Influenza virus backbone

Abstract

The present invention provides influenza viruses that exhibit enhanced growth in Vero cells. The influenza viruses include PB1, PB2, PA, NP, and NS gene segments that encode proteins having amino acid sequences that include selected amino acids. Optionally, at least one of the PB1, PB2, and PA gene segments includes a promoter cytosine to uracil mutation at nucleotide position 4. The present invention also provides pharmaceutical formulations containing the influenza viruses, methods for inducing an immune response in a mammal by administering the influenza viruses to a mammal, and methods for producing influenza viruses.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 858,737, filed June 7, 2019, which is incorporated by reference in its entirety.

[0002] Incorporation by Reference of Electronically Filed Materials The computer readable nucleotide / amino acid sequence listing, filed concurrently herewith and identified as follows, is hereby incorporated by reference in its entirety: One 73,600 byte ASCII (text) file entitled "749488SequenceListing_ST25.txt", created on June 2, 2020. [Background technology]

[0003] Background of the Invention Influenza, or "flu," is a highly contagious viral infection that kills hundreds of thousands of people worldwide each year. There are four types of influenza viruses (i.e., A, B, C, and D) classified based on their core proteins, but seasonal epidemics are mostly caused by circulating influenza A and B viruses.

[0004] Vaccines are the best way to prevent influenza, but influenza viruses are subject to antigenic drift and shift, requiring frequent reformulation of influenza vaccines. Furthermore, influenza viruses, especially influenza A, generally exhibit high rates of mutation and evolution, which can lead to mismatches between influenza vaccine strains and circulating strains, resulting in limited vaccine efficacy. However, if the circulating influenza virus is sufficiently matched to the influenza vaccine, vaccination can reduce the risk of influenza-related illness by 40% to 60% in the overall population. Therefore, researchers have been exploring vaccines that can induce cross-protective immunity across various influenza subtypes. One such example is a vaccine containing a live-attenuated influenza virus that does not express a functional M2 protein (e.g., the M2SR vaccine).

[0005] Influenza vaccines are preferably grown in Madin-Darby canine kidney (MDCK) cells and African green monkey (Vero) cells because mammalian cell culture offers advantages over egg-based production. These advantages include reduced costs, shorter production times, and a reduced risk of antigenic mutation in the virus. For example, M2SR vaccines are grown in Vero cells that stably express the M2 protein. However, vaccine production in cell culture often results in undesirable yields. Furthermore, MDCK cells are generally more permissive than Vero cells, making vaccine production in Vero cells relatively inefficient.

[0006] Modifications to six internal gene segments of the viral backbone, namely PB1, PB2, PA, NP, M, and NS, have been shown to enhance vaccine production. For example, the high-yield vaccine backbone "PR8-HY" developed and described in Ping et al., Nature Communications, 6:8148 (2015), contains specific amino acid mutations in the PB1, PB2, PA, NP, and NS1 proteins and was expected to improve the titer of pandemic and seasonal influenza vaccines in both cell culture and egg culture systems. However, these modifications described in the art were not effective in increasing virus growth in Vero cells, especially under favorable manufacturing conditions. Therefore, there is a need to enhance virus growth in Vero cells so that vaccines such as the M2SR vaccine can be produced more efficiently and effectively. Summary of the Invention

[0007] Summary of the Invention The present invention provides influenza viruses with enhanced growth in Vero cells. The influenza viruses include gene segments encoding proteins, e.g., PB1, PB2, PA, NP, and NS1 proteins, having amino acid sequences containing selected amino acids. For example, the PB1 protein contains a leucine at position 40 and a tryptophan at position 180, and at least one of an asparagine at position 464 or a serine at position 607. The PB2 protein contains a valine at position 504, and optionally contains an isoleucine at position 467 and a valine at position 529. The PA protein contains a lysine at position 401. The NP protein contains a leucine at position 116, and at least one of a lysine at position 294 or an arginine at position 311. The NS1 protein contains a proline at position 30 and a lysine at position 118. Furthermore, at least one of the PB1, PB2, and PA gene segments optionally contains a cytosine to uracil mutation at the promoter nucleotide position 4.

[0008] The invention also provides pharmaceutical formulations comprising the influenza viruses, methods of inducing an immune response in a mammal comprising administering the influenza viruses to a mammal, and methods of producing influenza viruses. [Brief explanation of the drawings]

