Influenza virus backbone

Abstract

The present invention provides influenza viruses with enhanced growth in Vero cells. The influenza viruses contain PA, NP, and NS gene segments that have selected nucleotides and encode proteins having amino acid sequences that include selected amino acids. The invention also provides pharmaceutical formulations containing the influenza viruses, as well as methods for eliciting an immune response in a mammal, comprising administering the influenza viruses to the mammal, and methods for producing the influenza viruses.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of United States Provisional Patent Application No. 63 / 054,676, filed on 21 July 2020, all of which are incorporated herein by reference.

[0002] Research or development supported by the federal government This invention was made with federal government support under authorization number AI109925 granted by the National Institutes of Health. The government has certain rights in this invention.

[0003] Incorporation by referencing electronically submitted materials The computer-readable nucleotide / amino acid sequence listings submitted with this specification and identified below are all incorporated herein by reference: a single 46,917-byte ASCII (Text) file created on July 20, 2021, named "755023SequenceListing.txt".

[0004] Background of the Invention Influenza, or "flu," is a highly contagious viral infection that claims hundreds of thousands of lives worldwide every year. Seasonal outbreaks are mostly caused by the prevalent influenza A and B viruses, but there are four types of influenza viruses (A, B, C, and D) classified based on their core proteins.

[0005] While vaccines are the best way to prevent influenza, influenza vaccines often need to be reformulated because influenza viruses are prone to both continuous and discontinuous antigenic mutations. Furthermore, because influenza viruses generally exhibit high rates of mutation and evolution, influenza vaccine strains may not match circulating strains, potentially minimizing vaccine effectiveness. However, if circulating influenza viruses closely match the influenza vaccine, vaccination can reduce the risk of influenza-related illness for 40% to 60% of the general population. Therefore, researchers are studying vaccines that can induce cross-protective immunity between different influenza subtypes. One such example is a live, attenuated influenza virus that does not express a functional M2 protein (e.g., an M2SR vaccine against influenza A).

[0006] Mammalian cell culture offers advantages over egg-based manufacturing, making it preferable to grow influenza vaccines in Madin-Darby canine kidney (MDCK) cells or African green monkey (Vero) cells. These advantages include lower costs, faster manufacturing times, and a reduced risk of antigenic mutation in the virus. For example, the M2SR vaccine is grown in Vero cells that stably express the M2 protein. However, vaccine production in cell culture has often resulted in undesirable yields. Furthermore, MDCK cells are generally more tolerant than Vero cells, and virus production in Vero cells is relatively inefficient.

[0007] Modifications to the viral backbone, i.e., the six internal gene segments consisting of PB1, PB2, PA, NP, M, and NS, have been shown to enhance vaccine production. For example, the high-yield influenza A vaccine backbone "PR8-HY," developed and described by Ping et al., Nature Communications, 6: 8148 (2015), and the high-yield influenza B vaccine backbone, developed and described by Ping et al., PNAS, 113(51): E8296-E8305 (2016), contain specific amino acid mutations in the PB1, PB2, PA, NP, M, and NS1 proteins, and were expected to improve the titer of pandemic and seasonal influenza vaccines in both cell and egg culture systems. However, these modifications described in the art were ineffective in increasing viral replication in Vero cells, especially under favorable manufacturing conditions. Therefore, there is a need to enhance viral replication in Vero cells so that vaccines can be manufactured more efficiently and effectively.

[0008] Brief summary of the invention The present invention provides an influenza virus with enhanced proliferation in Vero cells. The influenza virus comprises gene segments encoding proteins such as PB1, PB2, PA, NP, M, and NS1 proteins, wherein at least the PA, NP, and NS gene segments have amino acid sequences containing selected amino acids. In particular, the PA gene segment contains thymine at nucleotide position 2272. The NP protein contains serine at position 40, asparagine or glycine at position 161, threonine at position 204, and optionally valine at position 93. The NS gene segment contains guanine at nucleotide position 39, and the NS gene segment encodes an NS1 protein containing glutamine at position 176.

[0009] The present invention also provides a pharmaceutical preparation containing the influenza virus, a method for inducing an immune response in a mammal, which comprises administering the influenza virus to the mammal, and a method for producing the influenza virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] [Figure 1] FIG. 1 is a graph showing growth over time (days) in BM2Vero P3 of influenza BM2SR4 CA12 BM2SR HA and NP mutations. [Figure 2] FIG. 2 is a graph showing growth over time (days post-infection) in animal-origin free (AOF) medium of B influenza NP mutants. [Figure 3] FIG. 3 is a series of Sanger sequence chromatogram data showing that the mutation frequency of NS 569_570insA in the backbone of B influenza increased from about 20% to about 50% in subsequent generations. [Figure 4] FIG. 4 is a graph showing growth over time (days post-inoculation) in AOF in BM2Vero P3 of WA / 02 / 2019 N209S and K420E HA. [Figure 5] FIG. 5 is a graph showing growth over time (days) in BM2Vero P3 of influenza BM2SR4 CA12 HA2 mutations. [Figure 6] FIG. 6 is a graph showing growth over time (days post-infection) in AOF of B influenza CA12 N211T HA2 mutants. [Figure 7A] FIG. 7A is a graph showing the titer against anti-B influenza-Vic HA over time (days post-immunization) in sera from mice immunized with monovalent BM2SR-Vic, monovalent BM2SR-Yam, bivalent BM2SR, trivalent BM2SR+M2SR-H1N1, trivalent BM2SR+M2SR-H3N2, quadrivalent M2SR, or sucrose-phosphate-glutamate buffer ("SPG" control). [Figure 7B]Figure 7B is a graph showing the titer of anti-Influenza B - Yam HA in sera from mice immunized with monovalent BM2SR - Vic, monovalent BM2SR - Yam, bivalent BM2SR, trivalent BM2SR+M2SR - H1N1, trivalent BM2SR+M2SR - H3N2, quadrivalent M2SR, or SPG (control) against time (days after immunization). [Figure 8] Figure 8 is a graph showing the % body weight change in mice immunized with monovalent BM2SR - Vic, monovalent BM2SR - Yam, quadrivalent M2SR, or SPG (control) against time (days after administration) after administration of B / Malaysia / 2506 / 2004 (Vic) virus. [Figure 9] Figure 9 is a graph showing the % body weight change in mice after immunization with monovalent BM2SR - Vic, monovalent BM2SR - Yam, quadrivalent M2SR, FLUMIST™ Quadrivalent, FLUZONE™ Quadrivalent, FLUZONE™ High Dose, or SPG (control) against time (days after administration). [Figure 10A] Figure 10A is a graph showing the ELISA titer of anti - Influenza B - Vic HA serum IgG in sera from mice immunized with monovalent BM2SR - Vic, monovalent BM2SR - Yam, quadrivalent M2SR, FLUMIST™ Quadrivalent, FLUZONE™ Quadrivalent, FLUZONE™ High Dose, or SPG (control) against time (days after vaccination). [Figure 10B] Figure 10B is a graph showing the ELISA titer of anti - Influenza B - Yam HA serum IgG in sera from mice immunized with monovalent BM2SR - Vic, monovalent BM2SR - Yam, quadrivalent M2SR, FLUMIST™ Quadrivalent, FLUZONE™ Quadrivalent, FLUZONE™ High Dose, or SPG (control) against time (days after vaccination). [Figure 10C]Figure 10C is a graph showing the ELISA titer of anti-influenza A / H1 HA serum IgG against time (number of days after vaccination) in the serum of mice immunized with monovalent BM2SR-Vic, monovalent BM2SR-Yam, tetravalent M2SR, FLUMIST® Quadrivalent, FLUZONE® Quadrivalent, FLUZONE® High Dose, or SPG (control). [Figure 10D] Figure 10D is a graph showing the ELISA titer of anti-influenza A / H3 HA serum IgG against time (number of days after vaccination) in the serum of mice immunized with monovalent BM2SR-Vic, monovalent BM2SR-Yam, tetravalent M2SR, FLUMIST® Quadrivalent, FLUZONE® Quadrivalent, FLUZONE® High Dose, or SPG (control). [Figure 11A] Figure 11A is a graph showing the IgG ELISA titers for HA1 test antigens (A / H1, A / H3, B / Yam, or B / Vic) in tracheal-pulmonary lavage fluid from mice immunosensitized with monovalent BM2SR-Vic, monovalent BM2SR-Yam, tetravalent M2SR, FLUMIST® Quadrivalent, FLUZONE® Quadrivalent, FLUZONE® High Dose, or SPG (control). [Figure 11B] Figure 11B is a graph showing the ELISA titers of anti-influenza HA1 IgA against HA1 test antigens (A / H1, A / H3, B / Yam, or B / Vic) in tracheal-pulmonary lavage fluid from mice immunosensitized with monovalent BM2SR-Vic, monovalent BM2SR-Yam, tetravalent M2SR, FLUMIST® Quadrivalent, FLUZONE® Quadrivalent, FLUZONE® High Dose, or SPG (control). [Figure 12]Figure 12 is a graph showing the change in % body weight of mice immunized with monovalent BM2SR-Vic, monovalent BM2SR-Yam, tetravalent M2SR, FLUMIST® Quadrivalent, FLUZONE® Quadrivalent, FLUZONE® High Dose, or SPG (control) as a countermeasure to influenza B / Malaysia / 2506 / 2004 (Vic) over time (number of days after administration). [Figure 13A] Figure 13A is a graph showing the BM2SR-Vic viral titer as time (days after inoculation) in nasal lavage fluid derived from BM2SR-Vic donor ferrets, ferrets that had direct contact with them, and ferrets that had aerosol contact with them. The limit of viral detection was 1.67 log TCID50 / mL (dashed line). [Figure 13B] Figure 13B is a graph showing the wild-type influenza B (Vic) virus titer against time (days after inoculation) in nasal lavage fluids from influenza B (Vic) donor ferrets, ferrets that had direct contact with them, and ferrets that had aerosol contact with them. The virus titer of individual ferrets, the group mean, and the standard error of the mean are plotted. The limit of virus detection was 1.67 log TCID50 / mL (dashed line). [Figure 14A] Figure 14A is a graph showing the anti-influenza A / H1 HAI titer as time (test days) in the serum of 13 ferret groups that were immunosensitized as described in Table 10. The average titer for each group is plotted. The limit of detection was 7.5. [Figure 14B] Figure 14B is a graph showing the anti-influenza A / H3 HAI titer as time (test days) in the serum of 13 ferret groups that were immunosensitized as described in Table 10. The average titer for each group is plotted. The limit of detection was 7.5. [Figure 14C]Figure 14C is a graph showing the anti-influenza B / Yam HAI titer as time (test days) in the serum of 13 ferret groups that were immunosensitized as described in Table 10. The average titer for each group is plotted. The limit of detection was 7.5. [Figure 14D] Figure 14D is a graph showing the anti-influenza A / Vic HAI titer as time (test days) in the serum of 13 ferret groups immunosensitized as described in Table 10. The average titer for each group is plotted. The detection limit was 7.5. [Figure 15A] Figure 15A is a graph showing the anti-influenza A / H1 PRNT titer as time (test days) in the serum of 13 ferret groups that were immunosensitized as described in Table 10. The average titer for each group is plotted. The detection limit was 15. [Figure 15B] Figure 15B is a graph showing the anti-influenza A / H3 PRNT titer as time (test days) in the serum of 13 ferret groups that were immunosensitized as described in Table 10. The average titer for each group is plotted. The detection limit was 15. [Figure 15C] Figure 15C is a graph showing the anti-influenza B / Yam PRNT titer as time (test days) in the serum of 13 ferret groups that were immunosensitized as described in Table 10. The average titer for each group is plotted. The detection limit was 15. [Figure 15D] Figure 15D is a graph showing the anti-influenza A / Vic PRNT titer as time (test days) in the serum of 13 ferret groups that were immunosensitized as described in Table 10. The average titer for each group is plotted. The detection limit was 15. [Figure 16] Figure 16 is a graph showing the titer of administered virus in the nasal turbinates three days after administration of influenza B / Vic, as described in Example 14. The average titer for each group is plotted. The detection limit was 1.3 log FFU / g. [Figure 17]Figure 17 is a graph showing the titers of administered virus in nasal lavage fluid on days 1, 3, 5, and 7 after administration of influenza B / Vic, as described in Example 14. The average titer for each group is plotted. The detection limit was 1.6 log TCID50 / mL. [Figure 18A] Figure 18A is a graph showing the frequency of IFN-γ-producing PBMCs (spot-forming units per 10⁶ cells) after cells were stimulated with the A / Singapore / INFIMH-16-0019 / 2016 virus (cells were collected 28 and 37 days after vaccination). [Figure 18B] Figure 18B is a graph showing the frequency of IFN-γ-producing PBMCs (spot-forming units per 10⁶ cells) after stimulating cells with the A / California / 07 / 2009 HA peptide pool (cells were collected on days 28 and 37 after vaccination). [Figure 19A] Figure 19A is a graph showing the average percentage weight change in each ferret group after administration of influenza B / Vic. [Figure 19B] Figure 19B is a graph showing the average body temperature change in each ferret group after administration of influenza B / Vic. [Modes for carrying out the invention]

[0011] Detailed description of the invention The influenza virus of the present invention may be any type of influenza virus. For example, the influenza virus may be any subtype of influenza B (e.g., Victoria or Yamagata). In some embodiments, the influenza virus may be a seasonal influenza B virus. In some embodiments, the influenza virus may be a recombinant influenza virus. Recombinant influenza viruses (e.g., reassembled influenza viruses) as used herein are genetically distinct, i.e., influenza viruses containing genetic material (e.g., gene segments) derived from different influenza viruses (e.g., heterogeneous gene segments). The influenza virus may also be an isolated influenza virus.

[0012] As used herein, the term "gene segment" means a nucleotide sequence that codes for a viral protein. The gene segment can be represented by a cDNA (complementary DNA) sequence that codes for viral RNA (vRNA), i.e., sequence numbers 8-12 and 18 that code for one or more viral proteins.

[0013] As used herein, the term “backbone” refers to the influenza gene segments encoding PB1, PB2, PA, NP, NS1 and / or NS2, and the M protein. The gene segments of the present invention encode proteins that may have selected amino acids.

[0014] As used herein, the term “selected amino acid” means a specific amino acid at a particular position in an amino acid sequence. In some embodiments, the selected amino acid is the result of a genetic mutation in the original amino acid sequence. The original amino acid sequence may be identical to the amino acid sequence containing the selected amino acid, except for the position corresponding to the selected amino acid.

[0015] Influenza virus (A) Backbone protein In one embodiment of the present invention, the influenza virus comprises PA, NP, and NS gene segments, wherein (a) the PA gene segment contains thymine at nucleotide position 2272, (b) the NP gene segment encodes an NP protein having an amino acid sequence containing selected amino acids, the selected amino acids including serine at position 40, asparagine or glycine at position 161, threonine at position 204, and optionally valine at position 93, and (c) the NS gene segment contains guanine at nucleotide position 39, and the NS gene segment encodes an NS1 protein having an amino acid sequence containing selected amino acids, the selected amino acids including glutamine at position 176.

