residue N at position 494, residue A at position 534, g at nucleotide position 2272, and t at nucleotide position 2213; in PB2, residue S at position 16; or any combination thereof, which have amino acids that enhance replication in Vero cells.

[0099] In one embodiment, for viruses related to the B / Yamagata lineage, the recombinant or reassortant influenza B virus has in HA1 a non-K at position 129, a non-N at position 168, a non-N at position 194, a non-T at position 196, a non-D at position 112, or any combination thereof; in NA a non-T at position 76, a non-R at position 102, a non-E at position 105, a non-P at position 139, a non-G at position 434, a non-T at position 436, a non-D at position 457, or any combination thereof; in NP a non-E at position 52, a non-S at position 57, a non-P at position 343, or any combination thereof; in M1 an amino acid other than G at position 34, a non-R at position 77, or an I at position 97, or a nucleotide other than c at position 500 in the NP vRNA, or any combination thereof; in BM2, an amino acid other than H at position 58, other than R at position 80, other than H at position 27, other than G at position 26, or any combination thereof; in NS1, an amino acid other than M at position 117, other than S at position 252, and / or other than D at position 494 in PA, and / or a nucleotide other than a at position 2272, other than g at nucleotide position 2213, other than a at position 1406, and / or other than c at position 1445, or any combination thereof in PA vRNA; in PB2, a residue other than N at position 16; or any combination thereof. In one embodiment, the recombinant influenza B virus has in HA1 an E at position 129 and a D at position 168; a P at position 196 and a D at position 194, in HA2 at position 112, or any combination thereof; in NA an M at position 76, a K at position 102, a K at position 105, an S at position 139, an E at position 434, an M at position 436, and / or an N at position 457, or any combination thereof; in NP a K at position 52, a G at position 57, and a T at position 343, or any combination thereof; in M1 a V or N at position 34, a K at position 77, and an N at position 97, or any combination thereof; in BM2 an R at position 58, a G at position 80, an R at position 27, and an R at position 26, or any combination thereof; In NS1, Y at position 117 and T at position 252, or any combination thereof; in PA, N at position 494, t at position 2272, and a at position 2213; in PB2, S at position 16; or any combination thereof.

[0100] In one embodiment, for influenza B viruses related to the B / Victoria lineage, the recombinant or reassortant influenza B virus has in HA1 a residue other than T at position 34, other than R at position 98, other than T at position 196, and / or in HA2 a residue other than K at position 39, other than S at position 56, other than K at position 61, or any combination thereof; in NA a residue other than N at position 169 and / or other than D at position 457; in NP a residue other than A at position 28, other than P at position 40, other than P at position 51, other than M at position 204, or in NP vRNA a nucleotide other than g at position 1795 or other than c at position 500, or any combination thereof; in M1 a residue other than D at position 54 and / or other than M at position 86; in BM2 a residue other than R at position 80; in NS1 a nucleotide other than Y at position 42, other than K at position 176, other than a at position 39; In PA, it has an amino acid other than Y at position 387, other than V at position 434, or other than T at position 524, or in PA vRNA, it has a nucleotide other than a at nucleotide position 2272, other than g at nucleotide position 2213, other than a at position 1406, or other than c at position 1445, or any combination thereof. In one embodiment, the recombinant influenza B virus has in HA1 an I at position 34, a P, I, A or N at position 196, and a K at position 98, and in HA2 a G at position 39, a G at position 56, and an N at position 61, or any combination thereof; in NA a T at position 169 and / or an N at position 457; in NP a T at position 28, an S at position 40, a Q at position 51, a T at position 204, and an a at position 1795, or any combination thereof; in M1 a G at position 54 and / or a T at position 86; in BM2 a G at position 80; in NS1 an N at position 42, a Q at position 176, a g at position 39, and an additional g after position 38; In PA, it has an amino acid having H at position 387, A at position 434, A at position 534, t at position 2272, a at position 2213, or any combination thereof.

[0101] In one embodiment, the recombinant or reassortant influenza B virus has one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the following: a residue other than Y at position 387, a residue other than V at position 434, a residue other than D at position 494, a residue other than T at position 524, a nucleotide other than a2272, g2213, a1406, c1445, or any combination thereof; in PA or PA vRNA, for example, 2272t, 2213a, 1406g, 1445t, 387H, 434A, 494N, 524A, or any combination thereof (e.g., residue H at position 387, residue A at position 434, residue N at position 494, residue A at position 524, or any combination thereof); in HA1, other than T at position 34, other than R at position 98, other than K at position 129, other than N at position 168, other than N at position 194, and / or other than T at position 196; in HA2, other than K at position 39, other than S at position 56, other than K at position 61, or other than D at position 112, e.g., in HA1, 34I, K129E, 168D, 196P / I / A / N, 98K, or 194D, and in HA2, 39G, 56G, 61N, or 112E, or any combination thereof; in NA, a residue other than T at position 76, a residue other than R at position 102, a residue other than E at position 105, a residue other than P at position 139, a residue other than N at position 169, a residue other than G at position 434, a residue other than T at position 436, and / or a residue other than D at position 457, e.g., 169T, 434E, 76M, 102K, 105K, 139S, 436M, 457N, or any combination thereof; in BM2, a residue other than H at position 58, a residue other than R at position 80, a residue other than H at position 27, and a residue other than G at position 26, e.g., residue R at position 58, residue G at position 80, residue R at position 27, and / or residue R at position 26; other than A at position 28; in NP, other than P at position 40, other than P at position 51, other than E at position 52, other than S at position 57, other than M at position 204, and / or other than P at position 343, and / or g at position 1795, or any combination thereof, such as residue T at position 28, residue S at position 40, residue Q at position 51, residue K at position 52, residue G at position 57, residue T at position 214, residue T at position 343, or any combination thereof;In M1, a residue other than G at position 34, other than D at position 54, other than R at position 77, other than M at position 86, other than I at position 97, or any combination thereof, e.g., a residue V or N at position 34, a residue G at position 54, a residue K at position 77, a residue T at position 86, and / or a residue N at position 97; in PB2, a residue other than N at position 16. In one embodiment, the recombinant virus has M1 34V / I97N, BM2 58R / 80G, NP 40S, NS1 86T;

[0102] In one embodiment, the influenza viruses of the invention are recombinant or reassortant influenza viruses with two or more selected amino acid residues at specific positions in one or more segments for PA, NP, M (M1 and BM2), and / or NS that can be used with the HA and NA genes of interest. In one embodiment, the recombinant reassortant influenza virus has two or more of the following in NP: A28T, P40S, P51Q, E52K, S57G, M204T, and / or P343T, and / or g1795a; in M1, G34V / N, D54G, R77K, M86T, I97N; in BM2, H58R, R80G, H27R, G26R; in NS1, M117Y, K176Q, S252T, a39g, g addition after position 38, Y42N; and in PA, a2272t, g2213g, Y387H, V434A, D494N, T524A.

[0103] In one embodiment, the influenza viruses of the invention are recombinant or reassortant influenza viruses with two or more selected amino acid residues at specific positions in one or more segments of PA, BM2, NP, M1, and / or NS, which can be used with the HA and NA genes of interest. For example, in one embodiment, the recombinant influenza B virus has M1 34V / I97N, BM2 58R / 80G, NP 40S, M1 M86T, or has NP P40S or NP E52K, or has substitutions in NP, M1, and optionally NS1 and BM2 such as clones 1-8 in Table 3, and substitutions in NP, M1, and optionally NS1 such as clones 1-8 in Table 4.

[0104] In one embodiment, the influenza virus of the present invention is a recombinant or reassortant influenza virus having two or more of a1406g, c1445t, a2272t in PA vRNA, P40S or P40S / M204T in NP, c500t, M77K or M86T in NP vRNA, and a39g or 38(+1)g in NS vRNA, or K176 Q in NS, for example, an influenza virus having PA a1406g / c1445t / a2272t, NP P40S, c500t, M R77K, and NS a39g K176Q, or an influenza virus having PA a1406g / c1445t / a2272, NP P40S / M204T, c500t, M M86T, and NS 38(+1)g.

[0105] The invention is illustrated by the following non-limiting examples. [Example]

[0106] Example 1 Vaccine yield is important from an economic standpoint. More importantly, the ability to produce large quantities of vaccine on a tight schedule could potentially save many lives during a viral outbreak. Mutations that increase the replication capacity of viruses in cell culture and / or embryonated eggs are useful for amplifying influenza viruses and establishing a potent influenza vaccine platform. Currently, most influenza vaccines are produced in embryonated chicken eggs. Influenza vaccines produced in MDCK cells are currently approved for human use in the United States and Europe, and Vero cell-derived influenza vaccines are approved for human use in Europe.