[0009] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] FIG. 1A is a graph of virus titer (log TCID / ml) versus time (days post-infection) showing the growth curve in Vero cells for A / Massachusetts / 15 / 2013 M2SR virus (i.e., M2SR-MA15V virus) containing a Vero-adapted HA protein and comprising a UW-PR8 ("HG") and PR8-HY ("HY") backbone. [Figure 1B] FIG. 1B is a graph of virus titer (TCID50 / ml) versus time (days post-infection) showing the growth curve in Vero cells for A / Brisbane / 10 / 2007 M2SR virus (i.e., BrislO M2SR) containing HG and HY backbones. [Figure 1C] FIG. 1C is a graph of HA titer (HA / 50 μl) versus time (days post-infection) in Vero cells for Bris10 M2SR-HG and Bris10 M2SR-HY viruses. [Figure 2] Figure 2 is a graph of virus titer (log TCID / ml) versus time (days post-infection) showing the growth curves in Vero cells for FGHY1-M2SR-MA15V and FGHY2-M2SR-CA07 viruses compared with the growth curves of HY-M2SR-MA15V and HY-M2SR-CA07 viruses. [Figure 3] Figure 3 is a graph of virus titer (TCID / ml) versus time (days post-infection) showing the growth curves in Vero cells for FGHY1-M2SR-MA15V and FGHY2-M2SR-MA15V viruses compared to the growth curves of HG-M2SR-MA15V and HY-M2SR-MA15V viruses. [Figure 4A] Figure 4A is a graph of virus titer (TCID50 / ml) versus time (days post-infection) showing growth curves in Vero cells for FGHY1-M2SR-Bris10 and FGHY2-M2SR-Bris10 viruses compared with growth curves for HY-M2SR-Bris10 and HG-M2SR-Bris10 viruses. [Figure 4B] Figure 4B is a graph of HA titer (HA / 50 μl) versus time (days post-infection) in Vero cells for FGHY1-M2SR-Bris10 and FGHY2-M2SR-Bris10 viruses compared with HY-M2SR-Bris10 and HG-M2SR-Bris10 viruses. [Figure 5A] Figure 5A is a graph of virus titer (TCID / ml) versus time (days post-infection) showing the growth curve in Vero cells for the A / Michigan / 45 / 2015 M2SR virus, which contains a Vero-adapted HA protein and an FGHY1 backbone (i.e., the FGHY1-M2SR-MI45V virus), compared to the growth curves for the FGHY1-M2SR-MI45, HY-M2SR-MI45, and HG-M2SR-MI45 viruses. [Figure 5B] Figure 5B is a graph of virus titer (log TCID / ml) versus time (days post-infection) showing the growth curve in Vero cells for the A / Hong Kong / 4801 / 2014 M2SR virus containing the FGHY1 backbone (i.e., the FGHY1-M2SR-HK4801 virus) compared to the growth curves for the HY-M2SR-HK4801 and HG-M2SR-HK4801 viruses. [Figure 5C] Figure 5C is a graph of virus titer (log TCID / ml) versus time (days post-infection) showing the growth curve in Vero cells for the A / Vietnam / 1203 / 04 M2SR virus containing the FGHY1 backbone (i.e., the FGHY1-M2SR-avVN1203 virus) compared to the growth curves for the HY-M2SR-avVN1203 and HG-M2SR-avVN1203 viruses. [Figure 6] FIG. 6 is a graph showing viral titers (log TCID / ml) of seasonal and pandemic influenza subtype M2SR viruses containing HG (i.e., UW-PR8), HY (i.e., PR8-HY), and FGHY1 backbones in M2CK and M2VeroA cells. [Figure 7A] FIG. 7A is a table showing HA mutations in Vero-adapted H1N1 viruses. [Figure 7B] FIG. 7B is a table showing HA mutations in Vero-adapted H3N2 viruses. [Figure 7C] Figure 7C is a graph of virus titer (log10 M2CK TCID50 / ml) versus time (days post-infection) showing growth curves for A / Singapore / INFIMH-16-0019 / 2016 M2SR viruses (i.e., M2SR Sing2016 viruses) containing various Vero-adapted HA mutations. [Figure 8A] Figure 8A shows NP antigen production (i.e., NP expression levels) in human cell lines (i.e., A549, MRC-5, and CALU) on days 1 and 2 post-infection for viruses with various backbones (i.e., FGHY1, UW-PR8 ("HG"), and IVR-147) and containing HA and NA from A / Brisbane / 10 / 2007 (i.e., Brisl0 virus). [Figure 8B] Figure 8B shows NP antigen production (i.e., NP expression levels) in human cell lines (i.e., A549, MRC-5, and CALU) on days 1 and 2 post-infection for viruses with various backbones (i.e., FGHY1, UW-PR8 ("HG")) and containing HA and NA from A / Singapore / INFIMH-16-0019 / 2016 (i.e., Sing2016 virus). [Figure 9A] Figure 9A shows the ratio of cells with high NP expression levels to cells with low NP expression levels in various cell lines after infection with Bris10 viruses having different backbones (i.e., FGHY1, UW-PR8 ("HG"), and IVR-147) on day 1 post-infection. [Figure 9B] Figure 9B shows the ratio of cells with high NP expression levels to cells with low NP expression levels in various cell lines after infection with Sing10 viruses having different backbones (i.e., FGHY1, UW-PR8 ("HG")) on day 1 post-infection. [Figure 10] 10 is a graph showing the weight change rate versus time (days after inoculation) of mice after inoculation with the FGHY1-M2SR mutant. The mean % weight of the group and the standard error of the mean are shown. [Figure 11] FIG. 11 is a graph showing anti-H3 HA ELISA IgG titers in the serum of ferrets immunized with 1×10 8 TCID 50 H3N2 FGHY1-M2SR, 1×10 9 H3N2 FGHY1-M2SR, or SPG (control) on days 14, 30, and 49 of the study. [Figure 12] FIG. 12 is a graph showing anti-H3 HAI titers in the serum of ferrets immunized with 1×10 8 TCID 50 H3N2 FGHY1-M2SR, 1×10 9 H3N2 FGHY1-M2SR, or SPG (control) on days 14, 30, and 49 of the study. [Figure 13] FIG. 13 is a graph showing anti-H3 PRNT50 titers in the serum of ferrets immunized with 1×10 8 TCID 50 H3N2 FGHY1-M2SR, 1×10 9 H3N2 FGHY1-M2SR, or SPG (control) on days 14, 30, and 49 of the study. [Figure 14A] Figure 14A is a graph showing anti-H1 HA titers versus time (days post-immunization) in serum from mice immunized with monovalent H1N1 FGHY1-M2SR, monovalent H3N2 FGHY1-M2SR, bivalent FGHY1-M2SR, trivalent FGHY1-M2SR+BM2SR-Vic, trivalent FGHY1-M2SR+BM2SR-Yam, tetravalent, or SPG (control). [Figure 14B]Figure 14B is a graph showing anti-H3 HA titers versus time (days post-immunization) in serum from mice immunized with monovalent H1N1 FGHY1-M2SR, monovalent H3N2 FGHY1-M2SR, bivalent FGHY1-M2SR, trivalent FGHY1-M2SR+BM2SR-Vic, trivalent FGHY1-M2SR+BM2SR-Yam, tetravalent, or SPG (control). [Figure 15] Figure 15 is a graph showing the percentage change in mouse weight versus time (days post-challenge) following influenza A / California / 07 / 2009 (H1N1) virus inoculation of mice immunized with monovalent H1N1 FGHY1-M2SR, monovalent H3N2 FGHY1-M2SR, tetravalent FGHY1-M2SR, or SPG (control) versus time (days post-challenge). [Figure 16] FIG. 16 is a graph showing the percent weight change versus time (days post-vaccination) of mice after immunization with monovalent H3N2 FGHY1-M2SR, tetravalent FGHY1-M2SR, FluMist Quadrivalent, Fluzone Quadrivalent, Fluzone High Dose, or SPG (control). [Figure 17A] Figure 17A is a graph showing anti-influenza A / H1 HA serum IgG ELISA titers versus time (days post-vaccination) in the serum of mice immunized with monovalent H3N2 FGHY1-M2SR, tetravalent FGHY1-M2SR, FluMist Quadrivalent, Fluzone Quadrivalent, Fluzone High Dose, or SPG (control). [Figure 17B] Figure 17B is a graph showing anti-influenza A / H3 HA serum IgG ELISA titers versus time (days post-vaccination) in sera of mice immunized with monovalent H3N2 FGHY1-M2SR, tetravalent FGHY1-M2SR, FluMist Quadrivalent, Fluzone Quadrivalent, Fluzone High Dose, or SPG (control). [Figure 17C]Figure 17C is a graph showing anti-influenza B / Yam HA serum IgG ELISA titers versus time (days post-vaccination) in sera of mice immunized with monovalent H3N2 FGHY1-M2SR, tetravalent FGHY1-M2SR, FluMist Quadrivalent, Fluzone Quadrivalent, Fluzone High Dose, or SPG (control). [Figure 17D] Figure 17D is a graph showing anti-influenza B / VicHA serum IgG ELISA titers versus time (days post-vaccination) in sera of mice immunized with monovalent H3N2 FGHY1-M2SR, tetravalent FGHY1-M2SR, FluMist Quadrivalent, Fluzone Quadrivalent, Fluzone High Dose, or SPG (control). [Figure 18A] Figure 18A is a graph showing IgG ELISA titers in tracheal-lung lavage fluid from mice immunized with monovalent H3N2 FGHY1-M2SR, tetravalent FGHY1-M2SR, FluMist Quadrivalent, Fluzone Quadrivalent, Fluzone High Dose, or SPG (control) - HA1 test antigen (A / H1, A / H3, B / Yam, or B / Vic). [Figure 18B] Figure 18B is a graph showing anti-influenza HA1 IgA ELISA titers in tracheal-lung lavage fluid from mice immunized with monovalent H3N2 FGHY1-M2SR, tetravalent FGHY1-M2SR, FluMist Quadrivalent, Fluzone Quadrivalent, Fluzone High Dose, or SPG (control) versus HA1 test antigen (MI45, SingEgg, Phuket, or CO / 06). [Figure 19]Figure 19 is a graph showing the percentage change in mouse weight versus time (days post-inoculation) following influenza A / H1N1 inoculation for mice immunized with H3N2 FGHY1-M2SR, tetravalent FGHY1-M2SR, FluMist Quadrivalent, Fluzone Quadrivalent, Fluzone High Dose, or SPG (control). DETAILED DESCRIPTION OF THE INVENTION