[0016] The PB1 (polymerase basic protein 1) gene segment of the present invention may encode a protein containing at least one selected amino acid, i.e., a PB1 protein. The selected amino acid can be obtained by genetic mutation into the original PB1 sequence, for example, a sequence identical to the PB1 amino acid sequence of the present invention, except for the position corresponding to the selected amino acid. The PB2 (polymerase basic protein 2) gene segment of the present invention may also encode a protein containing at least one selected amino acid, i.e., a PB2 protein.

[0017] The PA (polymerase acid protein) gene segment of the present invention may also encode a protein containing at least one selected amino acid, i.e., a PA protein. In a preferred embodiment, the gene segment contains thymine at nucleotide position 2272.

[0018] The NP (nucleoprotein) gene segment of the present invention may also encode a protein containing at least one selected amino acid, i.e., an NP protein. In a preferred embodiment, the NP segment contains thymine at position 177, adenine at position 540, and thymine at position 670, and the NP gene segment encodes a protein having selected amino acids, including serine at position 40, asparagine or glycine at position 161, threonine at position 204, and optionally valine at position 93.

[0019] The NS (non-structural) gene segment of the present invention may also encode a protein containing at least one selected amino acid, i.e., an NS1 and / or NS2 protein. In a preferred embodiment, the NS segment contains guanine at nucleotide position 39 and cytosine at position 570, and the NS gene segment encodes an NS protein (NS1 protein) having selected amino acids including glutamine at position 176.

[0020] In one embodiment of the present invention, the influenza virus includes a PB1 gene segment encoding a protein having selected amino acids, i.e., a PB1 protein. The PB1 gene segment may have a nucleotide sequence represented by SEQ ID NO: 8. The PB1 gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 13, i.e., a PB1 protein. In another aspect of this embodiment, the influenza virus may include a PB2 gene segment encoding a protein having selected amino acids, i.e., a PB2 protein. The PB2 gene segment may have a nucleotide sequence represented by SEQ ID NO: 9. The PB2 gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 14, i.e., a PB2 protein. In another aspect of this embodiment, the influenza virus may include an NP gene segment encoding an NP protein, which has selected amino acids at positions 40, 161, and 204, i.e., serine at position 40, asparagine or glycine at position 161, threonine at position 204, and optionally valine at position 93. The NP gene segment may have a nucleotide sequence represented by SEQ ID NO: 11. The NP gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 16, i.e., an NP protein. In another aspect of this embodiment, the influenza virus may include an NS gene segment encoding a protein having a selected amino acid at position 176, i.e., glutamine at position 176, i.e., NS1 and / or NS2 proteins. The NS gene segment may contain guanine at nucleotide position 39 and cytosine at position 570. The NS gene segment may have the nucleotide sequence represented by SEQ ID NO: 12. The NP gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 17, i.e., NS1 and / or NS2 proteins. In another aspect of this embodiment, the influenza virus may include a PA gene segment encoding a protein, i.e., a PA protein. The PA gene segment may have the nucleotide sequence represented by SEQ ID NO: 10.The PA gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 15, i.e., the PA protein.

[0021] The selected amino acids in this embodiment confer enhanced growth characteristics to the influenza virus under the same conditions, particularly in most of the backbone proteins, compared to an influenza virus that is identical except for the absence of the selected amino acids. For example, the influenza virus of the present invention exhibits enhanced growth in Vero cells.

[0022] The influenza virus of the present invention may also include an M (matrix protein) gene segment. In one embodiment of the present invention, the M gene segment may be a mutant gene segment derived from influenza B such that the virus lacks the expression of a functional BM2 protein. Such a virus is referred to herein as a "BM2SR" virus. The BM2SR virus is a single-replicating influenza B virus. The M gene segment of the BM2SR virus may be represented by Sequence ID No. 18. The M gene segment may encode a protein, for example, a truncated BM2 protein having the amino acid sequence of Sequence ID No. 20. The BM2SR virus can be grown in Vero cells that stably express the BM2 protein (i.e., BM2Vero cells), enabling multi-cycle replication. High yield in Vero cells does not depend on mutations in the M gene segment. Therefore, the influenza virus of the present invention may include an M gene segment encoding a functional M2 protein.

[0023] (B) Surface protein In a further embodiment of the present invention, the influenza virus comprises NA (neuraminidase) and HA (hemagglutinin) gene segments. In one embodiment of the present invention, the HA gene segment may encode an HA protein having an amino acid sequence comprising at least one selected amino acid (e.g., an amino acid mutation) in the HA1 subunit of the HA protein and / or at least one selected amino acid (e.g., an amino acid mutation) in the HA2 subunit of the protein. For example, at least one amino acid mutation in the HA2 subunit may be glutamic acid at position 61. In another embodiment, at least one amino acid mutation in the HA2 subunit may be glutamic acid at position 112. The amino acid mutation may be present in any subtype or lineage of influenza B virus (i.e., Victoria or Yamagata). In a preferred embodiment, the amino acid mutation in the HA2 subunit may be glutamic acid at position 61 in the Victoria lineage of influenza B virus. In another preferred embodiment, the amino acid mutation in the HA2 subunit may be glutamic acid at position 112 in the Yamagata lineage of influenza B virus. Such mutations can also contribute to the enhanced replication of the virus during production.

[0024] In one embodiment of the present invention, the PA, NP, and NS gene segments are derived from a single influenza strain. In another embodiment of the present invention, the PB1, PB2, PA, NP, and NS gene segments are derived from a single influenza strain. In one embodiment, the HA gene segment may be derived from an influenza strain different from the single influenza strain from which the PA, NP, and NS gene segments are derived. In another embodiment, the HA gene segment may be derived from an influenza strain different from the single influenza strain from which the PB1, PB2, PA, NP, and NS gene segments are derived. Similarly, the NA gene segment may be derived from an influenza strain different from the single influenza strain from which the PA, NP, and NS gene segments are derived. In another embodiment, the NA gene segment may be derived from an influenza strain different from the single influenza strain from which the PB1, PB2, PA, NP, and NS gene segments are derived. Therefore, the influenza virus of the present invention may be a seasonal influenza virus (e.g., influenza B).

[0025] (C) Characteristics of the influenza virus The influenza virus backbone of the present invention confers high proliferation characteristics to influenza viruses, regardless of the type of influenza virus, particularly in Vero cells. The influenza virus of the present invention exhibits high yields even in manufacturing processes using low MOI (e.g., 0.001). MOI refers to the average number of pathogens (e.g., viruses) per infectious target (e.g., cell). Lower MOIs are used when multiple cycles of infection are required (e.g., viral vaccine production). With the enforcement of Good Manufacturing Practices (PGP) by the FDA, the use of the lowest MOI that still produces high-yield viruses is generally required. This is because master seed stocks are expensive, and toxicity from non-infectious particles and excess cellular proteins can reduce virus production.

[0026] In a further embodiment of the present invention, the influenza virus is genetically stable, and its backbone proteins, particularly PB1, PB2, PA, NP, and NS proteins, are highly conserved even when grown at a low MOI. For example, in one embodiment of the present invention, the selected amino acids are conserved in at least one of the PB1, PB2, NP, and NS proteins after at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or eleven or more consecutive passages in the Vero cell line. In one embodiment, the Vero cell line may include Vero cells that stably express the BM2 ion channel protein of influenza B virus (i.e., BM2 Vero cells). In a preferred embodiment of the present invention, the selected amino acids are conserved in at least one of the NP and NS proteins after at least ten consecutive passages in the Vero cell line that stably expresses the BM2 ion channel protein of influenza B virus. BM2 is thought to functionally correspond to M2 of influenza A virus. In such an embodiment, the selected amino acids can be conserved even if the influenza virus is influenza A virus.

[0027] Genetically modified Vero cells (i.e., those expressing influenza M2 or BM2 proteins) exhibit similar behavior to normal Vero cells and support replication of influenza A or B viruses comparable to that of normal Vero cells. The viral titer for M2SR virus in M2VeroA cells is comparable to that of replicating influenza viruses expressing functional M2 in unmodified Vero cell lines. Furthermore, the viral titer for BM2SR virus (i.e., influenza viruses containing a mutant M gene segment derived from influenza B, resulting in the non-expression of functional BM2 protein) in BM2Vero cells is comparable to that of replicating influenza viruses expressing functional BM2 in unmodified Vero cell lines. Therefore, M2SR and BM2SR viruses exhibit similar behavior to replicating influenza viruses in M2VeroA and BM2Vero cell lines.

[0028] In one embodiment of the present invention, the influenza virus can replicate within human cells.

[0029] Method for generating influenza virus The present invention also provides a method for generating influenza viruses, wherein in one embodiment the generated influenza virus comprises PA, NP, and NS gene segments expressing a protein having selected amino acids, i.e., recombinant influenza viruses of the present invention as disclosed herein. In another embodiment the generated influenza virus comprises PB1, PB2, NP, and NS gene segments expressing a protein having selected amino acids, i.e., another embodiment of the present invention as disclosed herein.

[0030] In one embodiment of the method of the present invention, a method for producing recombinant influenza virus comprises successively passage a recombinant influenza virus (e.g., a first influenza virus) in Vero cells to produce the resulting influenza virus (e.g., a second influenza virus). The first influenza virus may include PA, NP, and NS gene segments that express a protein, i.e., the PA, NP, and NS1 protein having selected amino acids as described with respect to the influenza virus of the present invention. For example, the first influenza virus may include a PA gene segment containing thymine at nucleotide position 2272. The PA gene segment may encode a protein, i.e., the PA protein. The NP gene segment of the first influenza virus may contain thymine at position 177, adenine at position 540, and thymine at position 670, serine at position 40, asparagine or glycine at position 161, threonine at position 204, and optionally valine at position 93, i.e., the NP protein. The NS gene segment of the first influenza virus may contain guanine at nucleotide position 39 and glutamine at position 176, thereby encoding the NS1 and / or NS2 proteins. In one embodiment of the present invention, the second influenza virus (e.g., the generated influenza virus) is produced after at least four or at least five consecutive passages of the first influenza virus in Vero cells. The first influenza virus may further comprise PB1 and PB2 gene segments expressing the proteins, i.e., the PB1 and PB2 proteins having selected amino acids as described with respect to the influenza virus of the present invention.

[0031] The influenza virus of the present invention can also be generated using standard virus rescue techniques. For example, in one embodiment of the present invention, one or more plasmids in which cDNAs for each of eight viral gene segments (i.e., PB1, PB2, PA, NP, M, NS, HA, and NA) are cloned, and each cDNA sequence is flanked by an RNA polymerase I promoter and an RNA polymerase I terminator (i.e., a pPolI plasmid), are transfected into a eukaryotic host cell. The gene segments encoding the PB1, PB2, PA, NP, and NS1 and / or NS2 proteins may encode proteins having selected amino acids of the present invention. The gene segment encoding the M2 or BM2 protein may include a mutant M2 or BM2 gene segment such that the gene segment does not encode a functional M2 or BM2. The host cell can also be transfected with one or more expression plasmids encoding viral proteins (e.g., at least one of PA, PB1, PB2, and NP proteins, or at least one of PB1, PB2, PA, NP, M, NS1 and / or NS2, HA, and NA proteins). Eight influenza vRNAs (i.e., gene segments) are then synthesized after transfection of the host cell with at least one plasmid. Co-transfected viral polymerase and nucleoprotein assemble the vRNAs into functional vRNPs (i.e., viral ribonucleoprotein complexes) that are replicated and transcribed, ultimately forming the recombinant influenza virus of the present invention. This plasmid-based reverse genetics system is further described by Neumann et al., PNAS, 96: 9345-9350 (1999). The influenza virus of the present invention can also be produced by other methods known in the art, such as, for example, a ribonucleoprotein (RNP) transfection system described in U.S. Patent No. 9,284,533 (incorporated herein by reference).

[0032] Pharmaceutical preparations The present invention provides pharmaceutical formulations (e.g., vaccines or other immunological compositions) containing the recombinant virus of the present invention as described herein.

[0033] The pharmaceutical formulation may further contain at least one pharmaceutically acceptable carrier or excipient. As used herein, the term “pharmaceutically acceptable carrier or excipient” means any component of the pharmaceutical formulation other than the influenza virus of the present invention. The pharmaceutically acceptable carrier or excipient can enhance the efficacy of the recombinant virus of the present invention or maintain the stability of the pharmaceutical formulation, preferably without significantly inactivating the recombinant virus of the present invention.

[0034] At least one pharmaceutically acceptable carrier or excipient may be any suitable pharmaceutically acceptable carrier or excipient, many of which are known in the art. Examples of pharmaceutically acceptable carriers or excipients include components that maintain the pH of a pharmaceutical formulation (e.g., buffers), components that adjust tension (e.g., isotonic agents such as inorganic salts), components that improve the stability and / or immunogenicity of proteins (e.g., viruses), components that improve mucosal adhesion, components that prevent protein aggregation, and / or components that preserve the pharmaceutical formulation (e.g., preservatives). For example, a pharmaceutically acceptable carrier or excipient may contain at least one of inorganic salts, surfactants, amino acids, polymers or polymeric compounds (e.g., proteins, polysaccharides, or hydrogels), chelating agents, sugars, polyols, and / or adjuvants (e.g., any substance that enhances a particular immune response), many of which are known in the art. A particular carrier or excipient may serve two or more purposes in a pharmaceutical formulation. Accordingly, the following embodiments are not limited to those described herein.

[0035] Any suitable buffer can be present in the pharmaceutical formulation. In one embodiment, the buffer comprises at least one of imidazole buffer, potassium phosphate buffer, phosphate-buffered saline (PBS), Dulbecco's phosphate-buffered saline (DPBS) (e.g., 1×DPBS), histidine buffer, sodium citrate buffer, and sucrose-phosphate-glutamate buffer (SPG). The PBS and / or DPBS preparation may contain, for example, sodium chloride, potassium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate, and optionally further contain calcium chloride and / or magnesium chloride. Any suitable PBS and / or DPBS preparation can be used as a buffer in the pharmaceutical formulation, many of which are known in the art, but in some embodiments, the PBS and / or DPBS preparation contains about 136.9 mM sodium chloride, about 2.67 mM potassium chloride, about 1.47 mM potassium dihydrogen phosphate, and about 8.1 mM disodium hydrogen phosphate.