[0107] To develop a high-yield influenza B virus backbone for vaccine virus propagation in these specific host cells, random mutagenesis of the internal genes of B / Yamagata / 1 / 73 was performed; the HA and NA genes of the mutant virus library were derived from B / Yokohama / UT-K31 / 2012 (Yamagata lineage) or B / Yokohama / UT-K1A / 2011 (Victoria lineage), representing two major influenza B virus lineages. The virus library created contained random mutations in "internal" viral genes and genes encoding the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), as well as non-coding mutations. Mutations that confer improved growth and are vaccine virus candidates were further investigated. Notably, these vaccine virus candidates gave higher yields in growth systems commonly used for influenza vaccine virus production: embryonated chicken eggs, Madin-Darby canine kidney cells, and African green monkey (Vero) cells. These vaccine candidates can be used to improve the influenza B virus vaccine manufacturing process.

[0108] Materials and Methods cell MDCK cells were grown in MEM containing 5% (vol / vol) newborn bovine serum. Vero cells were maintained in MEM containing 10% (vol / vol) FBS. 293T human embryonic kidney cells were grown in DMEM supplemented with 10% (vol / vol) FBS.

[0109] Plasmid construction Eight viral RNA sequences from B / Yamagata / 1 / 73 virus were used to design gBlocks gene fragments (Integrated DNA Technologies), which were amplified by PCR and ligated; the resulting viral cDNA was inserted into the RNA polymerase I vector pHH21 (Neumann et al., 1999). vRNAs from B / Yokohama / UT-K31 / 2012, B / Yokohama / UT-K1A / 2011, B / Yokohama / P-2922 / 2005, B / Tokyo / UTE2 / 2008, B / Tochigi / UT-T1 / 2011, B / Massachusetts / 2 / 2012, and B / Brisbane / 60 / 2008 viruses were extracted from virus stocks using the RNeasy Kit (Qiagen). The viral HA and NA genes were amplified with gene-specific oligonucleotides using the One-Step RT-PCR Kit (Invitrogen), and the PCR products were cloned into the pHH21 vector. The HA and NA genes of B / Yamagata / 16 / 1988 were synthesized by PCR amplification of the combined gBlockes gene fragments and subsequent cloning into pHH21. The A / B chimeric HA and NA genes of B / Yokohama / UT-K31 / 2012, B / Yokohama / UT-K1A / 2011, B / Massachusetts / 2 / 2012, and B / Brisbane / 60 / 2008 viruses were generated by overlapping PCR.

[0110] Plasmid library construction Random mutations (one to four) were introduced into each of six internal genes of the B / Yamagata / 1 / 73 virus by error-prone PCR using the GeneMorph II Random Mutagenesis Kit. The randomly mutated PCR products were inserted into the pHH21 vector, and the diversity of the resulting plasmid library was confirmed by sequence analysis of at least 24 E. coli colonies for each viral gene.

[0111] Virus rescue and virus library construction Wild-type viruses and viral libraries with random mutations in the internal genes were generated using a reverse genetics approach (Neumann et al., 1999). A viral library was generated by transfecting 293T cells with the mutant plasmid library instead of the wild-type construct. After 48 hours, the supernatant from the transfected 293T cells was collected and amplified in MDCK cells to generate viral stocks; the titer of the viral stock was determined using a plaque assay in MDCK cells.

[0112] Assessment of viral growth kinetics Wild-type or recombinant viruses were inoculated into MDCK cells in triplicate at a multiplicity of infection (MOI) of .0001. After infection, cells were incubated with MEM / BSA medium containing 0.6 μg / mL of TPCK-trypsin. Supernatants were collected at the indicated time points, and virus titers were assessed by plaque assay in MDCK cells.

[0113] To analyze virus replication in embryonated chicken eggs, 1 × 10 cells were placed in 10-day-old embryonated chicken eggs (4 per virus). 4 pfu of virus was inoculated and incubated at 35° C. Allantoic fluid was collected at the indicated time points and virus titers were determined by using a plaque assay on MDCK cells.

[0114] The hemagglutination titers of viruses amplified in MDCK cells or embryonated chicken eggs were determined using a hemagglutination assay. Briefly, 50 μL of virus sample was serially diluted two-fold in a 96-well U-bottom microtiter plate (Thermo Scientific) containing 50 μL of PBS per well. Next, 50 μL of 0.5% turkey red blood cells was added to each well, the plate was incubated at room temperature for 45 minutes, and the hemagglutination titer was calculated as the reciprocal of the highest dilution at which agglutination occurred.

[0115] Virus concentration and purification MDCK cells were amplified in 4-Layers Easy-Fill Cell Factories (Thermo Scientific) and infected with wild-type or high-yield influenza B virus at an MOI of 0.001 when the cells reached approximately 95% confluency. Cell culture supernatants were harvested 48 hours later and clarified by centrifugation (3,500 rpm, 15 min, 4°C in a Beckman SX4750 rotor). Virus was pelleted by ultracentrifugation (18,500 rpm, 90 min, 4°C in a Beckman Type 19 rotor), resuspended in 5 mL of PBS, and loaded onto a 20–50% (wt / vol) continuous sucrose gradient, which was centrifuged at 25,000 rpm for 90 min at 4°C in a Beckman SW32 rotor. The virus-containing band was collected, diluted with PBS, and pelleted again by centrifugation (25,000 rpm, 90 min, 4°C in a Beckman SW32 rotor). The virus pellet was resuspended in 400 μL of PBS, aliquoted, and stored at -80°C.

[0116] Total protein assay Total protein yield of the virus concentrate was determined using the Pierce BCA protein assay kit (Thermo Scientific) according to the manufacturer's instructions.

[0117] Glycosylation of viral proteins using PNGase F Ten microliters of virus concentrate was denatured to remove sugar moieties, and then the sample was incubated with 2 μL of 1 / 10 diluted PNGase F enzyme solution (in the buffer provided by the manufacturer) (New England Biolabs) at 37°C for 20 hours with a final concentration of 1% Nonidet P-40.

[0118] SDS / PAGE Two microliters of virus concentrate was mixed with PBS to a total volume of 10 μL, and 2.5 μL of loading dye containing 2% (vol / vol) β-mercaptoethanol (as a reducing agent) was added and heated at 95°C for 5 minutes. The samples were then loaded onto NuPage 4-12% (wt / vol) Bris-Tris precast gels (Life Technologies), which were run at 150 V for 120 minutes using 1x Mes buffer (Bio-Rad) and then stained with SYPRO-Ruby (Sigma). Protein quantification was performed using ImageJ software (NIH). HA content was calculated by dividing the HA amount (calculated by summing the amounts of HA1 and HA2) by the sum of the amounts of HA1, HA2, NP, and M1, and multiplying this value by the total viral protein amount.

[0119] Toxicity studies in mice Six-week-old female BALB / c mice (Jackson Laboratory) were anesthetized with isoflurane and then incubated for 10 min. 6 Influenza B virus was inoculated intranasally in a volume of 50 μL at pfu. Five mice were infected per group; this sample size was adequate to detect large effects between groups. Mice were randomized and the investigators were not blinded. Weight change and survival were monitored daily for 14 days. To assess viral replication in mice, 10 mice were infected with 10 virus per virus. 6 pfu; 5 mice from each group were sacrificed on days 3 and 6 post-infection and virus titers in the lungs were determined using a plaque assay on MDCK cells.

[0120] Genetic Stability Test To evaluate the genetic stability of the high-yield vaccine backbone, viruses carrying HY(Yam) and HY(Vic) combined with the HA and NA vRNAs of B / Yokohama / UT-K31 / 2012 and B / Yokohama / UT-K1A / 2011, respectively, were passaged 10 times in MDCK cells at an MOI of 0.01. Viruses recovered after each passage were sequenced by Sanger sequencing.

[0121] Minireplicon assay For the minireplicon assay, 293T and MDCK cells were transfected with 0.05 μg of either pPolI-B / Yamagata / 1 / 73-NS-Luc (encoding the firefly luciferase gene under the control of the human RNA polymerase I promoter) or pPolIC250-B / Yamagata / 1 / 73-NS-Luc (encoding the firefly luciferase gene under the control of the canine RNA polymerase I promoter) along with 0.25 μg each of plasmids expressing the B / Yamagata / 1 / 73 PB2, PB1, PA, and NP proteins. Cells were cotransfected with 0.025 μg of pGL4.74(hRluc / TK) (an internal control for monitoring transfection efficiency; Promega). The transfected cells were incubated at 35°C for 48 hours, lysed, and assayed for luciferase activity using the Dual Luciferase System Detection Kit (Promega) according to the manufacturer's protocol. Firefly luciferase expression levels were normalized to Renilla luciferase activity. Data shown are means ± SD of three independent experiments.