[0010] Detailed Description of the Invention The influenza virus of the present invention can be any type of influenza virus. For example, the influenza virus can be any subtype of influenza A. In some embodiments, the influenza virus can be a pandemic influenza A virus (e.g., H5N1). In other embodiments, the influenza virus can be a seasonal influenza A virus (e.g., H1N1 or H3N2). In some embodiments, the influenza virus can be a recombinant influenza virus. As used herein, a recombinant influenza virus (e.g., a reassortant influenza virus) is an influenza virus that contains genetic material (e.g., gene segments) from genetically distinct influenza viruses (e.g., heterologous gene segments). The influenza virus can also be an isolated influenza virus.

[0011] As used herein, the term "gene segment" refers to a nucleotide sequence encoding a viral protein. A gene segment can be represented by a cDNA (complementary DNA) sequence encoding viral RNA (vRNA), i.e., SEQ ID NOs: 1-5, 11, 14, 16, and 18, which encodes a viral protein.

[0012] As used herein, the term "backbone" refers to influenza gene segments that encode the PB1, PB2, PA, NP, NS1 and / or NS2, and M proteins. The gene segments of the present invention encode proteins having selected amino acids.

[0013] As used herein, the term "selected amino acid" refers to a specific amino acid at a specific position in an amino acid sequence. In some embodiments, the selected amino acid is the result of a genetic mutation in a parent amino acid sequence. The parent 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. influenza virus (A) Backbone protein

[0014] The PB1 (polymerase basic protein 1) gene segment of the present invention can encode a protein (i.e., a PB1 protein) containing at least one selected amino acid. In a preferred embodiment, the selected amino acids include leucine at position 40 and tryptophan at position 180. The selected amino acids of the PB1 protein further include at least one of asparagine at position 464 or serine at position 607. The PB1 gene segment can optionally contain a cytosine to uracil mutation at nucleotide position 4 in the promoter.