[0036] In the pharmaceutical formulation, the buffer solution may be present at any appropriate concentration. In the pharmaceutical formulation, the buffer solution may be present at concentrations of approximately 0.1 mM or higher, approximately 1 mM or higher, approximately 10 mM or higher, approximately 20 mM or higher, approximately 30 mM or higher, approximately 40 mM or higher, approximately 50 mM or higher, approximately 60 mM or higher, approximately 70 mM or higher, approximately 80 mM or higher, approximately 90 mM or higher, approximately 100 mM or higher, approximately 120 mM or higher, approximately 140 mM or higher, approximately 160 mM or higher, approximately 180 mM or higher, approximately 200 mM or higher, approximately 250 mM or higher, approximately 300 mM or higher, approximately 350 mM or higher, approximately 400 mM or higher, approximately 450 mM or higher, or approximately 500 mM or higher. Alternatively, or in addition, in the pharmaceutical formulation, the buffer may be present at concentrations of approximately 1,000 mM or less, approximately 500 mM or less, approximately 450 mM or less, approximately 400 mM or less, approximately 350 mM or less, approximately 300 mM or less, approximately 250 mM or less, approximately 200 mM or less, approximately 180 mM or less, approximately 160 mM or less, approximately 140 mM or less, approximately 120 mM or less, approximately 100 mM or less, approximately 90 mM or less, approximately 80 mM or less, approximately 70 mM or less, approximately 60 mM or less, approximately 50 mM or less, approximately 40 mM or less, approximately 30 mM or less, approximately 20 mM or less, approximately 10 mM or less, or approximately 1 mM or less. In the pharmaceutical formulation, the buffer may be present at any concentration within the range bounded by any of the aforementioned endpoints. For example, in the pharmaceutical formulation, the buffer solution may be present at concentrations such as approximately 0.1 mM to approximately 1000 mM, approximately 0.1 mM to approximately 500 mM, approximately 0.1 mM to approximately 100 mM, approximately 1 mM to approximately 1000 mM, approximately 1 mM to approximately 500 mM, approximately 1 mM to approximately 100 mM, approximately 100 mM to approximately 1000 mM, and approximately 100 mM to approximately 500 mM.

[0037] In a further embodiment, the buffer is present in the pharmaceutical formulation at a percentage concentration (e.g., volume / volume percentage (% v / v), weight / volume percentage (% w / v), or weight / weight percentage (% w / w)). In the pharmaceutical formulation, the buffer may be present at concentrations of about 0.1% or more, about 1% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, or about 50% or more. Alternatively, or in addition, in the pharmaceutical formulation, the buffer may be present at concentrations of about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, or about 1% or less. In the pharmaceutical formulation, the buffer may be present at any percentage concentration within the range bounded by any of the aforementioned endpoints. For example, in the pharmaceutical formulation, the buffer solution may be present in percentage concentrations such as approximately 0.1% to approximately 60%, approximately 1% to approximately 60%, approximately 10% to approximately 60%, approximately 0.1% to approximately 50%, approximately 1% to approximately 50%, approximately 10% to approximately 50%, approximately 20% to approximately 60%, approximately 20% to approximately 50%, approximately 20% to approximately 40%, approximately 20% to approximately 30%, approximately 30% to approximately 40%, and approximately 40% to approximately 50%.

[0038] The buffer can maintain the pH of the pharmaceutical formulation at any appropriate pH. The buffer can maintain the pH of the pharmaceutical formulation at, for example, a pH of approximately 4 or higher, approximately 4.5 or higher, approximately 5 or higher, approximately 5.5 or higher, approximately 6 or higher, approximately 6.5 or higher, approximately 7 or higher, or approximately 7.5 or higher. Alternatively, or in addition to the above, the buffer can maintain the pH of the pharmaceutical formulation at, for example, a pH of approximately 8 or lower, approximately 7.5 or lower, approximately 7 or lower, approximately 6.5 or lower, approximately 6 or lower, approximately 5.5 or lower, approximately 5 or lower, or approximately 4.5 or lower. The buffer can maintain the pH of the pharmaceutical formulation within a range bounded by any of the aforementioned endpoints. For example, a buffer solution can maintain the pH of a pharmaceutical preparation at pH levels such as approximately 4 to 8, 4.5 to 8, 5 to 8, 5.5 to 8, 6 to 8, 6.5 to 8, 7 to 8, 7.5 to 8, 4 to 7.5, 5 to 7.5, 6 to 7.5, 7 to 7.5, 4 to 7, 5 to 7, and 6 to 7.

[0039] Any suitable isotonic agent may be present in the pharmaceutical formulation. In one embodiment, one or more inorganic salts are present in the pharmaceutical formulation as isotonic agents. The inorganic salt may be at least one of sodium chloride (NaCl), magnesium sulfate (MgSO4), and magnesium chloride (MgCl2). The isotonic agent, such as an inorganic salt, may be present in the pharmaceutical formulation in any suitable amount. An isotonic agent, such as an inorganic salt, may be present in the pharmaceutical preparation at concentrations of approximately 0.1 mM or higher, approximately 0.2 mM or higher, approximately 0.4 mM or higher, approximately 0.6 mM or higher, approximately 0.8 mM or higher, approximately 1 mM or higher, approximately 1.2 mM or higher, approximately 1.4 mM or higher, approximately 1.6 mM or higher, approximately 1.8 mM or higher, approximately 2 mM or higher, approximately 3 mM or higher, approximately 4 mM or higher, approximately 5 mM or higher, approximately 6 mM or higher, approximately 7 mM or higher, approximately 8 mM or higher, approximately 9 mM or higher, approximately 10 mM or higher, approximately 20 mM or higher, approximately 30 mM or higher, approximately 40 mM or higher, approximately 50 mM or higher, approximately 100 mM or higher, approximately 200 mM or higher, approximately 300 mM or higher, approximately 400 mM or higher, approximately 500 mM or higher, approximately 600 mM or higher, approximately 700 mM or higher, approximately 800 mM or higher, approximately 900 mM or higher, approximately 1000 mM or higher, or approximately 1500 mM or higher. Alternatively, or in addition, an isotonic agent, such as an inorganic salt, may be present in the pharmaceutical formulation at concentrations of approximately 2000 mM or less, approximately 1500 mM or less, approximately 1000 mM or less, approximately 900 mM or less, approximately 800 mM or less, approximately 700 mM or less, approximately 600 mM or less, approximately 500 mM or less, approximately 450 mM or less, approximately 400 mM or less, approximately 350 mM or less, approximately 300 mM or less, approximately 250 mM or less, approximately 200 mM or less, approximately 150 mM or less, approximately 100 mM or less, approximately 50 mM or less, approximately 45 mM or less, approximately 40 mM or less, approximately 35 mM or less, approximately 30 mM or less, approximately 25 mM or less, approximately 20 mM or less, approximately 10 mM or less, approximately 9 mM or less, approximately 8 mM or less, approximately 7 mM or less, approximately 6 mM or less, approximately 5 mM or less, approximately 4 mM or less, approximately 3 mM or less, approximately 2 mM or less, approximately 1.8 mM or less, approximately 1.6 mM or less, and approximately 1.4 mM or less. It may be present at concentrations of mM or less, approximately 1.2 mM or less, approximately 1 mM or less, approximately 0.8 mM or less, approximately 0.6 mM or less, approximately 0.4 mM or less, or approximately 0.2 mM or less.An isotonic agent, such as an inorganic salt, may be present in the pharmaceutical formulation at any concentration within the range bounded by any of the aforementioned endpoints. For example, isotonic agents, such as inorganic salts, are present in the pharmaceutical formulation at concentrations of approximately 0.1 mM to approximately 2000 mM, approximately 0.1 mM to approximately 1500 mM, approximately 0.1 mM to approximately 1000 mM, approximately 0.1 mM to approximately 500 mM, approximately 0.1 mM to approximately 250 mM, approximately 0.1 mM to approximately 100 mM, approximately 0.1 mM to approximately 50 mM, approximately 0.1 mM to approximately 10 mM, approximately 1 mM to approximately 2000 mM, approximately 1 mM to approximately 1500 mM, approximately 1 mM to approximately 1000 mM, approximately 1 mM to approximately 500 mM, approximately 1 mM to approximately 250 mM, approximately 1 mM to approximately 100 mM, approximately 1 mM to approximately 500 mM, approximately 1 mM to approximately 100 mM, approximately 10 mM to approximately 2000 mM, approximately 10 mM to approximately 1500 mM, approximately 10 mM to approximately 1000 mM, approximately 10 mM to approximately 500 mM, and approximately 10 It can exist at concentrations such as mM to approximately 250 mM, approximately 10 mM to approximately 100 mM, approximately 10 mM to approximately 50 mM, approximately 100 mM to approximately 2000 mM, approximately 100 mM to approximately 1500 mM, approximately 100 mM to approximately 1000 mM, approximately 100 mM to approximately 500 mM, approximately 100 mM to approximately 250 mM, approximately 500 mM to approximately 2000 mM, approximately 500 mM to approximately 1500 mM, and approximately 500 mM to approximately 1000 mM.

[0040] In a further embodiment, the inorganic salt is present in the pharmaceutical formulation at a percentage concentration (e.g., volume / volume percentage (% v / v), weight / volume percentage (% w / v), or weight / weight percentage (% w / w)). The isotonic agent, e.g., the inorganic salt, is present in the pharmaceutical formulation at a percentage concentration of about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, or about 10% or more. Alternatively, or in addition, the isotonic agent, e.g., the inorganic salt, may be present in the pharmaceutical formulation at a percentage concentration of about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less. The isotonic agent, e.g., the inorganic salt, may be present in the pharmaceutical formulation at any percentage concentration within the range bounded by any of the above endpoints. For example, isotonic agents, such as inorganic salts, may be present in the pharmaceutical preparation at percentage concentrations of approximately 0.1% to 1%, approximately 0.1% to 2%, approximately 0.1% to 5%, approximately 0.1% to 10%, approximately 1% to 2%, approximately 1% to 5%, approximately 1% to 10%, approximately 2% to 10%, approximately 3% to 10%, approximately 4% to 10%, approximately 5% to 10%, and so on.

[0041] Any suitable surfactant may be present in the pharmaceutical formulation. In some embodiments, the surfactant may contain at least one of polysorbate 20, polysorbate 80, sodium deoxycholate, and poloxamer 188. The surfactant may be present in the pharmaceutical formulation in any suitable amount. In some embodiments, the surfactant is present in the pharmaceutical formulation at a percentage concentration (e.g., volume / volume percentage (% v / v), weight / volume percentage (% w / v), or weight / weight percentage (% w / w)). Surfactants may be present in the pharmaceutical formulation at a percentage concentration of approximately 0.01% or more, approximately 0.02% or more, approximately 0.03% or more, 0.04% or more, approximately 0.05% or more, approximately 0.06% or more, approximately 0.07% or more, approximately 0.08% or more, approximately 0.09% or more, approximately 0.1% or more, approximately 0.2% or more, approximately 0.3% or more, approximately 0.4% or more, approximately 0.5% or more, approximately 0.6% or more, approximately 0.7% or more, approximately 0.8% or more, approximately 0.9% or more, or approximately 1% or more. Alternatively, or in addition, surfactants may be present in the pharmaceutical formulation at a percentage concentration of approximately 1% or less, approximately 0.9% or less, approximately 0.8% or less, approximately 0.7% or less, approximately 0.6% or less, approximately 0.5% or less, approximately 0.4% or less, approximately 0.3% or less, approximately 0.2% or less, or approximately 0.1% or less. The surfactant may be present in the pharmaceutical formulation at any percentage concentration within the range bounded by any of the aforementioned endpoints. For example, the surfactant may be present in the pharmaceutical formulation at percentage concentrations such as approximately 0.01% to approximately 1%, approximately 0.01% to approximately 0.1%, approximately 0.05% to approximately 1%, approximately 0.05% to approximately 0.1%, approximately 0.1% to approximately 1%, approximately 0.1% to approximately 0.5%, approximately 0.2% to approximately 1%, and approximately 0.5% to approximately 1%.

[0042] Any suitable amino acid may be present in the pharmaceutical formulation. In one embodiment, the amino acid may be one or more of arginine, glutamic acid or glutamate, asparagine, histidine, and glycine. The amino acid may be present in the pharmaceutical formulation in any suitable amount. The amino acid may be present in the pharmaceutical formulation at concentrations of about 1 mM or more, about 2 mM or more, about 3 mM or more, about 5 mM or more, about 6 mM or more, about 7 mM or more, about 8 mM or more, about 9 mM or more, or about 10 mM or more. Alternatively, or in addition, the amino acid may be present in the pharmaceutical formulation at concentrations of about 100 mM or less, about 90 mM or less, about 80 mM or less, about 70 mM or less, about 60 mM or less, about 50 mM or less, about 40 mM or less, about 30 mM or less, about 20 mM or less, or about 10 mM or less. The amino acid may be present in the pharmaceutical formulation at any concentration within the range bounded by any of the above endpoints. For example, amino acids may be present in the pharmaceutical preparation at concentrations such as approximately 1 mM to approximately 10 mM, approximately 1 mM to approximately 50 mM, approximately 1 mM to approximately 100 mM, approximately 5 mM to approximately 50 mM, approximately 10 mM to approximately 50 mM, and approximately 20 mM to approximately 50 mM.

[0043] In some embodiments, amino acids are present in the pharmaceutical formulation at a percentage concentration (e.g., volume / volume percentage (% v / v), weight / volume percentage (% w / v), or weight / weight percentage (% w / w)). The amino acids may be present in the pharmaceutical formulation at a percentage concentration of about 0.1% or more, about 0.2% or more, about 0.3% or more, about 0.4% or more, about 0.5% or more, about 0.6% or more, about 0.7% or more, about 0.8% or more, about 0.9% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, or about 5% or more. Alternatively, or in addition, amino acids may be present in the pharmaceutical formulation at a percentage concentration of about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less. Amino acids may be present in the pharmaceutical formulation at any percentage concentration within the range bounded by any of the aforementioned endpoints. For example, amino acids may be present in the pharmaceutical formulation at percentage concentrations such as approximately 0.1% to 10%, approximately 0.2% to 10%, approximately 0.5% to 10%, approximately 0.1% to 5%, approximately 0.1% to 2%, approximately 0.2% to 2%, approximately 0.5% to 1%, etc.

[0044] Any suitable polymer or polymeric compound may be present in the pharmaceutical formulation. The polymer or polymeric compound may be, for example, a protein, polysaccharide, hydrogel, or any other suitable polymer or polymeric compound, many of which are known in the art. For example, the polymer or polymeric compound may be recombinant human serum albumin (rHSA), serum albumin (SA), gelatin, hydroxyethyl starch (HES), chitosan, dextran (DEX70K, DEX40K), and polyvinylpyrrolidone (PVP40K).

[0045] Polymers or polymeric compounds may be present in the pharmaceutical formulation in any appropriate amount. The polymers or polymeric compounds may be present in the pharmaceutical formulation at a percentage concentration (e.g., volume / volume percentage (% v / v), weight / volume percentage (% w / v), or weight / weight percentage (% w / w)). The polymers or polymeric compounds may be present in the pharmaceutical formulation at a percentage concentration of approximately 0.1% or more, approximately 0.2% or more, approximately 0.3% or more, approximately 0.4% or more, approximately 0.5% or more, approximately 0.6% or more, approximately 0.7% or more, approximately 0.8% or more, approximately 0.9% or more, approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, or approximately 5% or more. Alternatively, or in addition, polymers or polymeric compounds may be present in the pharmaceutical formulation at a percentage concentration of about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less. Polymers or polymeric compounds may be present in the pharmaceutical formulation at any percentage concentration within the range bounded by any of the aforementioned endpoints. For example, polymers or polymeric compounds may be present in the pharmaceutical formulation at percentage concentrations of about 0.1% to about 10%, about 0.2% to 10%, about 0.5% to about 10%, about 0.1% to about 5%, about 0.1% to about 2%, about 0.2% to about 2%, about 0.5% to about 2%, about 0.1% to about 1%, about 0.2% to about 1%, about 0.5% to about 1%, etc.