[0122] To examine the significance of mutations in the noncoding regions of B / Yamagata / 1 / 73 PA and NS1 vRNAs, cells were transfected as described above; however, pPolIC250-NP(0)Fluc(0) was replaced with a reporter construct in which the firefly luciferase gene was flanked by the wild-type or mutant noncoding regions of PA or NS vRNA, respectively. Forty-eight hours after transfection, luciferase activity was measured as described above.

[0123] VLP budding assay For VLP budding assays, 293T cells were transfected with 2 μg of each protein expression plasmid for wild-type or mutant B / Yamagata / 1 / 73 M1, HA, NA, NP, BM2, and NS2. Forty-eight hours after transfection, culture supernatants were collected, clarified, loaded onto a 20% (wt / vol) sucrose cushion, and ultracentrifuged at 60,000 rpm in a Beckman SW 60 Ti rotor for 2 hours; pelleted VLPs were resuspended in PBS overnight at 4°C. In parallel, transfected cells were lysed with RIPA buffer.

[0124] Purified VLPs and cell lysates were mixed separately with 5x loading dye buffer and fractionated on NuPage 4-12% (wt / vol) Bris-Tris precast gels (Life Technologies). Proteins were transferred to nitrocellulose membranes using the iBlot dry blotting system (Invitrogen). The membranes were then blocked for 3 hours at room temperature with PBS containing 5% (wt / vol) skim milk and 0.05% Tween 20 (PBS-T). They were then incubated overnight at 4°C with monoclonal antibodies against B / Brisbane / 60 / 2008 HA (1:1,000; BioSource, MBS430175) or influenza B virus NP (1:1,000; Abcam, ab47876) or M1 (1:2,000; Abcam, ab82608) proteins. After washing four times with PBS-T for 10 minutes each, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:2,000; Life Technology) at room temperature for 1 hour. After washing four times with PBS-T for 10 minutes each, the blots were developed using lumi-light Western blotting substrate (Roche Applied Science) and then visualized by autoradiography. Protein amounts were quantified using ImageJ software (NIH). The following formula was used to calculate the percentage of HA, NP, and M1 protein incorporation into VLPs: (ratio of the amount of protein in mutant VLPs to the total amount of mutant protein (VLPs + cell lysate) / ratio of the amount of protein in wild-type VLPs to the total amount of wild-type protein (VLPs + cell lysate)) × 100.

[0125] IFN antagonist assay To evaluate the IFN-antagonist activity of wild-type and mutant NS1 proteins, 293T cells were transfected with an NS1 protein expression plasmid and the reporter plasmid pGL-IFN-β (Bale et al., 2012), which encodes firefly luciferase under the control of the IFN-β promoter. Twenty-four hours after transfection, the cells were infected with Sendai virus at an MOI of 5 for 1 hour. The cells were incubated for 24 hours and lysed with Glo lysis buffer (Promega); Steady-Glo assay buffer (Promega) was then added, and luciferase expression was measured. In another set of experiments, 293T cells were transfected with wild-type or mutant NS1 protein expression plasmids and the reporter plasmid pISRE-Luc (Promega), which encodes firefly luciferase under the control of an IFN-regulated promoter. After 24 hours, the cells were treated with 104 U / mL human IFN-β for an additional 24 hours, followed by lysis and measurement of luciferase expression levels.

[0126] statistical analysis Statistical analysis of the data was performed using R software (www.r-project.org), v3.1. To compare multiple groups with measurements collected independently at several time points, a two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was used. To compare measurements from multiple groups collected at a single time point, a one-way analysis of variance (ANOVA) followed by Tukey's or Dunnett's post-hoc test was used. To compare multiple groups with dependent measurements (e.g., viral growth curves in cell culture where aliquots were collected from the same culture at different time points), the R package NLME was used to apply a linear mixed-effects model to the data, taking into account time, virus strain, and the interaction between these two factors. The R package PHIA was used to construct a contrast matrix for comparing strains in a pairwise manner at the same time point (e.g., group_1 vs. group_2 at 24 h post-infection, group_1 vs. group_3 at 24 h post-infection, group_2 vs. group_3 at 24 h post-infection). Comparisons were performed individually; therefore, final P values ​​were adjusted by accounting for multiple comparisons using the method of Holm.

[0127] Raw data from growth curves were transformed to a logarithmic scale before analysis; results were considered statistically significant at P (or adjusted P value) < 0.05. Differences between groups were assessed using the Levene test.

[0128] Ethics and Biosafety Experiments on mice were performed in accordance with the University of Wisconsin-Madison Animal Care and Use Protocol, as set forth in the "Basic Guidelines for Good Conduct of Animal Experiments" and related activities in the Animal Welfare Act and related animal welfare regulations and Public Health Service policies. All experiments were approved by the University of Wisconsin-Madison Animal Care and Use Committee, which recognizes and approves both the legal and ethical responsibilities of animals (Protocol No. V00806).

[0129] result Viral library screen for high-yield mutants in MDCK cells Mutations associated with high yields of influenza B virus were identified using a mutagenesis and screening approach similar to the strategy used to develop the high-yield influenza A virus PR8 vaccine backbone. The six internal vRNA fragments were derived from B / Yamagata / 1 / 73, which grows efficiently in MDCK cells and was isolated before the Victoria and Yamagata lineages were separated. Using an error-prone PCR-based mutagenesis method, cDNA libraries carrying one to four random amino acid changes in the viral proteins were generated (Figure 2). These cDNA libraries were then used to generate viral libraries. Six separate libraries representing each of the internal vRNAs (i.e., PB2, PB1, PA, NP, M, and NS) were created (Figure 2): three libraries for the combination of polymerase vRNA (PB2 + PB1, PB2 + PA, PB2 + PB1 + PA); one library for polymerase and nucleoprotein (NP) vRNA (i.e., PB2 + PB1 + PA + NP), because the PB2, PB1, PA, and NP proteins form the viral replication complex; one library for PB2 and NS vRNA (PB2 + NS), because the PB2 and NS1 proteins (encoded by NS vRNA) of influenza A virus are important determinants of host virulence (Wright et al., 2013); and one library for M and NS vRNA, because the M1 protein (encoded by M vRNA) of influenza A virus is associated with high growth properties. Each of these 12 viral libraries was constructed using HA and NA vRNAs from representative viruses of the Victoria (B / Yokohama / UT-K1A / 2011) or Yamagata (B / Yokohama / UTK31 / 2012) lineages, resulting in a total of 24 viral libraries. Libraries constructed using HA and NA vRNAs from the Victoria or Yamagata lineages are referred to as the "Victoria-lineage" or "Yamagata-lineage" libraries, respectively.

[0130] To select for mutants with enhanced growth properties, each library was passaged 12 times in MDCK cells. In parallel, after two passages in MDCK cells, the viral libraries were combined and then passaged 10 times in MDCK cells (Figure 2). More than 700 viral plaques were randomly selected from each of the Victoria and Yamagata lineage libraries, yielding a total of 1,472 plaque-purified viruses (Figure 2). The plaque-purified viruses were then amplified in MDCK cells, and their yields were assessed by hemagglutination assay (as a surrogate for high HA yield) and compared with those of the parental Victoria and Yamagata lineage viruses (containing the HA and NA vRNAs of the Victoria or Yamagata lineage combined with the six remaining vRNAs of the B / Yamagata / 1 / 73 virus). Twenty-nine Yamagata lineage and 28 Victoria lineage viruses were identified with hemagglutination titers at least twice those of their respective control viruses. These candidate viruses were reamplified in MDCK cells, and their high-yield properties were confirmed by assessing hemagglutination titers and replication kinetics in MDCK cells (used as another surrogate for high HA yield) (Figure 3).

[0131] Next, we sequenced the entire viral genomes of the top eight candidates from each lineage. We found distinct sets of mutations (evaluating amino acid changes and untranslated region changes) for the high-yielding candidates from the Yamagata and Victoria lineages (Tables 1 and 2). Seven of the eight high-yielding candidates isolated from the Yamagata lineage library harbored G34V and I97N mutations in the M1 matrix protein and H58R and R80G mutations in the BM2 ion channel protein (also encoded by the M gene) (Table 1), suggesting that these amino acid substitutions confer efficient replication in MDCK cells.