[0015] The selected amino acid can be obtained by genetic mutation into a parent PB1 sequence, e.g., a sequence identical to the PB1 amino acid sequence of the present invention except for the position corresponding to the selected amino acid. The amino acid at position 464 of the PB1 protein is located in the palm region of the influenza PB1 protein and connects the RNA-dependent RNA polymerase activity domain. In general, the aspartic acid at position 464 is highly conserved among influenza viruses isolated in eggs and MDCK cells. Although the role of this amino acid has not been identified, the observed amino acid change to asparagine (N) at this position may affect the conformation of the PB1 protein, its interaction with host cell factors, and therefore its polymerase activity in Vero cells. Furthermore, because the histidine at position 465 of the PB1 protein interacts with the glutamic acid at position 243 of the PA protein, changing the amino acid at position 464 of PB1 may alter the interaction between PB1 and PA. The function of the amino acid at position 607 of the PB1 protein is also unknown. However, this amino acid is located between the RNA-dependent RNA polymerase domain and the PB2 binding domain, suggesting that it may alter the interaction between PB1 and PB2 and thus affect polymerase activity in Vero cells.

[0016] The PB2 (polymerase basic protein 2) gene segment of the present invention may encode a protein (i.e., a PB2 protein) containing at least one selected amino acid. In a preferred embodiment, the selected amino acids include valine at position 504, optionally isoleucine at position 467, and valine at position 529. The PB2 gene segment may optionally include a cytosine to uracil mutation at nucleotide position 4 in the promoter. The amino acids at positions 467 and 529 of the PB2 protein are located in the PB2-C moiety. Specifically, the amino acid at position 467 is located in the cap-binding region of the PB2 protein, and the amino acid at position 529 is located in the cap-627 linker domain. In some influenza viruses, the PB2 protein binds to the cap structure of host capped RNA and utilizes the cap from the host RNA to generate influenza mRNA. This process is known as "cap snatching." Furthermore, the amino acid at position 627 of PB2 is known to be an important determinant in host range and viral pathogenicity. Therefore, changes in amino acids adjacent to the cap-binding region may affect the efficiency of viral mRNA synthesis.

[0017] The PA (polymerase acidic protein) gene segments of the present invention may encode proteins (i.e., PA proteins) that contain at least one selected amino acid. In a preferred embodiment, the selected amino acid comprises a lysine at position 401. The PA gene segment may optionally contain a cytosine to uracil mutation at nucleotide position 4 of the promoter.

[0018] The NP (nucleoprotein) gene segment of the present invention may encode a protein (i.e., an NP protein) that includes at least one selected amino acid. In a preferred embodiment, the selected amino acid includes at least one of leucine at position 116 and lysine at position 294 or arginine at position 311. Amino acid positions 294 and 311 of the NP protein are located in the body of the NP protein, and they do not function as a nuclear localization signal or a nuclear export signal.

[0019] The NS (nonstructural) gene segments of the present invention may encode proteins (i.e., NS1 and / or NS2 proteins) that contain at least one selected amino acid. In a preferred embodiment, the selected amino acids include proline at position 30 (NS1 protein) and lysine at position 118 (NS1 protein).

[0020] In one embodiment of the present invention, the influenza virus comprises a PB1 gene segment encoding a protein having selected amino acids at positions 40, 180, and 464 (i.e., a PB1 protein) i.e., a leucine at position 40, a tryptophan at position 180, and an asparagine at position 464. The PB1 gene segment may have a nucleotide sequence represented by SEQ ID NO:2. The PB1 gene segment may encode a protein having the amino acid sequence of SEQ ID NO:7 (i.e., a PB1 protein). In another aspect of the embodiment, the influenza virus may comprise a PB2 gene segment encoding a protein having a selected amino acid at position 504 (i.e., a valine at position 504) (i.e., a PB2 protein). The PB2 gene segment may have a nucleotide sequence represented by SEQ ID NO:14. The PB2 gene segment may encode a protein having the amino acid sequence of SEQ ID NO:15 (i.e., a PB2 protein). The NP gene segment of this embodiment may encode a protein having selected amino acids at positions 116 and 294, i.e., a leucine at position 116 and a lysine at position 294 (i.e., the NP protein). The NP gene segment may have a nucleotide sequence represented by SEQ ID NO: 1. The NP gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 6 (i.e., the NP protein). The PA and NS gene segments of this embodiment may also encode proteins (i.e., the PA protein and the NS1 and / or NS2 proteins) containing selected amino acids at positions 401 (PA protein), 30 (NS1 protein), and 118 (NS1 protein), i.e., a lysine at position 401 (PA protein), a proline at position 30 (NS1 protein), and a lysine at position 118 (NS1 protein). The PA gene segment may have a nucleotide sequence represented by SEQ ID NO: 16. The PA gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 17 (i.e., the PA protein). The NS gene segment can have the nucleotide sequence represented by SEQ ID NO: 18. The NS gene segment can encode a protein having the amino acid sequence of SEQ ID NO: 19 (i.e., the NS1 protein).The NS gene segment can encode a protein having the amino acid sequence of SEQ ID NO: 20 (i.e., the NS2 protein). The PB1, PB2, and PA gene segments of this embodiment can also include a cytosine to uracil mutation at nucleotide position 4 in the promoter.