[0046] Any suitable chelating agent may be present in the pharmaceutical formulation. The chelating agent may be, for example, ethylenediaminetetraacetic acid (EDTA), an amidoxime compound (AOX), and / or dithiothreitol (DTT). The chelating agent may be present in the pharmaceutical formulation at any suitable concentration. The chelating agent may be present in the pharmaceutical formulation at concentrations of 10 μM or more, about 20 μM or more, about 30 μM or more, about 40 μM or more, about 50 μM or more, about 60 μM or more, about 70 μM or more, about 80 μM or more, about 90 μM or more, about 100 μM or more, about 120 μM or more, or about 150 μM or more. Alternatively, or in addition, the chelating agent may be present in the pharmaceutical formulation at concentrations of approximately 500 μM or less, approximately 400 μM or less, approximately 300 μM or less, approximately 200 μM or less, approximately 150 μM or less, approximately 140 μM or less, approximately 130 μM or less, approximately 120 μM or less, approximately 110 μM or less, approximately 100 μM or less, approximately 80 μM or less, approximately 70 μM or less, approximately 60 μM or less, or approximately 50 μM or less. The chelating agent may be present in the pharmaceutical formulation at any concentration within the range bounded by any of the aforementioned endpoints. For example, the chelating agent may be present in the pharmaceutical preparation at concentrations such as approximately 10 μM to approximately 500 μM, approximately 10 μM to approximately 200 μM, approximately 10 μM to approximately 150 μM, approximately 10 μM to approximately 100 μM, approximately 50 μM to approximately 500 μM, approximately 50 μM to approximately 200 μM, approximately 50 μM to approximately 150 μM, and approximately 50 μM to approximately 100 μM.

[0047] Any suitable sugar may be present in the pharmaceutical formulation. The sugar may be, for example, one or more of sucrose, trehalose, mannose, and lactose, and the sugar may be present in the pharmaceutical formulation at any suitable concentration. The sugars in the pharmaceutical preparation are approximately 0.1 mM or more, approximately 0.2 mM or more, approximately 0.4 mM or more, approximately 0.6 mM or more, approximately 0.8 mM or more, approximately 1 mM or more, approximately 1.2 mM or more, approximately 1.4 mM or more, approximately 1.6 mM or more, approximately 1.8 mM or more, approximately 2 mM or more, approximately 3 mM or more, approximately 4 mM or more, approximately 5 mM or more, approximately 6 mM or more, approximately 7 mM or more, approximately 8 mM or more, approximately 9 mM or more, approximately 10 mM or more, approximately 20 mM or more, approximately 30 mM or more, approximately 40 mM or more, approximately 50 mM or more, approximately 60 mM or more, approximately 70 mM or more, approximately 80 mM or more, approximately 90 mM or more, approximately 100 mM or more, approximately 200 mM or more, approximately 300 mM or more, approximately 400 mM or more, approximately 500 mM or more, approximately 600 mM or more, approximately 700 mM or more, approximately 800 mM or more, approximately 900 mM or more, and approximately 1000 It may be present at concentrations of mM or higher, or approximately 1500 mM or higher. Alternatively, or in addition, sugars in the pharmaceutical preparation may be present in amounts of approximately 2000 mM or less, approximately 1500 mM or less, approximately 1000 mM or less, approximately 900 mM or less, approximately 800 mM or less, approximately 700 mM or less, approximately 600 mM or less, approximately 500 mM or less, approximately 450 mM or less, approximately 400 mM or less, approximately 350 mM or less, approximately 300 mM or less, approximately 250 mM or less, approximately 200 mM or less, approximately 150 mM or less, approximately 100 mM or less, approximately 50 mM or less, approximately 45 mM or less, approximately 40 mM or less, approximately 35 mM or less, approximately 30 mM or less, approximately 25 mM or less, approximately 20 mM or less, approximately 10 mM or less, approximately 9 mM or less, approximately 8 mM or less, approximately 7 mM or less, approximately 6 mM or less, approximately 5 mM or less, approximately 4 mM or less, approximately 3 mM or less, approximately 2 mM or less, approximately 1.8 mM or less, approximately 1.6 mM or less, approximately 1.4 mM or less, and approximately 1.2 The sugar may be present at concentrations of less than mM, about 1 mM or less, about 0.8 mM or less, about 0.6 mM or less, about 0.4 mM or less, or about 0.2 mM or less. The sugar may be present in the pharmaceutical formulation at any concentration within the range bounded by any of the aforementioned endpoints.For example, sugars in the pharmaceutical preparation are approximately 0.1 mM to approximately 2000 mM, approximately 0.1 mM to approximately 1500 mM, approximately 0.1 mM to approximately 1000 mM, approximately 0.1 mM to approximately 500 mM, approximately 0.1 mM to approximately 250 mM, approximately 0.1 mM to approximately 100 mM, approximately 0.1 mM to approximately 50 mM, approximately 0.1 mM to approximately 10 mM, approximately 1 mM to approximately 2000 mM, approximately 1 mM to approximately 1500 mM, approximately 1 mM to approximately 1000 mM, approximately 1 mM to approximately 500 mM, approximately 1 mM to approximately 250 mM, approximately 1 mM to approximately 100 mM, approximately 1 mM to approximately 500 mM, approximately 1 mM to approximately 2000 mM, approximately 10 mM to approximately 1500 mM, 10 mM to approximately 1000 mM, approximately 10 mM to approximately 500 mM, and approximately 10 mM to approximately 250 mM. It can exist at concentrations such as mM, approximately 10 mM to approximately 100 mM, approximately 10 mM to approximately 50 mM, approximately 100 mM to approximately 2000 mM, approximately 100 mM to approximately 1500 mM, approximately 100 mM to approximately 1000 mM, approximately 100 mM to approximately 500 mM, approximately 100 mM to approximately 250 mM, approximately 500 mM to approximately 2000 mM, approximately 500 mM to approximately 1500 mM, and approximately 500 mM to approximately 1000 mM.

[0048] In another embodiment, sugar may be present in the pharmaceutical formulation at a percentage concentration (e.g., volume / volume percentage (% v / v), weight / volume percentage (% w / v), or weight / weight percentage (% w / w)). Sugar may be present in the pharmaceutical formulation at a percentage concentration of about 1% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, or about 40% or more. Alternatively, or in addition, sugar may be present in the pharmaceutical formulation at a percentage concentration of about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, or about 1% or less. Sugar may be present in the pharmaceutical formulation at any percentage concentration within the range bounded by any of the aforementioned endpoints. For example, sugar may be present in the pharmaceutical preparation at percentage concentrations such as approximately 0.1% to approximately 50%, approximately 1% to approximately 50%, approximately 10% to approximately 50%, approximately 0.1% to approximately 20%, approximately 1% to approximately 20%, approximately 10% to approximately 20%, approximately 0.1% to approximately 10%, and approximately 1% to approximately 10%.

[0049] Any suitable polyol may be present in the pharmaceutical formulation. The polyol may be, for example, sorbitol and / or mannitol. The polyol may be present in the pharmaceutical formulation at any suitable concentration. The polyol may be present in the pharmaceutical formulation at concentrations of about 0.1 mM or more, about 1 mM or more, about 10 mM or more, about 20 mM or more, about 30 mM or more, about 40 mM or more, about 50 mM or more, about 60 mM or more, about 70 mM or more, about 80 mM or more, about 90 mM or more, about 100 mM or more, about 120 mM or more, about 140 mM or more, about 160 mM or more, about 180 mM or more, about 200 mM or more, about 250 mM or more, about 300 mM or more, about 350 mM or more, about 400 mM or more, about 450 mM or more, or about 500 mM or more. Alternatively, or in addition, polyols may be present in the pharmaceutical formulation at concentrations of approximately 1000 mM or less, approximately 500 mM or less, approximately 450 mM or less, approximately 400 mM or less, approximately 350 mM or less, approximately 300 mM or less, approximately 250 mM or less, approximately 200 mM or less, approximately 180 mM or less, approximately 160 mM or less, approximately 140 mM or less, approximately 120 mM or less, approximately 100 mM or less, approximately 90 mM or less, approximately 80 mM or less, approximately 70 mM or less, approximately 60 mM or less, approximately 50 mM or less, approximately 40 mM or less, approximately 30 mM or less, approximately 20 mM or less, approximately 10 mM or less, or approximately 1 mM or less. Polyols may be present in the pharmaceutical formulation at any concentration within the range bounded by any of the aforementioned endpoints. For example, polyols may be present in the pharmaceutical formulation at concentrations such as approximately 0.1 mM to approximately 1000 mM, approximately 0.1 mM to approximately 500 mM, approximately 0.1 mM to approximately 100 mM, approximately 1 mM to approximately 1000 mM, approximately 1 mM to approximately 500 mM, approximately 1 mM to approximately 100 mM, approximately 100 mM to approximately 1000 mM, and approximately 100 mM to approximately 500 mM.

[0050] In another embodiment, the polyol may be present in the pharmaceutical formulation at a percentage concentration (e.g., volume / volume percentage (% v / v), weight / volume percentage (% w / v), or weight / weight percentage (% w / w)). The polyol may be present in the pharmaceutical formulation at a percentage concentration of about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, or about 45% or more. Alternatively, or in addition, polyols may be present in the pharmaceutical formulation at a percentage concentration of about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less. Polyols may be present in the pharmaceutical formulation at any percentage concentration within the range bounded by any of the aforementioned endpoints. For example, polyols may be present in the pharmaceutical preparation at percentage concentrations such as approximately 0.1% to approximately 50%, approximately 1% to approximately 50%, approximately 5% to approximately 50%, approximately 10% to approximately 50%, approximately 15% to approximately 50%, approximately 0.1% to approximately 25%, approximately 1% to approximately 25%, approximately 5% to approximately 25%, approximately 10% to approximately 25%, approximately 15% to approximately 25%, approximately 0.1% to approximately 15%, approximately 1% to approximately 15%, approximately 5% to approximately 15%, approximately 10% to approximately 15%, approximately 0.1% to approximately 10%, approximately 1% to approximately 10%, approximately 5% to approximately 10%, approximately 0.1% to approximately 5%, and approximately 1% to approximately 5%.

[0051] In one embodiment, the pharmaceutical formulation contains the influenza virus of the present invention, about 0.5 M sucrose, about 0.1 M or about 0.5 M mannose, about 0.3 M or about 0.5 M trehalose, about 50% SPG, and about 0.05% polysorbate 20. In another embodiment, the pharmaceutical formulation contains the influenza virus of the present invention, about 0.5 M sucrose, about 0.3 M trehalose, and about 0.05% polysorbate 20.

[0052] At least one pharmaceutically acceptable carrier or excipient may be an ingredient that acts to bind the components of the pharmaceutical formulation together (e.g., a binder). Such binders include, but are not limited to, proteins (e.g., gelatin), polymers (e.g., polyethylene glycol, polyvinylpyrrolidone), and / or polysaccharides or their derivatives (e.g., starch and cellulose). At least one pharmaceutically acceptable carrier or excipient may be an ingredient that increases the bulk of the pharmaceutical formulation (e.g., a filler, diluent, and / or filler). Such fillers include, but are not limited to, polysaccharides or their derivatives, sugars, and / or inorganic compounds. A pharmaceutically acceptable carrier or excipient may be an ingredient that improves the taste and / or appearance of the pharmaceutical formulation (e.g., a flavoring, sweetener, and / or coloring). A pharmaceutically acceptable carrier or excipient may be an ingredient that protects the pharmaceutical formulation from moisture by adsorbing liquids or gases (e.g., an adsorbent). Examples of adsorbents include, but are not limited to, starch, calcium phosphate, and / or colloidal silicon dioxide. Pharmaceutically acceptable carriers or excipients may be components that promote the dissolution of the pharmaceutical formulation (e.g., disintegrants), such as starch, cellulose, and / or any other polymer known in the art, or derivatives thereof (e.g., cross-linked polyvinylpyrrolidone or carboxymethylcellulose).

[0053] In some embodiments, pharmaceutically acceptable carriers or excipients are components (e.g., flow promoters) that reduce interparticle adhesion in or during the manufacture of a pharmaceutical formulation and / or optimize the flow of substances. Examples of flow promoters include, but are not limited to, talc, colloidal silicon dioxide, and corn starch. Particularly when a pharmaceutical formulation is formulated as an oral preparation, pharmaceutically acceptable carriers or excipients may be components (e.g., anti-adhesives) that impart non-sticking properties, such as reducing adhesion between components and, for example, the tablet surface or a lubricant in or during the manufacture of the pharmaceutical formulation. For example, an anti-adhesive may be magnesium stearate. In another embodiment, pharmaceutically acceptable carriers or excipients may be components (lures) that reduce the aggregation of components and / or reduce friction between the surface of a pharmaceutical formulation, i.e., a pharmaceutical formulation formulated as an oral preparation, and the die wall during manufacturing. Water-soluble or water-insoluble lubricants such as magnesium stearate, stearic acid, vegetable oil, mineral oil, polyethylene glycol, and / or sodium lauryl sulfate can be used depending on the embodiment. Pharmacopoecitable carriers or excipients may be components acting as coating agents. Examples of coating agents include, but are not limited to, gelatin and / or cellulose-based coating agents (e.g., hydroxymethylcellulose).

[0054] Other suitable binders, fragrances, sweeteners, colorants, disintegrants, flow enhancers, anti-adhesives, lubricants, and coatings are well known and readily identifiable in the art.

[0055] The pharmaceutical formulation may further contain therapeutic agents (e.g., chemotherapeutic agents or anti-inflammatory agents). The pharmaceutical formulation may also contain chemical substances that elicit an immune response other than that of the influenza virus. Such additional components other than the influenza virus of the present invention may be present in any appropriate amount.

[0056] Additional components can be mixed with other components to form a pharmaceutical formulation prior to presentation to the immune system. Additional components can also be presented to the immune system separately from the pharmaceutical formulation. For example, additional components and the pharmaceutical formulation can be presented (administered to the body) separately to the immune system. When additional components and the pharmaceutical formulation are administered separately, they can be administered to the same site in the body being immunized.

[0057] In one embodiment of the pharmaceutical formulation, the pharmaceutical formulation is a viral vaccine. The viral vaccine may be a live, attenuated viral vaccine or an inactivated viral vaccine (e.g., a whole-virus vaccine, a split-virus vaccine, or a subunit vaccine). The viral vaccine may be formulated as a monovalent, bivalent, trivalent, or quaternary vaccine. For example, the virus may comprise the influenza virus of the present invention in several embodiments. In some embodiments, the vaccine may further comprise at least one influenza virus different from the influenza virus of the present invention.