[0132] All eight high-yielding candidates obtained from the Victoria lineage library encoded a P40S mutation in NP and an M86T mutation in M1 (Table 2). In addition, six of these eight high-yielding candidates encoded nucleotide changes in the noncoding region of the NS vRNA: a nucleotide addition after position 38 was detected in five viruses (NS-38(+1)g (all nucleotide changes in noncoding regions are italicized), and a39g nucleotide substitution was detected in one virus (Table 2). The g1795a mutation was identified in the noncoding region of the NP fragment in three high-yielding candidates (Table 2).

[0133] Although HA and NA vRNAs were not targeted by PCR-mediated random mutagenesis, several mutations were detected in the HA and NA proteins (Tables 1 and 2). Specifically, in seven of the eight high-yield candidates from the Victoria lineage library, the threonine at position 196 of HA was substituted with various other amino acids (e.g., alanine, isoleucine, proline, or asparagine) (Table 2), suggesting strong selective pressure at this position.

[0134] Potential combinatorial effects of mutations Next, we used a reverse genetics approach to generate viruses carrying various combinations of mutations found in the top eight high-yielding Yamagata and Victoria lineage candidates (Tables 3 and 4). For example, the NP-E52K and M1-R77K mutations found in high-yielding candidate #21 (which replicated to the highest titer in MDCK cells) (Figure 3) were combined with the NS1-M117Y / S252T mutations found in high-yielding candidates #23 and #26 (Figure 3). Because the internal vRNAs of the Yamagata and Victoria lineage libraries are derived from the same virus, mutations found in the high-yielding Yamagata and Victoria lineage candidates were also combined (Tables 3 and 4). The resulting viruses were tested for hemagglutination and viral titers in MDCK cells (Figure 4). The high-yield candidates, the Yamagata and Victoria lineages, all replicated more efficiently in MDCK cells and had higher hemagglutination titers than wild-type virus at one or more time points, and most of these differences were statistically significant (Figure 4).

[0135] Yamagata lineage RG (Yam) #8 (encoding NP-P40S, M1-R77K, NS1-K176Q, and NS-a39g mutations) and Victoria lineage RG (Vic) #2 (encoding NP-P40S / M204T, M1-M86T, and NS-38 (+1)g mutations) were selected as lead candidate vaccine backbones because they had the highest titers in each group.

[0136] Viral library screen for high-yield mutants in Vero cells An ideal vaccine virus backbone should give high yields in the three growth systems currently used for commercial production of human influenza vaccines: MDCK cells, Vero cells, and embryonated chicken eggs. In parallel with the development of a high-yield influenza B virus vaccine backbone in MDCK cells, all 24 virus libraries (Figure 2) were passaged in Vero cells. Because the titer of the influenza B virus library was low in Vero cells, the library was first passaged five times in a coculture of MDCK and Vero cells, followed by five passages in Vero cells. In parallel, after two passages in cocultured MDCK and Vero cells, the virus libraries were combined and passaged three more times in the coculture cells, followed by five passages in Vero cells. A total of 382 individual virus plaques were randomly picked from the various virus libraries and amplified in Vero cells. Based on the results of the hemagglutination assay, the top six candidates from the Yamagata and Victoria lineages were selected and their complete genome sequences were determined (Tables 5 and 6).

[0137] High-yield candidates harbored some of the mutations identified after passaging in MDCK cells, which may have been selected in the MDCK cells during coculture. These mutations included amino acid changes at positions M1-34 / 97, BM2-58 or -80, and HA1-196 (compare Tables 1 and 2 with Tables 5 and 6). Mutations at positions M1-34 / 97 and BM2-58 were detected only in the Yamagata lineage library, whereas mutations at position BM2-80 occurred in both Yamagata and Victoria lineage viruses. The mutation at position HA1-196 was predominant in Victoria lineage viruses but also occurred in one Yamagata lineage virus. In addition, mutations not previously observed after passaging in MDCK cells were observed in high-yield candidates of both viral lineages, most notably the a2272t nucleotide substitution in the noncoding region of PA (Tables 5 and 6).

[0138] Selection of high-yielding Yamagata-lineage and Victoria-lineage vaccine virus candidates Next, we tested whether the PA-a2272t mutation, found after subculture in Vero cells, enhanced the growth characteristics of RG(Yam)#8 and / or RG(Vic)#2. The resulting viruses (HY(Yam) and HY(Vic)) exhibited higher hemagglutination and viral titers compared with RG(Yam)#8 and RG(Vic)#2, respectively, and compared with the parental Yamagata lineage and Victoria lineage viruses; some of these differences were small (but statistically significant). Therefore, HY(Yam) (encoding NP-P40S, M1-R77K, NS1-K176Q, NS-a39g, and PA-a2272t) and HY(Vic) (encoding NP-P40S / M204T, M1-M86T, NS-(38+1)g, and PA-a2272t) were selected as high-yield candidates.

[0139] Evaluation of high-yield vaccine virus backbones containing different influenza B virus HA and NA genes HY(Yam) and HY(Vic) were developed using the HA and NA genes of B / Yamagata / UT-K31 / 2012 and B / Yokohama / UT-K1A / 2011, respectively. Because the high-yield vaccine virus backbones HY(Yam) and HY(Vic) vaccine viruses have a general growth-enhancing effect, we tested the HY(Yam) and HY(Vic) vaccine virus backbones carrying the HA and NA genes of six influenza B viruses isolated over several decades, including those in WHO-recommended vaccines (Figure 7). At one or more time points tested, the HY(Yam) and HY(Vic) vaccine virus backbones yielded higher virus or hemagglutination titers compared to the parental viruses, although not all differences were statistically significant. For the viruses tested, HY(Vic) demonstrated a greater growth-enhancing effect than HY(Yam).

[0140] HY(Yam) and HY(Vic) backbone exchange Next, we tested whether the HY(Yam) and HY(Vic) backbones support efficient replication of viruses carrying HA and NA genes from other influenza B virus lineages (Fig. 8). High virus titers and hemagglutination titers were detected for HY(Yam) viruses encoding HA and NA genes from the Victoria lineage and for HY(Vic) viruses encoding HA and NA genes from the Yamagata lineage. Overall, the HY(Yam) vaccine backbone yielded slightly higher virus titers or hemagglutination titers than the HY(Vic) vaccine backbone at some time points, although most of these differences were not statistically significant. These data indicate that the HY(Yam) and HY(Vic) vaccine backbones confer efficient replication to viruses of both lineages.

[0141] Comparison of influenza A and B virus vaccine backbones Chimeric influenza A viruses carrying the HA and NA genes of influenza B viruses have been previously constructed (Horimoto et al., 2004 and Flandorfer et al., 2003). The use of influenza A and B viruses may simplify the vaccine production process. Reassortants between influenza A and B viruses likely do not occur naturally due to the type-specific viral packaging signals located in the 5' and 3' terminal regions of influenza vRNA segments (Fujii et al., 2003; Baker et al., 2014). Therefore, we constructed a vRNA segment in which the extracellular domains of the influenza A PR8 virus HA and NA proteins were replaced with the corresponding portions of influenza B virus (Figure 5). We then compared the hemagglutination and viral titers of the following three viruses: wild-type Yamagata lineage influenza B virus; HY (Yam) virus, which contains the HA and NA genes of Yamagata lineage virus; and high-yield PR8 virus, which contains chimeric A / B HA and NA genes of Yamagata lineage virus (Figure 9A-B). Similar experiments were performed with Victoria lineage viruses (Figure 9C-D). At most time points tested, the high-yield influenza A or B vaccine virus backbone significantly increased hemagglutination and viral titers compared to the wild-type virus. Comparison of the influenza A and B vaccine backbones showed higher viral titers with the influenza B vaccine backbone, but interestingly, the influenza A vaccine backbone also showed higher hemagglutination titers. Additionally, for the two influenza B vaccines representing both lineages, the influenza A vaccine backbone was superior to the influenza B vaccine backbone in terms of hemagglutination titers and viral titers in embryonated chicken eggs (Figure 4E-F).Other influenza B viruses containing HA and NA from B / Yokohama / UT-K1A / 2011, B / Yokohama / P-2922 / 2005, B / Tokyo / UTE2 / 2008, or B / Tochigi / UT-T1 / 2014 did not grow well in embryonated chicken eggs, despite their backbone (wild-type or high-yield), suggesting that the HA and NA genes of these human viruses may limit their efficient growth in embryonated chicken eggs.