[0021] In another embodiment of the invention, an influenza virus comprises a PB1 gene segment encoding a protein having selected amino acids at positions 40, 180, and 607, i.e., leucine at position 40, tryptophan at position 180, and serine at position 607 (i.e., a PB1 protein). The PB1 gene segment may have a nucleotide sequence represented by SEQ ID NO:4. The PB1 gene segment may encode a protein having the amino acid sequence of SEQ ID NO:9 (i.e., a PB1 protein). In another aspect of the embodiment, an influenza virus may comprise a PB2 gene segment encoding a protein having selected amino acids at positions 504, 467, and 529, i.e., a valine at position 504, an isoleucine at position 467, and a valine at position 529 (i.e., a PB2 protein). The PB2 gene segment may have a nucleotide sequence represented by SEQ ID NO:5. The PB2 gene segment may encode a protein having the amino acid sequence of SEQ ID NO:10 (i.e., a PB2 protein). The NP gene segment of this embodiment may encode a protein having selected amino acids at positions 116 and 311, i.e., leucine at position 116 and arginine at position 311 (i.e., the NP protein). The NP gene segment may have a nucleotide sequence represented by SEQ ID NO:3. The NP gene segment may encode a protein having the amino acid sequence of SEQ ID NO:8 (i.e., the NP protein). The PA and NS gene segments may also encode proteins containing selected amino acids at positions 401 (PA protein), 30 (NS1 protein), and 118 (NS1 protein), i.e., lysine at position 401 (PA protein), proline at position 30 (NS1 protein), and lysine at position 118 (NS1 protein) (i.e., the PA protein and the NS1 and / or NS2 proteins). The PA gene segment may have a nucleotide sequence represented by SEQ ID NO:16. The PA gene segment may encode a protein having the amino acid sequence of SEQ ID NO:17 (i.e., the PA protein). The NS gene segment may have a nucleotide sequence represented by SEQ ID NO:18.The NS gene segment can encode a protein having the amino acid sequence of SEQ ID NO: 19 (i.e., an NS1 protein). The NS gene segment can encode a protein having the amino acid sequence of SEQ ID NO: 20 (i.e., an NS2 protein). The PB1, PB2, and PA gene segments of this embodiment can also include a cytosine to uracil mutation at nucleotide position 4 in the promoter.

[0022] The selected amino acids of the embodiments confer enhanced growth characteristics to the influenza virus, particularly in most proteins of the backbone, compared to an identical influenza virus except for the absence of the selected amino acid under identical conditions. For example, the influenza viruses of the invention exhibit enhanced growth in Vero cells.

[0023] Influenza viruses of the present invention may also contain an M (membrane protein) gene segment. In one embodiment of the present invention, the M gene segment may be a mutated gene segment from influenza A such that the virus lacks expression of a functional M2 protein. Such viruses are referred to herein as "M2SR" viruses. M2SR viruses are single-replication influenza viruses. The M gene segment of an M2SR virus may be represented by SEQ ID NO: 11. The M gene segment may encode a protein, e.g., a truncated M2 protein having the amino acid sequence of SEQ ID NO: 12. M2SR viruses may be propagated in Vero cells stably expressing the M2 protein (i.e., M2VeroA cells) and capable of multi-cycle replication. High yields in Vero cells are not dependent on mutations in the M gene segment. Thus, influenza viruses of the present invention may contain an M gene segment encoding a functional M2 protein. (B) Surface proteins

[0024] In a further embodiment of the invention, the influenza virus comprises NA (neuraminidase) and HA (hemagglutinin) gene segments. In one embodiment of the invention, the HA gene segment can encode an HA protein having an amino acid sequence that includes at least one selected amino acid (e.g., amino acid mutation) in the HA1 subunit of the protein and / or at least one selected amino acid (e.g., amino acid mutation) in the HA2 subunit of the protein. For example, the at least one amino acid mutation in the HA2 subunit can be an asparagine at position 107. Such mutations can also contribute to enhanced viral growth during production.

[0025] In one embodiment of the present invention, the PB1, PB2, PA, NP, and NS gene segments are derived from a single influenza strain. The HA gene segment can 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 can 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. Thus, the influenza viruses of the present invention can be pandemic influenza viruses (e.g., H5N1, H7N9) or seasonal influenza viruses (e.g., H1N1, H3N2, influenza B). (C) Properties of influenza viruses

[0026] The influenza virus backbone of the present invention confers high growth characteristics to the influenza virus, particularly in Vero cells, regardless of the influenza virus type (e.g., seasonal or pandemic influenza virus). The influenza viruses of the present invention exhibit high yields even in manufacturing processes using a low multiplicity of infection (MOI) (e.g., 0.001). MOI refers to the average number of pathogens (e.g., viruses) per infected target (e.g., cell). A lower MOI is used when multiple infection cycles are required (e.g., producing a viral vaccine). Current good manufacturing practice regulations, enforced by the FDA, generally require the use of the lowest MOI that still produces a high virus yield. This is because master seed stocks are costly and toxicity due to non-infectious particles and excess cellular proteins can reduce virus production.

[0027] In further embodiments of the present invention, the influenza virus is genetically stable such that selected amino acids in the backbone proteins, particularly the PB1, PB2, PA, NP, and NS1 proteins, are highly conserved even when propagated 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, and NP 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 more than ten serial passages in a Vero cell line. In one embodiment, the Vero cell line may comprise Vero cells stably expressing the M2 ion channel protein of influenza A virus (i.e., M2VeroA cells). In another embodiment of the present invention, the Vero cell line may comprise Vero cells stably expressing the BM2 ion channel protein of influenza B virus (i.e., BM2Vero cells). BM2 is believed to be the functional counterpart of influenza A virus M2. In such embodiments, the selected amino acids may be conserved even when the influenza virus is an influenza A virus.

[0028] Genetically modified Vero cells (i.e., those expressing influenza M2 or BM2 proteins) behave like normal Vero cells and support the growth of influenza A or B viruses comparable to normal Vero cells. Viral titers of M2SR viruses in M2VeroA cells are comparable to replicating influenza viruses expressing functional M2 in unmodified Vero cell lines. Furthermore, viral titers for BM2SR viruses (i.e., influenza viruses containing a mutant M gene segment from influenza B and thus not expressing functional BM2 protein) in BM2Vero cells are comparable to replicating influenza viruses expressing functional BM2 in unmodified Vero cell lines. Thus, M2SR and BM2SR viruses behave like replicating influenza viruses in M2VeroA and BM2Vero cell lines.

[0029] In one embodiment of the invention, the influenza virus is capable of replicating in human cells. How to make influenza viruses

[0030] Also provided herein are methods for producing influenza viruses, wherein the produced influenza viruses comprise PB1, PB2, PA, NP, and NS gene segments that express proteins having selected amino acids, i.e., recombinant influenza viruses of the invention, as disclosed herein.