[0058] Viral vaccines can be formulated into compositions for any suitable means of administration. For example, viral vaccines can be formulated as oral preparations (e.g., capsules, tablets, oral films), sprays (e.g., nasal sprays), or for intranasal administration, or as any composition suitable for parenteral administration, such as intravenous, intramuscular, intradermal, or subcutaneous administration, such as aqueous or non-aqueous emulsions, solutions, suspensions, etc.

[0059] Methods for inducing an immune response The present invention provides a method of inducing an immunization method in a mammal, which includes administering the influenza virus of the present invention to the mammal. In one embodiment, the influenza virus includes proteins, namely, PB1 gene segment, PB2 gene segment, PA gene segment, NP gene segment, and NS gene segment, each encoding PB1 protein, PB2 protein, PA protein, NP protein, and NS1 protein, which contain selected amino acids, that is, the influenza virus of the present invention described herein.

[0060] The mammal can be, for example, a human or a primate, but is not limited thereto.

[0061] In one embodiment of the present invention, the influenza virus of the present invention is administered in a pharmaceutical preparation (e.g., a vaccine or other immunogenic composition) described herein. The pharmaceutical preparation can be administered intranasally. In another embodiment, the pharmaceutical preparation is administered intramuscularly. The pharmaceutical preparation can also be administered subcutaneously or orally.

[0062] The administration schedule of the pharmaceutical preparation, such as a virus vaccine, can depend on the age, weight, gender, and medical history of the mammal. For example, in one embodiment, the single dose of the live attenuated virus vaccine for humans is about 10 11 , 9 , 10 , 7 , 8 , 5 , 6 , 12 , 50 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 、10 10 、10 11 、or 10 12 、or the particle-forming unit (PFU), focus-forming unit (FFU), or TCID of the influenza virus of the present invention within the range between any two of the above numerical values 50This may include. In some embodiments, a prophylactic or therapeutic regimen for influenza virus includes administering the pharmaceutical preparation as a single treatment. The pharmaceutical preparation may also be administered two or more times, for example, the regimen may include a booster dose. For example, a booster dose of the pharmaceutical preparation may be administered after the initial dose at intervals of 7 days or more, for example, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 3 weeks or more, 4 weeks or more, 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, 10 months or more, 11 months or more, 1 year or more, 2 years or more, 3 years or more, 4 years or more, or 5 years or more.

[0063] Embodiment The present invention provides the following embodiments:

[0064] (1) An influenza virus comprising PA, NP and NS gene segments, wherein (a) the PA gene segment comprises thymine at nucleotide position 2272; (b) the NP gene segment encodes an NP protein having an amino acid sequence comprising selected amino acids, the selected amino acids comprising serine at position 40, asparagine or glycine at position 161, threonine at position 204, and optionally valine at position 93; (c) the NS gene segment comprises guanine at nucleotide position 39, and the NS gene segment encodes an NS1 protein having an amino acid sequence comprising selected amino acids, the selected amino acid comprising glutamine at position 176.

[0065] (2) The influenza virus according to embodiment 1, wherein the PA gene segment has a nucleotide sequence represented by sequence number 10.

[0066] (3) An influenza virus according to embodiment 1 or 2, wherein the NP gene segment has a nucleotide sequence represented by SEQ ID NO: 11.

[0067] (4) Any one of embodiments 1 to 3 wherein the PA gene segment encodes a PA protein having the amino acid sequence represented by Sequence ID No. 15.

[0068] (5) Any one of the influenza viruses in any of embodiments 1 to 4, wherein the NP gene segment encodes an NP protein having the amino acid sequence represented by SEQ ID NO: 16.

[0069] (6) An influenza virus according to any one of embodiments 1 to 5, wherein the NS gene segment has the nucleotide sequence represented by Sequence ID No. 12.

[0070] (7) Any one of embodiments 1 to 6 wherein the NS gene segment encodes an NS1 protein having the amino acid sequence represented by SEQ ID NO: 17.

[0071] (8) Any one of the influenza viruses in any of embodiments 1 to 7, wherein the selected amino acid is conserved in at least one of the NP and NS proteins even after at least 10 consecutive passages in the Vero cell line.

[0072] (9) Any one of the influenza viruses in any of embodiments 1 to 8, wherein the selected amino acid is conserved in at least one of the NP and NS proteins even after at least 10 consecutive passages in a Vero cell line that stably expresses the BM2 ion channel protein of influenza B virus.

[0073] (10) An influenza virus according to any one of embodiments 1 to 9, wherein the influenza virus is a recombinant influenza virus.

[0074] (11) An influenza virus in any one of embodiments 1 to 10, wherein the virus further comprises a PB gene segment.

[0075] (12) An influenza virus in any one of embodiments 1 to 11, wherein the virus further comprises an NA gene segment and an HA gene segment.

[0076] (13) An influenza virus according to embodiment 12, wherein the HA gene segment encodes an HA protein having an amino acid sequence containing at least one amino acid mutation within HA2.

[0077] (14) The influenza virus of embodiment 13, wherein at least one amino acid mutation in HA2 is glutamic acid at position 61.

[0078] (15) An influenza virus according to embodiment 13, wherein at least one amino acid mutation in HA2 is glutamic acid at position 112.

[0079] (16) Any one of the influenza viruses from embodiments 1 to 15, wherein the PA, NP, and NS gene segments are derived from a single influenza strain.

[0080] (17) An influenza virus according to embodiment 16, wherein the HA gene segment is derived from an influenza strain different from the single influenza strain from which the PA, NP, and NS gene segments are derived.

[0081] (18) An influenza virus according to embodiment 16 or 17, wherein the NA gene segment is derived from an influenza strain different from the single influenza strain from which the PA, NP, and NS gene segments are derived.

[0082] (19) An influenza virus in any one of embodiments 1 to 18, further comprising a mutated M gene segment.

[0083] (20) An influenza virus according to embodiment 19, wherein the influenza virus does not encode a functional BM2 protein.

[0084] (21) An influenza virus according to any one of embodiments 1 to 20, wherein the virus is capable of replicating in human cells.

[0085] (22) Any one of the influenza viruses described in aspects 1 to 21, wherein the virus exhibits enhanced replication in Vero cells under identical conditions compared to an influenza virus that is identical except for the absence of selected amino acids.

[0086] (23) A pharmaceutical preparation comprising any one of the influenza viruses described in embodiments 1 to 22.

[0087] (24) A pharmaceutical preparation according to embodiment 23, wherein the pharmaceutical preparation is a vaccine.

[0088] (25) A pharmaceutical preparation according to embodiment 24, wherein the vaccine is formulated as a monovalent vaccine.

[0089] (26) A pharmaceutical preparation according to embodiment 24, wherein the vaccine is formulated as a bivalent vaccine.

[0090] (27) A pharmaceutical preparation according to embodiment 24, wherein the vaccine is formulated as a trivalent vaccine.

[0091] (28) A pharmaceutical preparation according to embodiment 24, wherein the vaccine is formulated as a quadrivalent vaccine.

[0092] (29) A method for inducing an immune response in a mammal, comprising administering to the mammal one influenza virus of any one of embodiments 1 to 22 or one pharmaceutical preparation of any one of embodiments 23 to 28, thereby inducing an immune response to the influenza virus in the mammal.

[0093] (30) The method of aspect 29, wherein the mammal is a human.

[0094] (31) A method for generating an influenza virus, any one of embodiments 1 to 22, comprising serially passage the influenza virus in Vero cells. [Examples]

[0095] The following embodiments further illustrate the present invention, but should not be construed as limiting its scope in any way.

[0096] [Example 1] This example demonstrates the successful isolation of the influenza B NP D161N mutation, which promotes proliferation across strains.

[0097] Two vaccine candidate influenza B BM2SR strains, containing a deletion of BM2 ion channel function essential for multicycle replication, were constructed using a plasmid-based virus rescue procedure in BM2Vero cells constitutively overexpressing cDNA encoding BM2 from influenza B / Lee / 40 (see, e.g., Neumann et al., PNAS, 96: 9345-9350 (1999)). The two segments encoding the major antigens, hemagglutinin (HA) and neuraminidase (NA), were obtained from two strains recommended by the WHO for use in vaccination against seasonal influenza B: the Victoria lineage (VL) strain B / Brisbane / 60 / 2008 (Bris60) and the Yamagata lineage (YL) strain B / Wisconsin / 010 (WI01). BM2-deficient segment 7 was obtained by manipulating the influenza B / Florida / 4 / 2006 M segment to (1) remove the BM2 transmembrane H+ channel, (2) introduce multiple stop codons into all translational reading frames, and (3) maintain the native RNA structure for genetic stability and proper expression of the M1 polypeptide. Five of the eight genomic segments (i.e., PA, PB1, PB2, NP, and NS) were obtained by selecting VL and YL strains from an influenza B / Yamagata / 1 / 73 viral library that were enhanced to grow in high yield during in vitro Vero cell culture (Ping et al. 2016, cited above). After three passages (P3) in BM2Vero, the complete nucleotide sequence of each BM2SR strain was verified by Sanger sequencing of cDNA amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from all eight segments. The sequences of all eight viral segments were identical to those of the eight RNA segment expression plasmids used to rescue the virus from both the young liver (YL) and the very long liver (VL).

[0098] Next, the vaccine candidates Bris60(VL) and WI01(YL)BM2SR P3 strains underwent 10 additional viral passages at a low MOI, for a total of 13 passages (P13). After each passage round, a 50% tissue culture infectious dose assay (TCID) was performed in modified MDCK cells (BM2CK, Hattast al., J. Virol., 78(11): 5576-5583(2004)) that were tolerant of BM2SR replication. 50 The viral titer was measured by ). Each subsequent passage was set to MOI = 0.001 TCID per BM2 Vero cell to allow for multi-cycle replication. 50 The procedure involved infecting the virus with a sufficient amount of material. After repeated viral replication through 10 passages, the nucleotide sequences of two P13 strains were obtained again. The sequences of the P13 VL and YL strains were compared with the sequence of the P3 strain to identify adaptive mutations. In the viral genomes obtained from three independent passage experiments, only one identical mutation was identified in VL and YL, or one cross-lineage mutation of influenza B. The unique cross-lineage mutation was g540a in influenza B genome segment 5, encoding the substitution of aspartic acid to asparagine at position 161 in the nucleoprotein (NP D161N).

[0099] The initial B / Yamagata / 1 / 73 high-yield (HY) segment sequences of VL and YL P3 strains differ in two segments: nucleoprotein (NP) segment 5 and non-structural protein (NS) segment 8. In P3, Bris60 (VL) encoded NP P40S / M204T and NS g38insg, while WI01 (YL)BM2SR contained NP-P40S, and NS a39g and NS1-K176Q (Ping et al. 2016, cited above). After passage, VL NP segment 5 was found to encode NP P40S / D161N / M204T, and YL segment 5 encoded NP P40S / D161N. The same D161N mutation was present in both strains. The segregation of the same substitution was not random, and therefore, D161N promotes the proliferation of influenza B in cultured Vero cells. This contrasts with the lineage-specific mutations described for highly proliferative viruses in Vero cells (Ping et al. 2016, see above).

[0100] Except for the HA and NA segments being obtained from B / CA / 12 / 2015(YL), the same BM2SR mono-replicating vaccine virus backbone was used for the third passage experiment. After six passages, another NP segment nucleotide mutation a541g was isolated, similarly resulting in the substitution of aspartic acid to glycine at position 161, D161G. This example demonstrates that site 161 is involved in high-yield growth in Vero culture.

[0101] The NP D161N mutation was added to B / CA / 12 / 2015 HA in combination with the HA1 N211T mutation, which is known to improve the proliferation of influenza B YL in culture. HA1 N211T improved proliferation rate and titer. The NP D161N mutation showed a synergistic effect with HA1 N211T, reducing the time to maximum titer by one day, as shown in Figure 1.

[0102] Another influenza B NP variant a336g encoding NP M93V was isolated multiple times, but only when HA and NA originated from VL. A mixture of g1164r encoding a mixture of WT and NP V369I was also isolated once.

[0103] [Example 2] This embodiment demonstrates the successful isolation of influenza B strains containing the influenza B NP D161N adaptive mutation.

[0104] In BM2Vero cells in a tolerant state constitutively overexpressing cDNA encoding BM2 from influenza B / Lee / 40, various vaccine candidate influenza B BM2SR strains, including those lacking BM2 ion channel function essential for multicycle replication, were constructed using plasmid-based virus rescue procedures (Neumann et al., see above). Two segments encoding hemagglutinin (HA) and neuraminidase (NA) antigens were obtained from the Victoria lineage (VL) strain B / CO / 06 / 2017 (CO06), a strain recommended by the World Health Organization (WHO) for use in vaccination against seasonal influenza B. The BM2-deficient segment 7 was obtained by manipulating the influenza B / Florida / 4 / 2006 M segment to (1) remove the BM2 transmembrane H+ channel, (2) introduce multiple stop codons into all translational reading frames, and (3) maintain the native RNA structure for genetic stability and proper expression of the M1 polypeptide. Four of the eight genomic segments (i.e., PA, PB1, PB2, and NS) were obtained by selecting VL strains from an influenza B / Yamagata / 1 / 73 virus library that were enhanced to grow in high yield during in vitro Vero cell culture (Ping et al. 2016, see above).

[0105] The six strains differed only in the nucleoprotein (NP) of segment 5 and contained permutations of three high-yield mutants isolated from influenza B NP sequences obtained after passage in BM2Vero cells. Three strains with one amino acid substitution in the NP were constructed: P40S, D161N, or M204T. A control wild-type NP strain without NP substitution was also constructed. Finally, strains with two mutations (D161N / M204T) and strains with all three mutations were constructed. After two passages (P2) in BM2Vero, the nucleotide sequences of the NP and BM2SR segments of each strain were validated by Sanger sequencing of RT-PCR-amplified cDNA from all eight segments.

[0106] To compare growth characteristics, third passage of BM2Vero was performed in triplicate for each virus. Culture samples were taken daily for 4 days after inoculation with MOI=0.001, and aliquots were collected. 50 The samples were frozen at -80°C for subsequent analysis by assay. The average of the triplicate measurements was calculated for each day and plotted in Figure 2. Each single mutation produced a growth effect on its own, but the D161N mutation had the greatest effect by itself. When D161N was combined with M204T, the double mutant achieved a faster growth rate compared to the wild type and single mutant. The triple mutant achieved a faster growth rate and peak maximal titer in 4 days compared to the double mutant. Thus, D161N is a growth-promoting NP mutation and, in synergy with other known NP substitutions, enables influenza B strains that grow faster and reach even higher maximal titers.

[0107] [Example 3] This example demonstrates the stability of influenza B NS segments during subculturing in culture.

[0108] The function of NS1 is known to be unnecessary for in vitro culture of influenza B virus in Vero cell cultures. During passage experiments, the NS segment accumulated various mutations. However, these mutations were not fully elucidated and were observed as admixture with the starting sequence.