[0142] Assessment of total viral protein yield and HA content Most preparations of inactivated influenza vaccines contain 15 μg each of H1 HA, H3 HA, and type B HA proteins. Therefore, for vaccine optimization, total viral protein yield and HA content are important parameters, prompting us to compare the total protein yield and HA content of the distinct HY(Yam) and HY(Vic) viruses with their respective wild-type viruses in MDCK cells. Cell culture supernatants were collected from infected cells, and the viruses were concentrated and purified using sucrose gradient centrifugation. Total viral protein yield was then determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). In parallel, purified virus samples were treated with PNGase F, resulting in HA deglycosylation, allowing for more easily detected HA2 (which, in its glycosylated form, is similar in size to M1). Samples were separated using SDS / PAGE (Figure 10A-B), and the amounts of HA1, HA2, NP, and M1 were determined based on densitometric analysis. HA content was calculated by dividing the amount of HA (calculated by adding up the amounts of HA1 and HA2) by the sum of the amounts of HA1, HA2, NP, and M1, and multiplying this value by the amount of total viral protein in the sample analyzed by gel electrophoresis (Figure 10A-B). For all viruses tested, total viral protein yield and HA content were significantly higher in the HY(Yam) and HY(Vic) vaccine backbones compared to the wild-type virus from which the HA and NA vRNAs were derived.

[0143] Virulence of PR8-HY-based vaccine viruses in mice The experimental approach was designed to select for mutations with increased replication capacity, which may increase their virulence in mammals. To address this question, five mice per group were inoculated with 100 ribosomal RNAs containing wild-type Yamagata lineage virus, a virus carrying the HA and NA vRNAs of Yamagata lineage virus in combination with the remaining six genes of wild-type B / Yamagata / 1 / 73 virus (used to generate the viral library), and a virus carrying the HA and NA vRNAs of Yamagata lineage virus in combination with the HY (Yam) vaccine virus backbone. 6 pfu (Fig. 11A-B). In parallel, groups of 10 mice were infected with 10 6 Animals were infected with pfu of the above-mentioned viruses and five animals were sacrificed on days 3 and 6 postinfection to assess lung virus titers (Figure 11C). Similar experiments were performed using Victoria lineage viruses and the HY (Vic) vaccine backbone (Figure 11D-F). The wild-type B / Yamagata / 1 / 73 virus backbone exhibited higher virulence than the B / Massachusetts / 2 / 2012 and B / Brisbane / 60 / 2008 backbones, respectively. However, the yield-enhancing mutations in HY (Yam) and HY (Vic) did not further increase virulence in mice; in fact, they showed a slightly reduced effect compared to the B / Yamagata / 1 / 73 backbone.

[0144] Genetic stability of the HY(Yam) and HY(Vic) vaccine backbones. Vaccine viruses must be genetically stable to maintain their desired properties. To test the genetic stability of high-yield candidates, 10 serial passages of HY(Yam) virus were performed in MDCK cells using HY(Vic) virus with HA and NA vRNAs from B / Yokohama / UTK31 / 2012 and B / Yokohama / UT-K1A / 2011. After each passage, the genome sequence of the virus was determined by Sanger sequencing. For Yamagata lineage viruses, no mutations were detected. For Victoria lineage viruses, no mutations in the internal genes that define the high-yield properties of HY(Vic) were detected. However, after five passages, a mixed population encoding HA1-196T and -196I was detected; after 10 passages, only the HA1-196I mutant was detected.

[0145] Similarly, several wild-type and high-yield influenza B viruses of both lineages were passaged in embryonated chicken eggs (Table 7). After 5 to 10 serial passages, no egg-adapted mutations were detected in internal genes. However, for viruses that retain glycosylation at amino acids 196-196 of the HA, a mutation occurred that abolished that glycosylation site. This result is consistent with the earlier discovery of mutations at glycosylation sites in viruses of the Victoria lineage (Table 2). Collectively, the data indicate that the yield-enhancing mutations in HY(Yam) and HY(Vic) were genetically stable for at least 5 to 10 serial passages in MDCK cells and embryonated chicken eggs.

[0146] Contribution of individual vRNAs to the high-yield properties of HY(Yam) and HY(Vic) The HY(Yam) and HY(Vic) vaccine backbones contain mutations in several vRNAs. To better understand the contribution of these mutations to the HY(Yam) and HY(Vic) phenotypes, we used reverse genetics to generate viruses carrying individual mutant vRNA segments of HY(Yam) or HY(Vic): for example, wild-type B / Yamagata / 1 / 73 (used to generate the virus library), wild-type B / Yamagata / 1 / 73 (which also encodes the NP-P40S found in HY(Yam)), or the six remaining vRNAs of HY(Yam) combined with the HA and NA vRNAs of Yamagata-lineage viruses (Figure 12). The resulting viruses were tested for their replication ability and hemagglutination titer in MDCK cells (Figure 12). When tested individually, the NP-P40S, M1-R77K, and NS-a39g + NS1-K176Q mutations significantly increased viral titers and hemagglutination titers of Yamagata lineage viruses at one or more time points compared with the reference virus. For Victoria lineage viruses, each of the tested mutations had a statistically significant growth-enhancing effect at one or more time points. Most viruses carrying individual mutations found in HY(Yam) or HY(Vic) did not replicate as efficiently as high-yield vaccine candidates, indicating that several mutation combinations are important for the high-yield properties of HY(Yam) and HY(Vic).

[0147] Effects of individual mutations in HY(Yam) and HY(Vic) on the activity of the viral replication complex. The NP-P40S mutation was selected individually in combination with NP-M204T from the Yamagata lineage and Victoria lineage libraries, respectively. Additionally, these mutations increased virus titers and hemagglutination titers when tested without other growth-enhancing mutations (Figure 12). To assess whether these mutations affected the activity of the viral replication complex, 293T and MDCK cells were transfected with plasmids expressing the three polymerase subunits of B / Yamagata / 1 / 73 virus, wild-type or mutant B / Yamagata / 1 / 73 NP, and influenza B virus-like RNA expressing the luciferase reporter protein (the NP-M204T mutation was not tested separately, as it was selected from the viral library only in combination with NP-P40S). Interestingly, the NP-P40S and -P40S / M204T mutations significantly reduced the replication activity of the viral replication complex (Figure 13A-B).

[0148] The PA-a2272t mutation, which arose during passaging of the viral library in Vero cells (Tables 5 and 6), significantly increased the viral titer and hemagglutination titer of the Victoria lineage vaccine candidate (Figure 6). This mutation was found to significantly increase viral-like RNA replication in a minireplicon assay (Figure 10C). HY(Yam) and HY(Vic) carry the NS-a39g and NS-38(+1)g mutations, respectively. These mutations were introduced into luciferase-expressing viral-like RNA. Both mutations resulted in a significant increase in the expression of the reporter protein from the viral-like RNA (Figure 13D); this effect was substantially greater for the NS-38(+1)g mutation compared to the NS-a39g mutation.

[0149] The effect of individual mutations in HY(Yam) and HY(Vic) on the composition of virus-like particles. When tested individually, the M1-R77K and M1-M86T mutations in HY(Yam) and HY(Vic) affected virus titers and hemagglutination titers (Figure 12). M1 is a major component of the virion, and mutations in this protein may affect virion composition. 293T cells were transfected with plasmids for expression of B / Yamagata / 1 / 73 HA, NA, NP, BM2, NS2, and wild-type or mutant M1 proteins; expression of this set of viral proteins results in efficient virus-like particle (VLP) formation and release (Gomez-Puertas et al., 1999). Cell culture supernatants were harvested, and the efficiency of VLP incorporation of viral proteins was assessed (Figure 14). Both mutations in M1 significantly increased the amounts of viral HA, NP, and M1 proteins in the culture supernatant, which likely increased the virus titer and hemagglutination titer conferred by the M1-R77K and -86T mutations.