[0031] In one embodiment of the methods of the invention, a method for producing a recombinant influenza virus includes serially passage a recombinant influenza virus (e.g., a first influenza virus) in Vero cells to produce an engineered influenza virus (e.g., a second influenza virus). The first influenza virus can include PB1, PB2, PA, NP, and NS gene segments that express proteins, i.e., PB1, PB2, PA, NP, and NS1 proteins, having selected amino acids, as described for the influenza viruses of the invention. For example, the first influenza virus can include a PB1 gene segment encoding a protein comprising leucine at position 40, tryptophan at position 180, aspartic acid at position 464, and proline at position 607 (i.e., the PB1 protein). The PB2 gene segment of the first influenza virus can encode a protein comprising methionine at position 467, valine at position 504, and isoleucine at position 529 (i.e., the PB2 protein). The PA gene segment of the first influenza virus may encode a protein comprising a lysine at position 401 (i.e., a PA protein). The NP gene segment of the first influenza virus may encode a protein comprising a leucine at position 116, a glutamic acid at position 294, and a glutamine at position 311 (i.e., an NP protein). The NS gene segment of the first influenza virus may encode a protein comprising a proline at position 30 and a lysine at position 118 (i.e., an NS1 protein). The PB1, PB2, and PA gene segments of the first influenza virus optionally comprise a uracil at nucleotide position 4. In one embodiment of the present invention, the second influenza virus (e.g., an engineered influenza virus) is generated after serial passage of the first influenza virus in Vero cells at least four or at least five times.

[0032] Influenza viruses of the present invention may be produced using standard virus rescue techniques. For example, in one embodiment of the present invention, one or more plasmids (i.e., pPolI plasmids) into which cDNAs for each of the eight viral gene segments (i.e., PB1, PB2, PA, NP, M, NS, HA, and NA) have been cloned, with each cDNA sequence flanked by an RNA polymerase I promoter and RNA polymerase I terminator, are transfected into eukaryotic host cells. Gene segments encoding PB1, PB2, PA, NP, and NS1 and / or NS2 proteins may encode proteins having selected amino acids of the present invention. Gene segments encoding M2 or BM2 proteins may include mutant M2 or BM2 gene segments such that the gene segments do not encode functional M2 or BM2. Host cells may be transfected with one or more expression plasmids encoding viral proteins (e.g., at least one or more of the PA, PB1, PB2, and NP proteins, or at least one or more of the PB1, PB2, PA, NP, M, NS1 and / or NS2, HA, and NA proteins). Subsequently, after transfection of at least one or more plasmids into a host cell, eight influenza vRNAs (i.e., gene segments) are synthesized. 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 viruses of the invention. This plasmid-based reverse genetics system is further detailed in Neumann et al., Proc. Natl. Acad. Sci. USA, 96:9345-9350 (1999). Influenza viruses of the invention may also be produced using other methods known in the art, such as, but not limited to, the ribonucleoprotein (RNP) transfection system, as described in U.S. Pat. No. 9,284,533. Pharmaceutical preparations

[0033] The invention provides pharmaceutical formulations (eg, vaccines or other immunogenic compositions) comprising the influenza viruses of the invention as described herein.

[0034] The pharmaceutical formulation may further comprise at least one pharmaceutically acceptable carrier or excipient. As used herein, "pharmaceutically acceptable carrier or excipient" refers to any component of the pharmaceutical formulation other than the influenza virus of the present invention. The pharmaceutically acceptable carrier or excipient may enhance the efficacy of the influenza virus of the present invention or maintain the stability of the pharmaceutical formulation, preferably without significantly inactivating the influenza virus of the present invention.

[0035] The at least one pharmaceutically acceptable carrier or excipient can be any suitable pharmaceutically acceptable carrier or excipient, many of which are known in the art. Exemplary pharmaceutically acceptable carriers or excipients include components that maintain the pH of the pharmaceutical formulation (e.g., a buffer), adjust tonicity (e.g., a tonicity modifier such as an inorganic salt), improve the stability and / or immunogenicity of a protein (e.g., a virus), improve mucoadhesion, prevent protein aggregation, and / or preserve the pharmaceutical formulation (e.g., a preservative). For example, the pharmaceutically acceptable carrier or excipient can include at least one of an inorganic salt, a surfactant, an amino acid, a polymer or polymeric compound (e.g., a protein, a polysaccharide, or a hydrogel), a chelating agent, a sugar, a polyol, and / or an adjuvant (e.g., any substance that enhances a specific immune response), many of which are known in the art. Because a particular carrier or excipient may serve more than one purpose in a formulation, the following embodiments are not limited to the descriptions set forth herein.

[0036] Any suitable buffer may 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., 1x DPBS), histidine buffer, sodium citrate buffer, and sucrose phosphate glutamate (SPG) buffer. PBS and / or DPBS preparations may comprise, for example, sodium chloride, potassium chloride, potassium phosphate monobasic, and sodium phosphate dibasic, and may optionally further comprise calcium chloride and / or magnesium chloride. In some embodiments, the PBS and / or DPBS preparation comprises 136.9 mM sodium chloride, 2.67 mM potassium chloride, 1.47 mM potassium phosphate monobasic, and 8.1 mM sodium phosphate dibasic, although any suitable PBS and / or DPBS preparation, many of which are known in the art, may be used as a buffer in the pharmaceutical formulation.