[0109] Many of the mutations are expansions of the polynucleotide tract. Within the sequence reads of the B / Brisbane / 60 / 2008 BM2SR4 P13 NS segment containing g38insg, there is a mixture of polyA7 to polyA8 expansions in the UTR (mRNA sense) just 5' of AUG (SEQ ID NO: 1), AAAAAAAUG (SEQ ID NO: 2) to AAAAAAAUG (SEQ ID NO: 3). The B / CO / 06 / 2017 strain with NS g38insg grew to P6 and also had the polyA7 to A8 expansion combined with the reversion of the published g38insg HY promoter mutation. These sequence data show a complex mixture of sequences in the 5' UTR, suggesting that NS g38insg was unstable in these experiments.

[0110] The B / CA / 12 / 2015 BM2SR strain, possessing an NS segment with g38insg, was passaged six times, and a mixture was observed between the starting WT NS1 sequence and a polyA6 tract at position 569 that was extended to polyA7. The 569_570insA mutation results in a 176-amino acid NS1 truncation protein with a 4-residue C-terminal extension. This mutation is located in the same polyA6 tract region as a570c, which encodes the previously identified NS HY mutation, K176Q. After passages P3, P5, and P6, genomic RNA was extracted from the viral culture supernatant, and cDNA was amplified from all eight genomic segments by RT-PCR. The sequence chromatogram data shown in Figure 3 indicates that the polyA stretch at base pairs 570-574 of NS segment 8 lengthens by 5 to 6 nucleotides over time during passage. The frequency of the NS 569_570insA mutation increased from approximately 20% to approximately 50% in subsequent passages, indicating that this mutation is advantageous for proliferation in Vero cells.

[0111] The result of the NS 569_570insA mutation (SEQ ID NO: 4) is a translational frameshift in the NS1 open reading frame (ORF) starting from amino acid K176. This frameshift fuses four new amino acids, KGYT (SEQ ID NO: 5), followed by the TAA stop codon (SEQ ID NO: 6). Thus, the 176-amino acid NS truncation protein with a 4-residue C-terminal extension is expressed as a 180-amino acid peptide (SEQ ID NO: 7, where X is any amino acid including G, R, or V). In this example, X was G. Splicing and expression of the NEP ORF are not affected by this mutation.

[0112] Cleaving the C-terminal 105 amino acids of the 281-residue influenza B strain appears to result in the loss of some or all of NS1 function. NS1 plays a role in the reuse of host mRNA metabolism, such as polyadenylation and splicing, and inhibits interferon-dependent innate immune responses in host cells, such as the induction of RIG-I. Because Vero cells in culture lack an interferon response, even influenza strains with complete NS1 deletion can grow in this cell line. The deletion of the NS1 C-terminus is favorable for the proliferation of the BM2SR backbone in Vero, and this mutation is thought to promote the production of vaccine material. The K176Q mutation may enhance the genetic stability of this site by inserting G into the polyA6 rune.

[0113] Passing B / WI / 01 / 2010 BM2SR4 containing K176Q up to P13 yielded a mixture of NP sequences. The sequence of segment 8 was approximately 70% identical to the starting plasmid containing K176Q. In the remaining sequences, approximately 30% of the sequences were deleted at 25 bp, 416del25, from the NS1 ORF. This deletion resulted in cleavage of 158 residues from P123 onwards, and in the 135 AA NS1 truncation mutant, 12 new amino acids (AA) were added. These data suggest that the NS segment a39g K176Q is generally more stable. The combination of the NS a39g K176Q segment and the NP D161N HY mutation was investigated to improve stability in BM2Vero cells. The B / CA / 12 / 2015 strain, containing the NS a39g K176Q and NP P40S D161N M204T segments, was passaged 10 times in BM2Vero cells. No mutations were observed in the NS or NP segments, suggesting that combining the two HY segments improves stability.

[0114] [Example 4] This example demonstrates the isolation of adaptive influenza B HA segments during subculturing in culture.

[0115] Multiple BM2SR strains obtained from both VL and YL cells were passaged in BM2Vero cells, and the nucleotide sequences of the HA segment were determined. No silent mutations that did not alter the coding sequence were identified. Eighteen mutations altering amino acids at 14 locations were identified in the HA segment (Table 1). The mutations could be broadly divided into two groups. Mutations in HA1 often occurred directly at the well-known N-linked glycosylation site N209 (WA02 VL) / N210 (CO / 06 VL) / N211 (YL), or near residues important for glycosylase recognition. The majority of isolated mutations were located in the HA2 coiled-coil region. Two HA2 mutations stood out. The HA a1294g mutation was observed in multiple experiments passaged from the B / CO / 06 / 2017 BM2SR strain, independently of NP or NS segment identity. These adaptations of glycosylation sites are thought to affect the affinity of HA binding to target Vero cells. Changes in the HA2 coiled coil are thought to influence the pH-dependent structural changes necessary for HA to fuse with the membrane of target cells.

[0116] [Table 1]

[0117] Different strains of influenza B may have different amino acid sequences compared to influenza B / Bris / 60 / 2008 HA1. Influenza B VL has sequential deletions of 1, 2, or 3 amino acids in the HA1 gene segment (this varies by year), while influenza B YL has a 1AA deletion in the HA1 gene segment. In Table 1, amino acids marked with an asterisk indicate that mutations may occur at different locations depending on the strain. Table 2 shows examples of different amino acid mutation locations across strains. Some of the isolated mutations were introduced into the HA of other strains to investigate their growth-promoting effects.

[0118] [Table 2]

[0119] [Example 5] This example demonstrates the rescue of the influenza B / WA / 02 / 2019 BM2SR(VL) strain, which possesses an adaptive mutation in the influenza B HA.

[0120] Three versions of cDNA encoding influenza HA segment 5 of the WHO-recommended vaccine strain B / WA / 02 / 2019(VL) were synthesized and inserted into an influenza B plasmid-based virus rescue RNA expression plasmid vector. The first HA version encoded the wild-type (WT) HA protein sequence obtained directly from primary isolates of influenza patients. WT HA retains the N209 glycosylation site of HA1 in its entirety. Passaging the influenza B / WA / 02 / 2019(VL) strain in cell or egg culture results in loss of glycosylation at this site, primarily due to N209S substitution (GISAID). The second plasmid encodes the mutant HA2 K61E (K421E) obtained from passaging the B / CO / 06 / 2017 strain. A double mutant was also constructed, adding a glycosylation-negative N209S HA1 variant to the HA2 K61E variant.

[0121] Using a standard plasmid-based virus rescue procedure with three types of HA plasmids, B / WA / 02 / 2019 BM2SR(VL) virus was constructed by transfection into BM2Vero cells. Both single and double mutant HA plasmids supported the rescue of BM2SR virus, but the complete WT HA sequence did not generate viable virus (assay LOD = log 10 0.67 TCID 50 ( / mL). All successful vaccine candidates were passaged to P2 in BM2Vero and the HA sequence was confirmed. The virus was passaged to P3 with MOI = 0.01, and the TCID of viral titers was determined as shown in Figure 4 and Table 3. 50Aliquots were frozen daily for evaluation. The single mutant strain with the K420E (K61E HA2) mutation grew well, and the glycosylated (+) strain reached a maximum log of 7.67 in 3 days. 10 TCID 50 It reached / mL. The double mutant, GlycoHA strain, grew faster but logged in 2 days. 10 A similar peak titer of 7.50 was reached.

[0122] [Table 3]

[0123] [Example 6] This example demonstrates the propagation of the influenza B / CA / 12 / 2015 BM2SR(YL) strain, which possesses an adaptive mutation in influenza B HA.

[0124] Eight versions of cDNA encoding influenza HA segment 5 of the WHO-recommended vaccine strain B / CA / 12 / 2015(YL) were synthesized and inserted into an RNA expression plasmid vector for influenza B reverse gene rescue to create four HA mutant strains. These four HA mutant strains derived from Vero passages contain five mutations: four mutations in four HA2 residues: 1.) N51D and D61E; 2.) D90N; 3.) D112E; and 4.) D112K, all of which are combined with the HA1 glycosylation site mutation N211T. The HA2 N51D and K61E mutations obtained from passages of B / CO / 06 / 2017 aided proliferation, but only when combined with N211T. The HA2 D90N and D112K mutations obtained from passages of influenza A H3N2 in Vero either harmed proliferation or had no effect when combined with N211T. As shown in Figure 5, D112E (D473E) was advantageous in that it resulted in faster growth to maximum titer, regardless of the glycosylation site N211.

[0125] [Example 7] This example further demonstrates the propagation of the influenza B / CA / 12 / 2015 BM2SR strain, which possesses an adaptive mutation for influenza B type HA.

[0126] The HA2 D112 site in influenza B YL significantly affected growth in culture. Independent of the glycosylation site N211, the HA2 D112 mutation D473E (D112E) was favorable for influenza BM2SR, resulting in faster growth to maximal titer than other B / CA / 12 / 2015(YL)HA2 112 variants; therefore, additional substitutions HA2 D112G and HA2 D112N were performed. These HA2 mutations are influenza A HA2 mutations at position E112 that confer amantadine resistance and significantly increase the pH at which structural changes in the HA spike protein can occur (Byrd-Leotis et al., J. Virol., 89(8): 4504-4516 (2015)). HA2 D112G has been shown to function in all previously tested influenza A HAs. As shown in Figure 6, for influenza B, HA2 D112G and HA2 D112N, when combined with N211, were advantageous and synergistic, resulting in faster proliferation to maximum titer.

[0127] [Example 8] This example further demonstrates the success of isolating other adaptive influenza B HA mutations during subculturing.

[0128] Following previous examples, further adaptive influenza B HA mutations were isolated during subculturing in the culture. These mutations are summarized in Table 4 below.

[0129] [Table 4]

[0130] [Example 9] This example demonstrates that the BM2SR virus elicits an antibody response against influenza B virus incorporated into a multivalent vaccine.

[0131] Influenza BM2SR-Vic or BM2SR-Yam viruses (viruses containing PB1, PB2, PA, NP, NS, and M gene segments, each having the nucleotide sequences represented by SEQ ID NOs: 8, 9, 10, 11, 12, and 18, respectively) induce an antibody response when prescribed as a monovalent, bivalent, trivalent, or quadrivalent vaccine.

[0132] Seven-week-old female BALB / c mice (N=8) were immunized intranasally with either monovalent BM2SR-Vic, monovalent BM2SR-Yam, bivalent BM2SR, trivalent BM2SR-Vic+BM2SR-Yam+M2SR-H1N1, trivalent BM2SR-Vic+BM2SR-Yam+M2SR-H3N2, or quadrivalent BM2SR-Vic and -Yam, and M2SR-H1N1 and -H3N2 vaccines. Control mice were mock-immunized with SPG buffer (sucrose phosphate glutamate buffer). Twenty-eight days after vaccination, mice were intranasally immunized with a booster immunization consisting of the same vaccine administered for primary immunization. Twenty-eight days after vaccination, mice were orally administered a booster immunization consisting of the same vaccine administered for primary immunization. Serum samples were collected on days 7, 14, and 21 after the initial immunization, and on days 35, 42, and 49 after the booster immunization (day 28). Anti-influenza B-Vic HA and anti-influenza B-Yam HA serum IgG antibody titers were measured from the serum samples by ELISA.

[0133] The obtained anti-influenza B-Vic HA data is shown in Figure 7A. The obtained anti-influenza B-Yam HA data is shown in Figure 7B. As a result, all vaccines were able to increase anti-influenza virus antibodies compared to the SPG control, and the amount of increase was comparable among the vaccine formulations. These results demonstrate that there is no interference between monovalent components when combined in a multivalent vaccine.

[0134] [Example 10] This embodiment demonstrates that intranasal administration of a monovalent or tetravalent M2SR vaccine protects mice from lethal influenza B viruses not included in the vaccine.

[0135] In Example 9, female BALB / c mice were immunized with a lethal dose of influenza B / Malaysia / 2506 / 2004 (Vic) virus (>10 mice, 50% lethal dose (MLD)) 70 days after the initial immunization (6 weeks after the booster immunization). 50 The mice were challenged with the following vaccines: monovalent BM2SR-Vic (influenza B / CO / 06 / 2017) or BM2SR-Yam (influenza B / Phuket / 3073 / 2013) and quadrivalent M2SR vaccine. All mice immunized with monovalent BM2SR-Vic, monovalent BM2SR-Yam, and quadrivalent M2SR vaccine remained healthy without weight loss. The weight loss data obtained is shown in Figure 8. Control mice mock-immunized with SPG only experienced weight loss, and 4 out of 8 mice became infected within 10 days after the challenge. Three days after the challenge, lung samples were taken from 3 mice per group, and viral load was measured by plaque assay using MDCK cells. As shown in Table 5, viral titers in the lungs of mice immunized with monovalent BM2SR-Vic, monovalent BM2SR-Yam, and quadrivalent M2SR vaccines were below the detection limit (less than 76 plaque-forming units (PFUs) per lung). In mice immunized with SPG control alone, viral load was detectable (mean 6.86 log PFU / g). These results indicate that monovalent and quadrivalent BM2SR vaccines provide cross-protection and limit the replication of challenge viruses that do not match any vaccine component.

[0136] [Table 5]

[0137] [Example 11] This example demonstrates that bivalent, trivalent, or tetravalent M2SRs containing the BM2SR component offer a favorable safety profile compared to approved nasal influenza vaccines and provide superior protection against influenza viruses not included in the vaccine compared to approved intramuscular inactivated influenza vaccines. Antigenically distinct monovalent BM2SR-Vic and monovalent BM2SR-Yam vaccines provide comparable protection to antigenically matched approved vaccines in protecting mice from lethal influenza B viruses.

[0138] Seven-week-old female BALB / c mice (N=13) were immunized with one of the following vaccines: monovalent BM2SR-Vic, monovalent BM2SR-Yam, tetravalent FGHY-M2SR, FLUMIST® Quadrivalent (AstraZeneca, Wilmington, DE), FLUZONE® Quadrivalent (Sanofi, Bridgewater, NJ), or FLUZONE® High Dose (Sanofi). The strain composition of each vaccine is shown in Table 6. BM2SR, M2SR, and FLUMIST® were administered intranasally, while both FLUZONE® vaccines were administered intramuscularly. Control mice were immunized intranasally with SPG (Sinofi Protein Glycerin). Body weight changes in the mice were observed for 14 days after immunization.

[0139] [Table 6]

[0140] The obtained weight loss data is shown in Figure 9. Mice immunized with monovalent BM2SR-Vic, monovalent BM2SR-Yam, tetravalent M2SR, FLUZONE® Quadrivalent, FLUZONE® High Dose, or SPG control showed no weight loss over a 14-day period. These data indicate that the BM2SR vaccine has a safety profile comparable to that of approved influenza vaccines.