[0150] Effect of mutations in the HY(Yam)NS1 protein on IFN antagonist activity The NS1 protein is the major influenza virus IFN antagonist (Yuan et al., 2001; Dauber et al., 2004). To evaluate the effect of the HY(Yam)NS1-K176Q mutation on the ability of NS1 to block IFN-β synthesis, 293T cells were transfected with wild-type or mutant NS1 protein expression plasmids and the reporter plasmid pGL-IFN-β (Bale et al., 2012), which encodes firefly luciferase under the control of the IFN-β promoter. The cells were then infected with Sendai virus to stimulate IFN-β synthesis and increase reporter gene expression. Wild-type and mutant NS1 had comparable abilities to downregulate IFN-β (Figure 12A). To determine the ability of wild-type and mutant NS1 to interfere with IFN-β-stimulated gene expression, 293T cells were transfected with wild-type and mutant NS1 protein expression plasmids and the reporter plasmid pISRE-Luc (Promega), encoding firefly luciferase under the control of an IFN-regulated promoter. Cells were stimulated with IFN-β for 24 hours, incubated again for 24 hours, and then assayed for luciferase expression. The NS1-K176Q protein was slightly less potent than wild-type NS1 in suppressing gene synthesis from the IFN-regulated promoter (Figure 12B). This suggests that other mechanisms account for its growth-enhancing effect.

[0151] Consideration To date, no systematic efforts have been made to develop a high-yield influenza B vaccine backbone. Vodeiko et al. (2003) compared two influenza B viruses with different replication capacities in embryonated chicken eggs. Reassortant experiments revealed several vRNAs that contribute to phenotypic differences (Vodeiko et al., 2003); however, specific amino acids determining growth characteristics were not identified. Kim et al. (2015) found that cold adaptation of influenza B viruses enhanced their growth characteristics. Several amino acid changes in the HA, NA, and NP (not the mutations reported here) were responsible for increased virus titers (Kim et al., 2015). Le et al. (2015) tested reassortant sequences between B / Lee / 40 and influenza B viruses of the Yamagata and Victoria lineages, isolated between 2002 and 2007; all 14 high-yield candidates possessed the NP vRNA of the B / Lee / 40 virus, suggesting that this vRNA confers efficient replication capability. Ping et al. (2015) presented a more comprehensive strategy for developing high-yield influenza A virus backbones. This was applied to influenza B virus: from a virus library containing random mutations in internal genes, candidates with improved influenza B virus replication in MDCK and Vero cells were selected. Combinations of mutations yielded high-yield Yamagata-lineage and Victoria-lineage vaccine candidates in MDCK and Vero cells, as well as in embryonated chicken eggs (mutations encoding NP-P40S, M1-R77K, NS1-K176Q, NS-a39g, and PA-a2272t for the Yamagata-lineage vaccine, and mutations encoding NP-P40S / M204T, M1-M86T, NS-(38+1)g, and PA-a2272t for the Victoria-lineage vaccine).Further studies in (semi)industrial settings will be required to determine whether lineage-specific vaccine backbones offer advantages over a single backbone used for viruses of both lineages.

[0152] The total viral protein yields and HA contents obtained from HY(Yam) and HY(Vic) were substantially higher than those derived from wild-type virus (Figure 10). High viral and HA yields are important for cost-effective vaccine production. More importantly, increased vaccine viral yields are essential given the high year-round vaccine demand and shortened virus production vaccine manufacturing times. However, some of the observed differences in viral titer or HA yield were small (although statistically significant), and the extent to which HY(Yam) and HY(Vic) increase vaccine virus yield in industrial vaccine production is currently unknown.

[0153] Evaluation of influenza B virus sequences revealed that amino acid changes in HY(Yam) and HY(Vic) are rare among natural influenza B viruses (NP-P40S, NP-M204T, and M1-M86T mutations have each been found in one isolate; the M1-R77K mutation has been reported in 14 isolates; and the NS1-K176Q mutation has not been detected). Although the 3D structure of the influenza B virus NP protein has been solved (Ng et al., 2012), position 40 (a P to S mutation selected from Yamagata and Victoria lineage libraries) is the N-terminal 71 amino acid sequence for which no structural data are available, suggesting that this region is highly flexible. Sequence analysis of influenza B virus NP proteins revealed that the proline at position 40 is highly conserved: only one of 3,234 sequences does not encode NP-40P (the only exception, B / Tennessee / 01 / 2015, encodes NP-40S, as seen in this study). Interestingly, the NP P40S mutation reduced the activity of the viral replication complex in minireplicon assays, suggesting that the yield-enhancing effect of this mutation is mediated by functions other than replication and transcription. For example, this region of NP may interact with viral or cellular proteins during transport of the viral ribonucleoprotein complex from the nucleus to the cytoplasm or during virion assembly.

[0154] Several mutations were also identified in the noncoding regions of the vRNA segment that increased virus yield. Some of these mutations resulted in increased replication and transcription levels, as measured by minireplicon assays. These mutations may, for example, increase vRNA stability or affect its interaction with the viral polymerase complex. Further studies are needed to elucidate the precise mechanistic functions of these mutations.

[0155] In summary, an influenza B virus backbone has been developed that can increase the titer of seasonal influenza B vaccines in growth systems currently used for human influenza vaccine virus production.

[0156] Table 1: Amino acid changes in selected high-yielding clones of the Yamagata lineage. [Table 1]

[0157] Table 2: Amino acid changes in selected high-yielding clones of the Victoria lineage. [Table 2] Mutations in lower case and italics in this and the following tables indicate nucleotide changes in non-coding regions.

[0158] Table 3: Amino acid changes in Yamagata lineage HY vaccine virus candidates generated using reverse genetics. [Table 3]

[0159] Table 4: Amino acid changes in Victoria lineage HY vaccine virus candidates generated using reverse genetics. [Table 4] The viral library was passaged a total of 12 times in MDCK cells (e.g., the libraries were mixed after two passages and passaged an additional 10 times (Figure 2)). After passage in MDCK cells, a plaque assay was performed and over 1,400 individual plaques were collected. High-yield candidates are listed in Tables 1-4.

[0160] Tables 5 and 6 show changes that enhance the proliferation of Vero cells using a similar protocol. Table 5: Amino acid changes in Vero cells adapted to HY Yamagata lineage virus. [Table 5]

[0161] Table 6: Amino acid changes in Vero cells adapted to HY Victoria lineage virus. [Table 6]

[0162] Table 7. Amino acid changes detected after successive virus passages in embryonated chicken eggs. [Table 7-1] [Table 7-2] [Table 7-3]

[0163] All publications, patents, and patent applications are incorporated herein by reference. In the foregoing specification, the invention has been described with reference to particularly preferred embodiments thereof, but many details have been set forth for purposes of illustration, and it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that several of the details set forth herein may be significantly modified without departing from the basic principles of the invention. [1] An isolated recombinant influenza B virus having PA, PB1, PB2, NP, NS, and M viral segments, a heterologous or chimeric influenza virus NA viral segment, and a heterologous or chimeric influenza virus HA viral segment, wherein the NS viral segment encodes a residue other than Y at position 42, a residue other than M at position 117, a residue other than K at position 176, and / or a residue other than S at position 252, and / or has a nucleotide other than a at nucleotide position 39 or a nucleotide insertion after position 38, or any combination thereof; or wherein the M viral segment encodes an M1 polypeptide having a residue other than G at position 34, a residue other than D at position 54, a residue other than R at position 77, a residue other than M at position 86, or a residue other than I at position 97, or any combination thereof; or the M viral segment encodes a BM2 polypeptide having a residue other than H at position 58, a residue other than R at position 80, a residue other than H at position 27, or a residue other than G at position 26, or any combination thereof; the NP viral segment encodes an NP polypeptide having a residue other than A at position 28, a residue other than P at position 40, a residue other than P at position 51, a residue other than E at position 52, a residue other than S at position 57, a residue other than M at position 204, and / or a residue other than P at position 343, and / or having a nucleotide other than g at nucleotide position 1795 or a nucleotide other than c at nucleotide position 500, or any combination thereof; or the PA viral segment has a residue other than Y at position 387, a residue other than V at position 434, a residue other than D at position 494, and / or a residue other than T at position 524, and / or a nucleotide other than a at nucleotide position 2272, a nucleotide other than a at position 1406, a nucleotide other than c at position 1445, or a nucleotide other than g at position 2213, or any combination thereof; or the PB2 viral segment encodes a PB2 polypeptide having a residue other than N at position 16; or any combination thereof. [2] The isolated virus of item 1, wherein the NP polypeptide has at least one of the following: T at position 28, S at position 40, Q at position 51, K at position 52, G at position 57, T at position 204, T at position 343, a at position 1795, or the NP viral segment has a t at position 500. [3] The isolated virus of item 1 or 2, wherein the M1 polypeptide has at least one of the following: V or N at position 34, G at position 54, K at position 77, T at position 86, or N at position 97. [4] The isolated virus of paragraph 1, 2, or 3, wherein the BM2 polypeptide has at least one of the following: R at position 58, G at position 80, R at position 27, or R at position 26. [5] The isolated virus of any one of items 1 to 4, wherein the NS1 polypeptide has at least one of the following: N at position 42, Y at position 117, Q at position 176, or T at position 252, or the NS segment has an insertion of g after nucleotide position 38 or g at nucleotide position 39. [6] The isolated virus of any one of paragraphs 1 to 5, wherein the PA viral segment has at least one of the following: H at position 387, A at position 434, N at position 494, A at position 524, g at position 1406, t at position 2272, t at position 1445, or any combination thereof. [7] The isolated virus of any one of items 1 to 6, wherein the NP polypeptide has an S at position 40 and the NP vRNA has a t at nucleotide position 500, the M1 polypeptide has a K at position 77, the NS1 polypeptide has a Q at position 176 and the NS vRNA has a g at nucleotide position 39, and the PA vRNA has a g at nucleotide position 1406, a t at nucleotide position 1445, and a t at nucleotide position 2272. [8] The isolated virus of any one of items 1 to 7, wherein the NP polypeptide has an S at position 40 and a T at position 204, and the NP vRNA has a t at nucleotide position 500, the M1 polypeptide has a T at position 86, the NS vRNA has a g insertion after nucleotide position 38, and the PA vRNA has a g at nucleotide position 1406, a t at nucleotide position 1445, and a t at nucleotide position 2272. [9] The isolated virus according to any one of items 1 to 8, wherein the NA gene segment and the HA gene segment are derived from the same influenza virus isolate.