[0037] The buffer may be present in the pharmaceutical formulation at any suitable concentration, such as at least 0.1 mM, at least 1 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 120 mM, at least 140 mM, at least 160 mM, at least 180 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 450 mM, or at least 500 mM. Alternatively, or additionally, the buffer may be present in the pharmaceutical formulation at a concentration of 1000 mM or less, 500 mM or less, 450 mM or less, 400 mM or less, 350 mM or less, 300 mM or less, 250 mM or less, 200 mM or less, 180 mM or less, 160 mM or less, 140 mM or less, 120 mM or less, 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, 10 mM or less, or 1 mM or less. The buffer may be present in the pharmaceutical formulation at any concentration within a range bounded by either of the foregoing endpoints. For example, the buffer may be present in the pharmaceutical formulation at a concentration of 0.1 mM to 1000 mM, 0.1 mM to 500 mM, 0.1 mM to 100 mM, 1 mM to 1000 mM, 1 mM to 500 mM, 1 mM to 100 mM, 100 mM to 1000 mM, 100 mM to 500 mM, etc.

[0038] In further embodiments, 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)). The buffer may be present in the pharmaceutical formulation at a percentage concentration of 0.1% or more, 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, or 50% or more. Alternatively, or additionally, the buffer may be present in the pharmaceutical formulation at a percentage concentration of 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less. The buffer may be present in the pharmaceutical formulation at any concentration within a range bounded by any of the aforementioned endpoints. For example, the buffer may be present in the pharmaceutical formulation at a percentage concentration of 0.1% to 60%, 1% to 60%, 10% to 60%, 0.1% to 50%, 1% to 50%, 10% to 50%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 40%, 40% to 50%, etc.

[0039] The buffer can maintain the pH of the pharmaceutical formulation at any suitable pH. For example, the buffer can maintain the pH of the pharmaceutical formulation at a pH of 4 or higher, 4.5 or higher, 5 or higher, 5.5 or higher, 6 or higher, 6.5 or higher, 7 or higher, or 7.5 or higher. Alternatively, or additionally, the buffer can maintain the pH of the pharmaceutical formulation at a pH of 8 or lower, 7.5 or lower, 7 or lower, 6.5 or lower, 6 or lower, 5.5 or lower, 5 or lower, or 4.5 or lower. The buffer can maintain the pH of the pharmaceutical formulation within a pH range bounded by any of the aforementioned endpoints. For example, the buffer can maintain the pH of the pharmaceutical formulation at a pH of 4-8, 4.5-8, 5-8, 5.5-8, 6-8, 6.5-8, 7-8, 7.5-8, 4-7.5, 5-7.5, 6-7.5, 7-7.5, 4-7, 5-7, 6-7, etc.

[0040] Any suitable tonicity modifying agent may be present in the pharmaceutical formulation. In certain embodiments, one or more inorganic salts are present in the pharmaceutical formulation as tonicity modifying agents. The inorganic salt(s) may be at least one of sodium chloride (NaCl), magnesium sulfate (MgSO4), and magnesium chloride (MgCl2). The tonicity modifying agent, e.g., inorganic salt(s), may be present in the pharmaceutical formulation in any suitable amount. The tonicity modifying agent, e.g., inorganic salt(s), may be present in the pharmaceutical formulation at a concentration of 0.1 mM or more, 0.2 mM or more, 0.4 mM or more, 0.6 mM or more, 0.8 mM or more, 1 mM or more, 1.2 mM or more, 1.4 mM or more, 1.6 mM or more, 1.8 mM or more, 2 mM or more, 3 mM or more, 4 mM or more, 5 mM or more, 6 mM or more, 7 mM or more, 8 mM or more, 9 mM or more, 10 mM or more, 20 mM or more, 30 mM or more, 40 mM or more, 50 mM or more, 100 mM or more, 200 mM or more, 300 mM or more, 400 mM or more, 500 mM or more, 600 mM or more, 700 mM or more, 800 mM or more, 900 mM or more, 1000 mM or more, or 1500 mM or more. Alternatively, or in addition, the tonicity modifying agent, e.g., inorganic salt(s), may be present in the pharmaceutical formulation at a concentration of 2000 mM or less, 1500 mM or less, 1000 mM or less, 900 mM or less, 800 mM or less, 700 mM or less, 600 mM or less, 500 mM or less, 450 mM or less, 400 mM or less, 350 mM or less, 300 mM or less, 250 mM or less, 200 mM or less, 150 mM or less, 100 mM or less, 50 mM or less, 4 It may be present at a concentration of 5 mM or less, 40 mM or less, 35 mM or less, 30 mM or less, 25 mM or less, 20 mM or less, 10 mM or less, 9 mM or less, 8 mM or less, 7 mM or less, 6 mM or less, 5 mM or less, 4 mM or less, 3 mM or less, 2 mM or less, 1.8 mM or less, 1.6 mM or less, 1.4 mM or less, 1.2 mM or less, 1 mM or less, 0.8 mM or less, 0.6 mM or less, 0.4 mM or less, or 0.2 mM or less. Tonicity modifying agents, e.g., inorganic salt(s), may be present in the pharmaceutical formulation at any concentration within a range bounded by any of the aforementioned endpoints.For example, the tonicity modifying agent, e.g., inorganic salt(s), may be present in the pharmaceutical formulation at concentrations of 0.1 mM to 2000 mM, 0.1 mM to 1500 mM, 0.1 mM to 1000 mM, 0.1 mM to 500 mM, 0.1 mM to 250 mM, 0.1 mM to 100 mM, 0.1 to 50 mM, 0.1 mM to 10 mM, 1 mM to 2000 mM, 1 mM to 1500 mM, 1 mM to 1000 mM, 1 mM to 500 mM, 1 mM to 250 mM, 1 mM to 100 mM, 1 mM to 50 mM, 1 mM to 100 mM, It may be present at a concentration of 10 mM to 10 mM, 10 mM to 2000 mM, 10 mM to 1500 mM, 10 mM to 1000 mM, 10 mM to 500 mM, 10 mM to 250 mM, 10 mM to 100 mM, 10 mM to 50 mM, 100 mM to 2000 mM, 100 mM to 1500 mM, 100 mM to 1000 mM, 100 mM to 500 mM, 100 mM to 250 mM, 500 mM to 2000 mM, 500 mM to 1500 mM, 500 mM to 1000 mM, etc.