[0141] Mice were immunized with a booster immunization 28 days after primary immunization. The booster immunization consisted of the same vaccine used to immunize the mice during primary immunization. Serum samples were collected weekly after primary and booster immunization, and pooled serum IgG titers for each vaccine component were measured by ELISA. The obtained anti-influenza B-Vic HA serum IgG ELISA titer data is shown in Figure 10A. The obtained anti-influenza B-Yam HA serum IgG ELISA titer data is shown in Figure 10B. The obtained anti-influenza A / H1 HA serum IgG ELISA titer data is shown in Figure 10C. The obtained anti-influenza A / H3 HA serum IgG ELISA titer data is shown in Figure 10D. These results indicate that all vaccines were able to increase serum IgG titers for influenza B-Vic HA and B-Yam HA compared to the SPG control, and these increases were comparable among the vaccine formulations. FLUZONE® Quadrivalent and FLUZONE® High Dose reduced serum IgG titers against influenza B HA antigen compared to live vaccines.

[0142] 49 days after initial immunization (21 days after booster immunization), tracheal-pulmonary lavage fluid was obtained from four mice per group, and IgG and IgA titers were measured by ELISA to evaluate the mucosal immune response. The obtained IgG titer data are shown in Figure 11A, and the obtained IgA titer data are shown in Figure 11B. Quadrivalent M2SR and FLUMIST® Quadrivalent induced both IgG and IgA titers for all tested antigens. Mice immunized with monovalent BM2SR-Vic and BM2SR-Yam showed IgG and IgA titers for both B-Vic and B-Yam HA antigens, but not for H1 or H3 HA antigens. In the groups immunized with FLUZONE® Quadrivalent and FLUZONE® High Dose vaccine, IgG titers increased for all four antigens, but no IgA antibody titers were detected for any of the antigens. These data demonstrate that the BM2SR vaccine elicits a mucosal immune response comparable to that of FLUMIST® Quadrivalent, an approved live attenuated influenza vaccine.

[0143] Six weeks after booster immunization, mice were challenged with a lethal dose of influenza B / Malaysia / 2506 / 2004 (Vic). Of the eight mock-immunized mice in the SPG control group, four died from infection within 10 days after challenge, but all vaccine groups survived. Post-challenge body weight changes are shown in Figure 12. Mice receiving monovalent BM2SR, bivalent M2SR, and the approved vaccine remained healthy and did not lose weight, while surviving mock-immunized mice lost approximately 25% of their body weight. In the immunization groups, except for FLUZONE® Quadrivalent, the challenged virus was not detected in the lungs 3 days after challenge (Table 7). BM2SR-Yam provided protection against lethal infection by drifted influenza B / Malaysia Victoria strain. Infectious viruses were not detected in the lungs of mice immunized with BM2SR-Vic or BM2SR-Yam, or in the lungs of any multivalent vaccine containing one or more BM2SR components (Table 7). Infectious viruses were detected in the lungs of naive mock-immunized mice. Viruses were also detected in the nasal turbinates after challenge. As shown in Table 7, the BM2SR groups (monovalent, bivalent, trivalent, or tetravalent) showed more than 2 log less virus in the nasal turbinates than mock-immunized mice, indicating that intranasal-administered vaccines provided better protection against challenge viruses than intramuscular vaccines (FLUZONE® Quadrivalent and High Dose). Furthermore, BM2SR-Yam (monovalent, or formulated with H1N1 or H3N2) provided superior protection against Victoria strain challenge compared to FLUZONE® High Dose (containing Yam) or FLUZONE® Quadrivalent (containing both Vic and Yam).

[0144] These data demonstrate that intranasal administration of the BM2SR vaccine provides better protection against challenge in mice than intramuscular administration of an approved inactivated vaccine.

[0145] [Table 7]

[0146] [Example 12] This example demonstrates that BM2SR virus production is scalable in BM2Vero cells used for production.

[0147] BM2Vero cells (Vero cells that stably express the influenza B BM2 protein) were cultured in OptiVero medium (InVitria, Aurora, CO) in a humidified incubator at 37°C with a 5% CO2 atmosphere. Approximately 242 million cells per lot in 2-4 culture chambers of CELLSTACK® culture chamber-5 chambers (CS5; Corning, Corning, NY) were infected in OptiVero medium with either the BM2SR-Vic virus encoding the HA and NA genes of influenza B / Colorado / 06 / 2017 (Vic) or the BM2SR-Yam virus encoding the HA and NA genes of influenza B / California / 12 / 2015 (Yam) at a MOI of 0.01. After 2-3 days at 35°C and 5% CO2, when the HA titer of the supernatant reached at least 32 HAU / 50μL, the culture medium was collected and cells and cellular debris were removed by slow centrifugation. The supernatant was further clarified by vacuum filtration through a 0.2 μM pore PES membrane and sterilized. The clarified supernatant was then treated with a nonspecific RNA and DNA nuclease (BENZONASE® benzonase, 5 units / mL) at 37°C for 2 hours to digest / hydrolyze any remaining cellular RNA and DNA. The BENZONASE® benzonase digest was then 235 cm³. 2The virus was purified by tangential flow filtration (TFF) using a 300kD MWCO-modified polyethersulfone (mPES) MIDIKROS® hollow fiber filter module (Repligen, Waltham, MA). The digest was concentrated 10 to 20 times. Next, contaminating host cell proteins (HCPs) and residual DNA fragments were removed by diafiltration using SPG with at least 15 column volume. The purified virus in SPG buffer was further concentrated 10 to 100 times by ultracentrifugation at 25,000 rpm through a 25% sucrose phosphate-buffered saline (PBS) cushion. The resulting virus pellet was resuspended in SPG, aliquoted, rapidly frozen in liquid N2, and stored at -80°C.

[0148] The sequence homology of the concentrated and purified BM2SR-Vic and BM2SR-Yam genes was compared with that of the reference sequence in the viral seed stock. Viral RNA extracted from the purified virus was subjected to RT-PCR to generate cDNA, which was then Sanger sequenced. Analysis of ORFs encoded by eight viral segments showed that all segments had 100% nucleotide sequence identity with respect to the reference.

[0149] The infectivity titers of concentrated and purified BM2SR-Vic and BM2SR-Yam viruses were measured by at least three independent measurements using BM2CK cells (MDCK cells that stably express influenza B BM2) to determine the 50% tissue culture infectious dose (TCID). 50 The titer was determined by assay. In this procedure, vaccine samples were serially diluted to replicate BM2CK cells in 96-well plates and cultured at 35°C in a 5% CO2 atmosphere for 4 days. On day 4 post-inoculation, the cell monolayer was visually observed and CPE was recorded. Viral titers were calculated using the Reed and Muench method and TCID. 50The titer was expressed as / mL. Furthermore, to verify the viral titer determined by CPE, HA activity was tested in aliquots of the supernatant from each well. As shown in Table 8, after concentration and purification, the BM2SR virus consistently showed a titer of 10. 8.6 TCID 50 It reached a high titer exceeding / mL.

[0150] [Table 8]

[0151] To confirm that the BM2SR vaccine virus maintained the replication-deficient phenotype after concentration and purification, the presence of replicating virus was evaluated by three consecutive passages of the test sample in MDCK cells, which are tolerant of wild-type influenza virus but not of BM2SR virus. In the first round of infection, the test virus was graded and seeded onto a cell monolayer. The infected cells were then cultured at 35°C in a 5% CO2 atmosphere for 4 days. The supernatant of the infected cells was then transferred to a fresh MDCK monolayer and incubated at 35°C in a 5% CO2 atmosphere for 4 days. In the third and final rounds of passage, this infected cell culture medium was transferred to another fresh MDCK monolayer and incubated at 35°C in a 5% CO2 atmosphere for a further 4 days. MDCK cells were observed for CPE after each 4-day culture at 35°C, and HA activity in the culture supernatant was determined to confirm the presence of progeny virions. For each round of infection, the previous lot's replication-deficient reference virus, B / California / 12 / 2015 BM2SR, was tested as a positive control. Only the culture medium was used as the negative control inoculum. The results showed that the control functioned as expected, and no infectious progeny were detected after inoculation of any of the four test products into normal cells. Therefore, the BM2SR vaccine virus preparations were demonstrated to be non-replicating and unable to replicate.

[0152] The sterility of the vaccine preparations was verified according to procedures based on WHO specifications for medicinal products. The vaccine preparations were inoculated under sterile conditions into three liquid media: Luria-Bertani broth (LB), trypsin-soybean broth (TSB), and thioglycolic acid medium (TGM). The cultures were grown at 37°C (LB and TSB) and ambient temperature (TGM). After 14 days, microbial growth was visually observed. No microbial growth was observed in any of the tested preparations under all growth conditions.

[0153] [Example 13] This embodiment demonstrates that in a ferret model, the BM2SR backbone becomes less toxic but does not detach and does not propagate.

[0154] M2SR viruses are recombinant influenza A viruses that encode the HA and NA genes of, for example, influenza A / Brisbane / 10 / 2007-like A / Uruguay / 716 / 2007 (H3N2) or influenza A / California / 07 / 2009 (H1N1pdm), but do not express the functional M2 protein. BM2SR viruses are recombinant influenza B viruses that encode the HA and NA genes of, for example, B / Brisbane / 60 / 2008 (Victoria) or B / Wisconsin / 01 / 2010 (Yamagata), but do not express the functional BM2 protein. A tetravalent M2SR, also referred to herein as an M2SR Quad, consists of two M2SR and two BM2SR viruses encoding A / H1N1, A / H3N2, B / Victoria, and B / Yamagata HA and NA.

[0155] Animals and Animal Management. A male ferret purchased from Triple F Farms was placed in this study. The animal was approximately 4 months old at the start of the study. The animal was certified by the supplier to be healthy and free of antibodies against infectious diseases. Upon arrival, the animal was housed alone in a suspended wire cage with a slatted bottom, suspended over a tray lined with paper. The animal room and cage were cleaned and disinfected prior to the receipt of the animals according to generally accepted animal management practices and associated standard operating procedures. Certified Teklad Global Ferret Diet #2072 (Teklad Diets, Madison, Wisconsin) and water were freely provided and replaced with fresh ones at least three times a week. The fluorescent lights in the animal room were maintained on a 12-hour light / dark cycle. The room temperature and relative humidity in the animal room were within the limits of each protocol, ranging from 20.0–25.0°C and 30–63%, respectively, during this study.

[0156] Animal quarantine and randomization. Ferrets were isolated for 7 days and observed daily prior to randomization. Based on daily observations indicating that the animals were generally healthy, the ferrets were released from quarantine for randomization and testing. After quarantine, the ferrets were weighed and assigned to treatment groups using a computerized randomization procedure (TOXDATA® version 2.1.E.11 (PDS Pathology Data Systems, Inc., Basel, Switzerland)) based on weight to generate similar group mean values. Within each group, all weights were within 20% of the mean. Animals selected for this study received permanent identification numbers via ear tags and transponders, and were also identified by identification numbers and identification groups via identification cage cards. The assigned identification numbers were unique within this study.

[0157] Pre-procedures for research animals. All animal procedures were performed in a biosafety level 2 facility for animals, following protocols approved by the IIT Laboratory's Animal Experimentation Committee. Prior to vaccination, ferrets were monitored for 3 days, their weight measured, and baseline body temperature established. Temperature readings were recorded daily via a subcutaneously implanted transponder (BioMedic data systems, Seaford, Delaware) in each ferret. Blood was collected before the start of the study, and serum was tested for influenza antibodies. Pre-vaccination serum samples were treated with receptor-destroying enzymes (RDEs) to remove nonspecific inhibitors, then serially diluted and mixed with specified amounts of influenza virus A / California / 07 / 2009 (H1N1pdm), influenza virus A / Switzerland / 9715293 / 2013 (H3N2), influenza virus B / Brisbane / 60 / 2008 (Victoria strain), and influenza virus B / Wisconsin / 01 / 2010 (Yamagata strain) with 0.5% turkey erythrocytes or 0.75–1.0% guinea pig erythrocytes. Antibody titer was defined by the lowest serum dilution that induced inhibition of hemagglutination. Only ferrets with HAI (hemagglutination inhibitory) titers less than 40 were considered seronegative and used in this study. Research animals were randomized and divided into groups as outlined below.

[0158] To evaluate the attenuation of the BM2SR vaccine virus, a group of three ferrets were anesthetized on day 0 of the study and administered a single dose of 1 x 10⁻¹⁰⁴⁻¹ 8.2 TCID 50 The B / CO / 06 / 2017(Vic)BM2SR virus was administered intranasally. The control group (3 ferrets) received a single targeted dose of 1x10⁻¹⁶. 7.4 TCID 50The animals were inoculated with wild-type B / Brisbane / 60 / 2008(Vic) virus. After exposure on day 0 of the study, body weight was recorded once daily, and changes in body temperature were monitored 6 hours after exposure and once daily thereafter. Survival checks were recorded twice daily. On day 3 of the study, the animals (3 animals per group) were euthanized, and tissue samples were collected from the following organs: nasal turbinates, trachea, lungs, kidneys, olfactory bulbs, brain, liver, spleen, and intestines. Some of the collected samples were fixed in buffered neutral formalin for histological evaluation, and other parts of the samples were stored below -65°C for viral titer measurement. Tissues collected for titer analysis include: the right nasal turbinate, the upper one-third of the trachea, the right cranial lung lobe (if of sufficient size), or a lung lobe with macroscopic lesions (if only one lobe is affected, that lobe is processed for histology and the viral titer is determined from the other lobes; if multiple lobes are affected, one lobe is collected for viral titer analysis and another for histology), the right kidney, the right olfactory bulb, the right brain, the right lateral lobe of the liver, the right half of the spleen (the end of the spleen visible when the abdominal cavity is opened), the small intestine, and the large intestine.

[0159] 1x10 7.4 TCID 50 One ferret vaccinated with wild-type influenza B / Brisbane / 60 / 2008(Vic) showed an elevated body temperature (2.2°C) on the first day after vaccination, but compared to two other ferrets vaccinated with the wild-type virus, 1x10 8.2 TCID 50 Three ferrets inoculated with the B / CO / 06 / 2017(Vic)BM2SR virus did not develop fever for three days after administration. The ferrets used in this study did not experience a decrease or increase in body weight exceeding 2.8%. All six ferrets were euthanized, and organ viral load was measured by titer. As shown in Table 9, wild-type influenza B / Brisbane / 60 / 2008(Vic) was detected in the upper respiratory tract, including the nasal turbinates and trachea, as well as in the brain. No virus was detected in organs derived from the inoculated ferrets.

[0160] [Table 9]

[0161] To evaluate the potential for BM2SR transmission in a ferret model, naive donor ferrets were given 1 x 10⁻¹⁶ samples. 8.2 TCID 50 Influenza B / CO / 06 / 2017(Vic)BM2SR vaccine at the following dosage, or 1x10 7.4 TCID 50 Control wild-type influenza B / Brisbane / 60 / 2008(Vic) virus was intranasally inoculated at the specified dose. 24 hours later, each donor ferret was placed in the same wire cage (double containment) as one naive ferret (direct contact). Additional naive ferrets (aerosol contact) were placed in a separate adjacent wire cage (single containment) within a transmission chamber, separated from the donor cage at a distance of 10-12 cm. This distance allowed only the passage of air and respiratory droplets to be shared between these ferrets, while preventing aerosol contact and physical contact between infected animals and direct contacts. For 7 days post-inoculation, the ferrets' body temperature, weight, and clinical symptoms were monitored daily. Nasal lavage fluid was collected from all inoculated donor ferrets on days 1, 3, 5, 7, and 9, and from all contact ferrets on days 2, 4, 6, 8, and 10. Nasal lavage fluid and serum were collected from all ferrets on day 14.