[10] The isolated virus of any one of Items 1 to 9, wherein the PA, PB1, PB2, NP, NS, and M viral segments comprise a sequence encoding at least one of the following: PB1 having the amino acid sequence encoded by SEQ ID NO:2 or having at least 80% sequence identity to the PB1 encoded by SEQ ID NO:2; PB2 having the amino acid sequence encoded by SEQ ID NO:3 or having at least 80% sequence identity to the PB2 encoded by SEQ ID NO:3; PA having the amino acid sequence encoded by SEQ ID NO:1 or PA having at least 80% amino acid sequence identity to the PA encoded by SEQ ID NO:1; NP having the amino acid sequence encoded by SEQ ID NO:4 or NP having at least 80% sequence identity to the NP encoded by SEQ ID NO:4; M having the amino acid sequence encoded by SEQ ID NO:5 or M having at least 80% amino acid sequence identity to the M encoded by SEQ ID NO:5; or NS having the amino acid sequence encoded by SEQ ID NO:6 or having at least 95% sequence identity to the NS encoded by SEQ ID NO:6.

[11] The isolated virus of any one of items 1 to 10, having a heterologous HA gene segment or a heterologous NA gene segment.

[12] The isolated virus of any one of items 1 to 11, wherein the M1 polypeptide has a V at position 34, an N at position 97, or a T at position 86, or any combination thereof; or the BM2 polypeptide has an R at position 58 and / or a G at position 80; or the NP polypeptide has an S at position 40 or a K at position 52.

[13] A vaccine comprising the recombinant virus described in any one of items 1 to 12.

[14] A method for preparing influenza virus, comprising: a vector for producing vRNA or cRNA comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence, a vector for producing vRNA or cRNA comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence, a vector for producing vRNA or cRNA comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence, a vector for producing vRNA or cRNA comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence, a vector for producing vRNA or cRNA comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence, a vector for producing vRNA or cRNA comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence, a vector for producing vRNA or cRNA comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence, and a vector for producing vRNA or cRNA comprising a promoter operably linked to influenza virus NS DNA linked to a transcription termination sequence; wherein the PB1, PB2, PA, NP, NS, and M DNA in the vector for vRNA or cRNA production is derived from one or more influenza vaccine virus isolates, wherein the NA DNA in the vector for NA vRNA or cRNA production has a heterologous or chimeric NA sequence, and wherein the HA DNA in the vector for HA vRNA or cRNA production has a heterologous or chimeric HA sequence, and wherein the NS viral segment encodes an NS1 polypeptide having a residue other than Y at position 42, a residue other than M at position 117, a residue other than K at position 176, and / or a residue other than S at position 252, and / or has a nucleotide other than a at position 39 or an insertion of a nucleotide after position 38, or any combination thereof; or the M viral segment encodes an M1 polypeptide having a residue other than G at position 34, a residue other than D at position 54, a residue other than R at position 77, a residue other than M at position 86, a residue other than I at position 97, or any combination thereof, or the M viral segment encodes a BM2 polypeptide having a residue other than H at position 58, a residue other than R at position 80, a residue other than H at position 27, a residue other than G at position 26, or any combination thereof; or the NP viral segment encodes an NP polypeptide having a residue other than A at position 28, a residue other than P at position 40, a residue other than P at position 51, a residue other than E at position 52, a residue other than S at position 57, a residue other than M at position 204, and / or a residue other than P at position 343, and / or having a nucleotide other than g at position 1795 or a nucleotide other than c at position 500, or any combination thereof; or the PA viral segment has a residue other than Y at position 387, a residue other than V at position 434, a residue other than D at position 494, and / or a residue other than T at position 524, and / or a nucleotide other than a at nucleotide position 2272, a nucleotide other than a at nucleotide position 1406, a nucleotide other than c at nucleotide position 1445, and / or a nucleotide other than g at nucleotide position 2213, or any combination thereof; or the PB2 viral segment encodes a PB2 polypeptide having a residue other than N at position 16; or any combination thereof; a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NP; and 1. A method for preparing an influenza virus comprising: contacting in an amount effective to yield infectious influenza virus.

[15] The method of item 14, wherein the cell is an avian cell or a mammalian cell.

[16] The method of item 15, wherein the cells are Vero cells, human cells, or MDCK cells.

[17] The method of any one of Items 14 to 16, wherein the NP polypeptide has S at position 40 and the NP vRNA has t at nucleotide position 500, the M1 polypeptide has K at position 77, the NS1 polypeptide has Q at position 176 and the NS vRNA has g at nucleotide position 39, and the PA vRNA has g at nucleotide position 1406, t at nucleotide position 1445, and t at nucleotide position 2272.

[18] The method of any one of Items 14 to 16, wherein the NP polypeptide has an S at position 40 and a T at position 204, and the NP vRNA has a t at nucleotide position 500, the M1 polypeptide has a T at position 86, the NS vRNA has a g insertion after nucleotide position 38, and the PA vRNA has a g at nucleotide position 1406, a t at nucleotide position 1445, and a t at nucleotide position 2272.

[19] The method of any one of items 14 to 18, further comprising isolating the infectious influenza virus.

[20] a) an influenza B virus NS1 having at least 85% amino acid sequence identity to a polypeptide encoded by SEQ ID NO: 6, 18, or 19 and at least one of the following: an NS vRNA having an insertion in NS1 of N at position 42, Y at position 117, Q at position 176, or T at position 252, g at nucleotide position 39, or g after nucleotide position 38; b) an influenza B virus M1 having at least 85% amino acid sequence identity to a polypeptide encoded by SEQ ID NO: 5, 16 or 17 and having at least one of the following in M1: V or N at position 34, G at position 54, K at position 77, T at position 86, or N at position 97; c) influenza B virus BM2 having at least 85% amino acid sequence identity to the polypeptide encoded by SEQ ID NO: 5, 16 or 17 and having R at position 58, G at position 80, R at position 27 or R at position 26; d) an influenza B virus NP having at least 85% amino acid sequence identity to a polypeptide encoded by SEQ ID NO: 4, 14, or 15 and at least one of the following: an NP vRNA having a T at position 28, an S at position 40, a Q at position 51, a K at position 52, a G at position 57, a T at position 204, or a T at position 343, or a t at nucleotide position 500 in NP; or e) an influenza B virus PA having at least 85% amino acid sequence identity to the polypeptide encoded by SEQ ID NO: 1 or 13 and having at least one of the following: a PA vRNA having H at position 387, A at position 434, N at position 494, or A at position 524, g at nucleotide position 1406, g at nucleotide position 1445, or t at nucleotide position 2272; Vectors for vRNA, cRNA, or mRNA expression.

Claims

1. 1. An isolated recombinant influenza B virus having PA, PB1, PB2, NP, NS, and M viral segments, a heterologous or chimeric influenza virus NA viral segment, and a heterologous or chimeric HA viral segment, wherein the NS viral segment encodes an NS1 polypeptide, the M viral segment encodes an M1 polypeptide and a BM2 polypeptide, the NP viral segment encodes an NP polypeptide, the PA viral segment encodes a PA polypeptide, or the PB2 viral segment encodes a PB2 polypeptide, wherein the NP polypeptide has an S at position 40, or an S at position 40 and a T at position 204.