[0162] No virus was detected at any point in time in nasal lavage fluids from three BM2SR vaccine-vaccinated virus donor ferrets, or from ferrets that had direct or aerosol contact with them (Figure 13A). Ferrets in the BM2SR vaccine-vaccinated virus group did not exhibit fever, weight loss, or clinical symptoms. Wild-type influenza B / Bris / 60 / 2008 was detected in nasal lavage fluids from wild-type virus donor ferrets 1, 3, and 5 days after vaccination (Figure 13B). Although none of the ferrets showed changes in body temperature, weight, or clinical symptoms, all ferrets that had direct or aerosol contact with the wild-type virus donor ferrets were infected and shed the virus.

[0163] These data indicate that the test setup was appropriate and that the replication and transmissibility of a replicable and infectious virus were demonstrated. In the same test setup, the BM2SR virus did not replicate in ferrets and was not detected in animals that had direct or aerosol contact, indicating that the BM2SR virus is highly attenuated, does not replicate in ferrets, and is therefore not transmissible. In the ferret model, BM2SR maintained its attenuation, and ferrets inoculated with BM2SR showed no clinical signs of disease and no viral replication in the respiratory tract or other organs. Furthermore, BM2SR was not detected in animals that had direct or aerosol contact, indicating that BM2SR is not transmissible.

[0164] [Example 14] This embodiment demonstrates that immune responses induced by trivalent M2SR (also known herein as M2SR Tri-Yam) vaccine and quadrivalent M2SR (also known herein as M2SR Quad) vaccine provide protection against drift challenge virus in both naive and preimmune ferret models.

[0165] Animal management, quarantine, randomization, and pre-study procedures were outlined in Example 13.

[0166] [Table 10]

[0167] The experimental design is shown in Table 10. On day 0 of the study, ferrets were either pre-infected with wild-type virus or inoculated intranasally with M2SR Tri-Yam, M2SR Quad, FLUMIST® Quadrivalent (also known herein as FLUMIST® Quad), or PBS, or intramuscularly with FLUZONE® Quadrivalent (also known as FLUZONE® Quad). On day 30 of the study, ferrets were vaccinated intranasally with M2SR Tri-Yam, M2SR Quad, FLUMIST® Quad, and PBS, or intramuscularly with FLUZONE® Quad. The bacterial strain components of each vaccine are shown in Table 6. Serum was collected from all animals on days 8 through 3, 28, 37, 51, and 65 after vaccination / exposure. Peripheral blood mononuclear cells (PBMCs) were collected from all animals on days 28 and 37 of the study. On day 72 of the study, ferrets were challenged with B / Brisbane / 60 / 2008 (B Victoria strain). For 14 days after the challenge, the ferrets' weight, body temperature, and clinical symptoms were monitored. On days 1, 3, 5, and 7 after the challenge (days 73, 75, 77, and 79 of the study), nasal lavage fluid was collected from all challenged ferrets (except those assigned to organ collection), and the samples were stored at -65°C or below for viral titer analysis. On day 75 of the study, four ferrets from each group were euthanized and necropsy performed. Nasal turbinates, trachea, and lungs were collected for viral titer analysis. On day 86 of the study, final blood collection was performed from all remaining animals for serum recovery.

[0168] To demonstrate the functional activity of the antibodies, serum HAI and PRNT (plaque reduction neutralization test) titers were measured for influenza A / Montana / 50 / 2016 (H1N1), A / Singapore / INFIMH-16-0019 / 2016 (H3N2), B / California / 12 / 2015 (Yam), and B / Colorado / 06 / 2017 (Vic) (Figures 14A-D and 15A-D). Vaccination with M2SR Tri-Yam, including BM2SR-Yam, induced anti-influenza HAI and neutralizing antibodies against all vaccine components in naive animals. M2SR Tri-Yam induced inter-strain influenza B antibody production (against B Victoria) in naive animals that received booster immunization after a second dose. Animals that received a single or double dose of M2SR Quad produced HAI and neutralizing antibodies against all four strain components. Furthermore, the level of antibody production from M2SR Quad was comparable to that of M2SR Tri-Yam, indicating that the addition of the second BM2SR component did not affect vaccine performance and that the M2SR Quad formulation did not exhibit strain interference (Figures 14A-D and 15A-D).

[0169] M2SR Quad and FLUMIST® Quad each induced HAI and neutralizing antibodies in naive animals, but FLUZONE® Quad vaccine failed to induce corresponding antibodies even after two consecutive doses. However, pre-immunized animals vaccinated with FLUMIST® Quad produced lower levels of HAI and neutralizing antibodies against several strains compared to M2SR Quad. Antibody production was also inhibited in animals vaccinated with FLUMIST® Quad twice compared to animals vaccinated with M2SR Quad twice (Figures 14A-D and 15A-D). These results indicate that M2SR Quad is not affected by pre-existing influenza immunity.

[0170] As a marker of the cellular immune response, influenza-specific IFN-γ secretion from whole blood PBMCs was measured using a ferret-specific enzyme immunospot (ELISpot). In contrast to only 6 out of 10 ferrets in the FLUMIST® Quad and only 2 out of 10 ferrets in the FLUZONE® Quad, 9 out of 10 ferrets in the M2SR Quad-vaccinated pre-immunized animals showed an increased cellular immune response after H3N2 influenza virus stimulation (Figure 18A). Similar responses were observed after stimulation with H1N1 HA peptide, with all 10 animals in the M2SR Quad showing an increased cellular immune response, but the frequency of increase was much lower in the FLUMIST® Quad and FLUZONE® Quad (6 out of 10 and 2 out of 10, respectively, Figure 18B). These results indicate that M2SR induces a strong T-cell response in addition to an antibody response.

[0171] Following the influenza B-Vic challenge, naive animals treated with mock treatment showed a temporary weight loss of approximately 5-6% and an increase in body temperature. Naive ferrets that received a single dose of FLUZONE® Quad showed a temporary weight loss of approximately 4-5%, similar to animals treated with mock treatment, but the M2SR Tri-Yam and M2SR Quad groups did not show weight loss. Among the pre-immune vaccine groups, FLUZONE® Quad ferrets showed similar weight loss and an increase in body temperature as the mock group, while the Quad M2SR group did not show weight loss or an increase in body temperature (Figure 19).

[0172] Nasal lavage fluid was collected on days 1, 3, 5, and 7 after the challenge, and TCID was analyzed in MDCK cells. 50Titer was measured by assay (Figure 17). M2SR Tri-Yam provided inter-strain influenza B protection in naive ferrets, showing a decrease in viral titer on all sampling days, reaching undetectable levels in control ferrets. Viral titers in naive ferrets administered M2SR Quad were comparable to or slightly lower than those of M2SR Tri-Yam, again demonstrating that the M2SR Quad formulation does not interfere. In pre-immunized ferrets, both M2SR Quad and FLUMIST™ Quad rapidly controlled challenge virus replication, which was only detectable at low levels on day 3 post-challenge. A decrease in viral levels in nasal lavage fluid was also observed in ferrets administered two doses of M2SR Quad or FLUMIST™ Quad, but titers in ferrets administered two doses of FLUZONE™ Quad remained high for 5 days post-challenge (Figure 17).

[0173] Respiratory tissue (nasal turbinates, trachea, and lungs) was collected from four ferrets per group on day 3 post-infection, and viral titers were measured by plaque assay using MDCK cells. Influenza B challenge virus was not recovered from the lungs and trachea of ​​any ferret in any of the vaccinated groups. M2SR Tri-Yam demonstrated inter-lineage influenza B protection in naive ferrets, particularly a decrease in viral titer in the nasal turbinates after two consecutive doses. Viral titers were further reduced in naive ferrets treated with M2SR Quad. In pre-immunized ferrets, no viral titer was detected on day 3 post-challenge as a result of vaccination with M2SR and FLUMIST® Quad. Interestingly, the high neutralizing antibody titers observed in HAI and PRNT serological analysis of pre-immunized ferrets vaccinated with FLUZONE® Quad did not inhibit challenge virus replication in the nasal turbinates. Furthermore, no viral titer was detected in ferrets administered M2SR Quad or FLUMIST® Quad twice, while ferrets administered FLUZONE® Quad twice were not protected.

[0174] This example demonstrates that intranasal administration of the M2SR Tri-Yam or M2SR Quad formulations is not associated with vaccine-related adverse events (e.g., elevated body temperature, weight loss, or clinical signs) in ferret models. The M2SR Tri-Yam vaccine induced the production of interstrain influenza B antibodies and post-challenge protection in naive animals. In M2SR Quad ferrets, high HAI and neutralization responses to all four strain components were maintained, indicating no interference with the M2SR multivalent formulation. Furthermore, unlike the approved vaccine FLUMIST® Quad, antibody production for M2SR Quad did not decrease in pre-immunized animals. The cellular immune response after influenza-specific stimulation was higher and more frequent in ferrets vaccinated with M2SR Quad, further distinguishing it from the currently approved vaccines FLUMIST® Quad and FLUZONE® Quad.

[0175] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as those contained herein, provided that each reference is specifically and individually indicated as being incorporated by reference.

[0176] In the context describing the present invention (particularly in the context of the following claims), the terms “a” and “an,” as well as “the” and “at least one” and similar reference subjects, should be interpreted as encompassing both singular and plural forms unless otherwise clearly indicated herein. The use of the term “at least one” followed by a list of one or more items (e.g., “at least one of A and B”) should be interpreted as meaning one item selected from the listed items (A or B), or any combination of two or more listed items (A and B). The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., “including, but not limited to”) unless otherwise indicated herein. Enumerations of value ranges herein are merely intended to serve as a concise way of referring individually to the individual values ​​that fall within the range, unless otherwise indicated herein, and the individual values ​​are incorporated herein as if they were individually enumerated. All methods described herein may be carried out in any suitable order, unless otherwise indicated herein or unless it is clearly inconsistent with the context. Any use of any examples or illustrative words provided herein (e.g., "etc.") is intended solely to better illustrate the invention and, unless otherwise claimed, does not limit the scope of the invention. The language of the specification should not be construed as indicating that any unclaimed element is essential for carrying out the invention.

[0177] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. By reading the foregoing description, variations of these preferred embodiments may become apparent to those skilled in the art. The inventors expect that those skilled in the art will appropriately adopt such variations, and they have in mind that the invention may be carried out in ways other than those specifically described herein. Accordingly, the invention encompasses all modifications and equivalents of the subject matter described in the claims appended herein, as permitted by applicable law. Furthermore, unless otherwise indicated herein, or unless clearly inconsistent with the context, any combination of all possible variations of the above elements is encompassed within the invention.

[0178] The sequencing rules are based on DNA that references four nucleotides: adenine (a), guanine (G), cytosine (C), and thymine (T). When referring to RNA or influenza viruses, T represents uracil (U).

Claims

1. A type B influenza virus comprising PA, NP, and NS gene segments, (a) The PA gene segment encodes the PA protein having the amino acid sequence of SEQ ID NO: 15; (b) The NP gene segment encodes an NP protein having the amino acid sequence of SEQ ID NO: 16; (c) The NS gene segment encodes the NS1 protein having the amino acid sequence of SEQ ID NO: 17, an influenza B virus.

2. The influenza B virus according to claim 1, wherein the PA gene segment has the nucleotide sequence of SEQ ID NO:

10.

3. The influenza B virus according to claim 1 or 2, wherein the NP gene segment has the nucleotide sequence of SEQ ID NO:

11.

4. The influenza B virus according to any one of claims 1 to 3, wherein the NS gene segment has the nucleotide sequence of SEQ ID NO:

12.

5. (a) serine at position 40, asparagine at position 161, and threonine at position 204 in the NP protein, or (b) glutamine at position 176 in the NS protein is conserved after at least 10 consecutive passages in the Vero cell line, according to any one of claims 1 to 4.

6. (a) serine at position 40, asparagine at position 161, and threonine at position 204 in the NP protein, or (b) glutamine at position 176 in the NS protein are preserved even after at least 10 consecutive passages in a Vero cell line that stably expresses the BM2 ion channel protein of influenza B virus, according to any one of claims 1 to 5.

7. The influenza B virus according to any one of claims 1 to 6, wherein the influenza B virus is a recombinant influenza virus.

8. The influenza B virus according to any one of claims 1 to 7, further comprising a PB gene segment.

9. The influenza B virus according to any one of claims 1 to 8, further comprising an NA gene segment and an HA gene segment.

10. The influenza B virus according to claim 9, wherein the HA gene segment encodes an HA protein having an amino acid sequence containing at least one amino acid mutation within HA2.

11. The influenza B virus according to claim 10, wherein at least one amino acid mutation in HA2 is glutamic acid at position 61.

12. The influenza B virus according to claim 10, wherein at least one amino acid mutation in HA2 is glutamic acid at position 112.

13. The influenza B virus according to any one of claims 1 to 12, wherein the PA, NP, and NS gene segments are derived from a single influenza strain.

14. The influenza B virus according to claim 13, wherein the HA gene segment is derived from an influenza strain different from the single influenza strain from which the PA, NP, and NS gene segments are derived.

15. The influenza B virus according to claim 13 or 14, wherein the NA gene segment is derived from an influenza strain different from the single influenza strain from which the PA, NP, and NS gene segments are derived.

16. The influenza B virus according to any one of claims 1 to 15, wherein the influenza B virus does not encode a functional BM2 protein.

17. The influenza B virus according to any one of claims 1 to 16, wherein the influenza B virus is capable of replicating in human cells.

18. The influenza B virus according to any one of claims 1 to 17, wherein, under the same conditions, the proliferation of the influenza B virus in Vero cells is enhanced compared to an influenza B virus that is identical except for (a) serine at position 40, asparagine at position 161, and threonine at position 204 in the NP protein, and (b) glutamine at position 176 in the NS protein.

19. A pharmaceutical preparation comprising the influenza B virus described in any one of claims 1 to 18.

20. The pharmaceutical preparation according to claim 19, wherein the pharmaceutical preparation is a vaccine.

21. The pharmaceutical formulation according to claim 20, wherein the vaccine is formulated as a monovalent vaccine.

22. The pharmaceutical formulation according to claim 20, wherein the vaccine is formulated as a bivalent vaccine.

23. The pharmaceutical formulation according to claim 20, wherein the vaccine is formulated as a trivalent vaccine.

24. The pharmaceutical formulation according to claim 20, wherein the vaccine is formulated as a quadrivalent vaccine.

25. A pharmaceutical preparation according to any one of claims 19 to 24, for use in inducing an immune response to the influenza B virus in mammals.

26. The pharmaceutical preparation according to claim 25, wherein the mammal is a human.

27. A method for producing influenza B virus according to any one of claims 1 to 18, comprising serially passage the influenza B virus in a Vero cell line.

Images