2. The isolated virus of claim 1, wherein the NP polypeptide further comprises one or more of Q at position 51, K at position 52, G at position 57, or T at position 343, or the NP viral segment has a at position 1795.

3. 3. The isolated virus of claim 1 or 2, wherein the M1 polypeptide has at least one of the following: V or N at position 34, G at position 54, K at position 77, T at position 86, or N at position 97.

4. 4. The isolated virus of any one of claims 1 to 3, wherein the BM2 polypeptide has at least one of the following: R at position 58, G at position 80, R at position 27, or R at position 26.

5. 5. The isolated virus of any one of claims 1 to 4, wherein the NS1 polypeptide has an N at position 42, a Q at position 176, or a T at position 252, or the NS viral segment has a nucleotide insertion of g after nucleotide position 38, or has a g at position 39, or any combination thereof.

6. 6. The isolated virus of any one of claims 1 to 5, wherein the PA polypeptide has at least one of H at position 387, A at position 434, N at position 494, or A at position 524, or the PA viral segment has a t at position 2272.

7. 7. The isolated virus of any one of claims 1 to 6, wherein the NP polypeptide has an S at position 40 and a T at position 204, the M1 polypeptide has a K at position 77, the NS1 polypeptide has a Q at position 176, the NS viral segment has a g at nucleotide position 39, and the PA viral segment has a t at nucleotide position 2272.

8. 8. The isolated virus of any one of claims 1 to 7, wherein the NP polypeptide has an S at position 40 and a T at position 204, and the M1 polypeptide has a T at position 86, the NS viral segment has a g insertion after nucleotide 38, and the PA viral segment has a t at nucleotide position 2272.

9. 9. The isolated virus of any one of claims 1 to 8, wherein the NA viral segment and the HA viral segment are derived from the same influenza virus isolate.

10. The PA, PB1, PB2, NP, NS, and M viral segments are selected from the following: PB1 having the amino acid sequence encoded by SEQ ID NO:2, or PB1 having at least 90% amino acid sequence identity to PB1 encoded by SEQ ID NO:2; PA having an amino acid sequence encoded by SEQ ID NO:3, or PA having at least 90% amino acid sequence identity to PB2 encoded by SEQ ID NO:3; PB2 having the amino acid sequence encoded by SEQ ID NO:1, or PB2 having at least 90% amino acid sequence identity to PA encoded by SEQ ID NO:1; an NP having an amino acid sequence encoded by SEQ ID NO:4, or an NP having at least 90% amino acid sequence identity to an NP encoded by SEQ ID NO:4; M having an amino acid sequence encoded by SEQ ID NO:5, or M having at least 90% 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.

10. The isolated virus of any one of claims 1 to 9, comprising a sequence encoding at least one of:

11. 1 to 3, which contain a heterologous HA or NA viral segment.

11. An isolated virus according to any one of claims 10.

12. 12. The isolated virus of any one of claims 1 to 11, wherein the M1 polypeptide has a V at position 34, an N at position 97, or a T at position 86, or any combination thereof; or the BM2 polypeptide has an R at position 58 and / or a G at position 80; or the NP polypeptide has an S at position 40.

13. 1. A method for producing influenza B virus in vitro, comprising the steps of: In vitro, mammalian cells are treated with an effective amount of one of the following: a vector for producing influenza virus PA vRNA or cRNA, comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence; a vector for producing PB1 vRNA or cRNA, comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence; a vector for producing PB2 vRNA or cRNA, comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence; a vector for producing influenza virus HA vRNA or cRNA, comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence; a vector for producing NP vRNA or cRNA, comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence; a vector for producing influenza virus NA vRNA or cRNA, comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence; a vector for producing influenza virus M vRNA or cRNA, comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence; and Vector for producing influenza virus NS vRNA or cRNA comprising a promoter operably linked to influenza virus NS DNA linked to a transcription termination sequence wherein the PB1, PB2, PA, NP, NS, and M DNA in the vector for vRNA or cRNA production are derived from one or more influenza vaccine virus isolates, and the NA DNA in the vector for NA vRNA or cRNA production has a heterologous or chimeric NA sequence; The HA DNA in the vector for producing HA vRNA or cRNA has a heterologous or chimeric HA sequence; the NP polypeptide encoded by the NP vRNA or cRNA has S at position 40, or has S at position 40 and T at position 204; the position in the NP polypeptide is relative to the NP polypeptide encoded by SEQ ID NO:4; and a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PA; a vector for producing mRNA, comprising a promoter operably linked to a DNA segment encoding influenza virus PB1; a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB2; and Vector for producing mRNA containing a promoter operably linked to a DNA segment encoding influenza virus NP contacting with The method comprising:

14. The method of claim 13, wherein the cell is a human cell.

15. 14. The method of claim 13, wherein the cell is a Vero cell, a human cell, or an MDCK cell.

16. 16. The method of any one of claims 13 to 15, wherein the NP polypeptide has an S at position 40, the M1 polypeptide encoded by the M vRNA or cRNA has a K at position 77, the NS1 polypeptide encoded by the NS vRNA or cRNA has a Q at position 176, and the NS vRNA or cRNA has a g at nucleotide position 39, and the PA vRNA or cRNA has a t at nucleotide position 2272.

17. the NP polypeptide has an S at position 40 and a T at position 204, and the M1 polypeptide encoded by the M vRNA or cRNA has a T at position 86; the NS vRNA or cRNA has a g insertion after nucleotide position 38; The method of any one of claims 13 to 15, wherein the PA vRNA or cRNA has a t at nucleotide 2272.

18. 18. The method of any one of claims 13 to 17, further comprising isolating the infectious influenza virus.

19. The vRNA or cRNA of PA, PB1, PB2, NP, NS, and M is selected from the following: PB1 having the amino acid sequence encoded by SEQ ID NO:2, or PB1 having at least 90% amino acid sequence identity to PB1 encoded by SEQ ID NO:2; a PA having an amino acid sequence encoded by SEQ ID NO:3 or a PA having at least 90% amino acid sequence identity to a PA encoded by SEQ ID NO:3; PB2 having the amino acid sequence encoded by SEQ ID NO:1 or PB2 having at least 90% amino acid sequence identity to PB2 encoded by SEQ ID NO:1; NP having an amino acid sequence encoded by SEQ ID NO:4 or NP having at least 90% amino acid sequence identity to NP encoded by SEQ ID NO:4; M having an amino acid sequence encoded by SEQ ID NO:5 or M having at least 90% 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 17. The method of any one of claims 13 to 16, comprising a sequence encoding at least one of:

20. the NS virus segment has a nucleotide insertion after position 38; or the M1 polypeptide encoded by the M viral segment has a T at position 86; or the NP polypeptide encoded by the NP viral segment has an S at position 40; or 2. The isolated virus of claim 1, wherein the PA viral segment has a nucleotide other than a at nucleotide position 2272.

21. the NS1 polypeptide encoded by said NS viral segment has a Q at position 176 and said NS viral segment has a nucleotide other than a at nucleotide position 39; or the M1 polypeptide encoded by the M viral segment has a K at position 77; or the NP polypeptide encoded by said NP viral segment has an S at position 40 and a T at position 204; or 2. The isolated virus of claim 1, wherein the PA viral segment has a nucleotide other than a at nucleotide position 2272.

22. the M1 polypeptide encoded by the M viral segment has a V at position 34 and / or an N at position 97; or the BM2 polypeptide encoded by the M viral segment has an R at position 58, an R at position 27, and / or an R at position 26; or the NP polypeptide encoded by the NP viral segment has a T at position 343; or the PA polypeptide encoded by said PA viral segment has an N at position 494, and / or said PA viral segment has a nucleotide other than a at nucleotide position 2272 or a nucleotide other than g at nucleotide position 2213; or 2. The isolated virus of claim 1, wherein the PB2 polypeptide encoded by the PB2 viral segment has an S at position 16.

23. the NS1 polypeptide encoded by the NS virus segment has an N at position 42; or the BM2 polypeptide encoded by the M viral segment has a G at position 80; or the PA polypeptide encoded by said PA viral segment has an H at position 387, an A at position 434, and / or an A at position 524; or 2. The isolated virus of claim 1, wherein the PA viral segment has a nucleotide other than a at nucleotide position 2272 or a nucleotide other than g at nucleotide position 2213.

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