[0041] In further embodiments, 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 tonicity modifying agent, e.g., inorganic salt(s), is present in the pharmaceutical formulation at a percentage concentration of 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more. Alternatively, or additionally, the tonicity modifying agent, e.g., inorganic salt(s), may be present in the pharmaceutical formulation at a percentage concentration of 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. The tonicity modifying agent, e.g., inorganic salt(s), may be present in the pharmaceutical formulation at any percentage concentration within a range bounded by any of the aforementioned endpoints. For example, the tonicity modifying agent, e.g., inorganic salt(s), may be present in the pharmaceutical formulation at a percentage concentration of 0.1% to 1%, 0.1% to 2%, 0.1% to 5%, 0.1% to 10%, 1% to 2%, 1% to 5%, 1% to 10%, 2% to 10%, 3% to 10%, 4% to 10%, 5% to 10%, etc.

[0042] Any suitable surfactant may be present in the pharmaceutical formulation. In certain embodiments, the surfactant may include 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). The surfactant may be present in the pharmaceutical formulation at a percentage concentration of 0.01% or more, 0.02% or more, 0.03% or more, 0.04% or more, 0.05% or more, 0.06% or more, 0.07% or more, 0.08% or more, 0.09% or more, 0.1% or more, 0.2% or more, 0.3% or more, 0.4% or more, 0.5% or more, 0.6% or more, 0.7% or more, 0.8% or more, 0.9% or more, or 1% or more. Alternatively, or additionally, the surfactant may be present in the pharmaceutical formulation at a percentage concentration of 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less. The surfactant may be present in the pharmaceutical formulation at any percentage concentration within a range bounded by any of the aforementioned endpoints. For example, the surfactant may be present in the pharmaceutical formulation at a percentage concentration of 0.01% to 1%, 0.01% to 0.1%, 0.05% to 1%, 0.05% to 0.1%, 0.1% to 1%, 0.1% to 0.5%, 0.2% to 1%, 0.5% to 1%, etc.

[0043] Any suitable amino acid may be present in the pharmaceutical formulation. In certain embodiments, the amino acid may be one or more of arginine, glutamic acid or glutamate, asparagine, histidine, and glycine. The amino acid(s) may be present in the pharmaceutical formulation in any suitable amount. The amino acid(s) may be present in the pharmaceutical formulation at a concentration of 1 mM or more, 2 mM or more, 3 mM or more, 5 mM or more, 6 mM or more, 7 mM or more, 8 mM, 9 mM or more, or 10 mM or more. Alternatively, or additionally, the amino acid(s) may be present in the pharmaceutical formulation at a concentration of about 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, or 10 mM or less. The amino acid(s) may be present in the pharmaceutical formulation at any concentration within a range bounded by any of the aforementioned endpoints. For example, the amino acid(s) may be present in the pharmaceutical formulation at a concentration of 1 mM to 10 mM, 1 mM to 50 mM, 1 mM to 100 mM, 5 mM to 50 mM, 10 mM to 50 mM, 20 mM to 50 mM, etc.

[0044] In some embodiments, the amino acid(s) 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 acid(s) may be present in the pharmaceutical formulation at a percentage concentration of 0.1% or more, 0.2% or more, 0.3% or more, 0.4% or more, 0.5% or more, 0.6% or more, 0.7% or more, 0.8% or more, 0.9% or more, 1% or more, 2% or more, 3% or more, 4% or more, or 5% or more. Alternatively, or additionally, the amino acid(s) may be present in the pharmaceutical formulation at a percentage concentration of 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. The amino acid(s) can be present in the pharmaceutical formulation at any percentage concentration within a range bounded by any of the aforementioned endpoints. For example, the amino acid(s) can be present in the pharmaceutical formulation at a percentage concentration of 0.1% to 10%, 0.2% to 10%, 0.5% to 10%, 0.1% to 5%, 0.1% to 2%, 0.2% to 2%, 0.5% to 1%, etc.

[0045] Any suitable polymer or polymer compound can be present in the pharmaceutical formulation. The polymer or polymer compound can be, for example, a protein, a polysaccharide, a hydrogel, or any other suitable polymer or polymer compound, many of which are known in the art. For example, the polymer or polymer compound can be recombinant human serum albumin (rHSA), serum albumin (SA), gelatin, hydroxyethyl starch (HES), chitosan, dextran (DEX70K, DEX40K), and polyvinylpyrrolidone (PVP40K).

[0046] The polymer(s) or polymeric compound(s) can be present in the pharmaceutical formulation in any suitable amount. The polymer(s) or polymeric compound(s) can 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 polymer(s) or polymeric compound(s) can be present in the pharmaceutical formulation at a percentage concentration of 0.1% or more, 0.2% or more, 0.3% or more, 0.4% or more, 0.5% or more, 0.6% or more, 0.7% or more, 0.8% or more, 0.9% or more, 1% or more, 2% or more, 3% or more, 4% or more, or 5% or more. Alternatively, or additionally, the polymer(s) or polymeric compound(s) may be present in the pharmaceutical formulation at a percentage concentration of 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. The polymer(s) or polymeric compound(s) may be present in the pharmaceutical formulation at any percentage concentration within a range bounded by any of the aforementioned endpoints. For example, the polymer(s) or polymeric compound(s) may be present in the pharmaceutical formulation at a percentage concentration of 0.1% to 10%, 0.2% to 10%, 0.5% to 10%, 0.1% to 5%, 0.1% to 2%, 0.2% to 2%, 0.5% to 2%, 0.1% to 1%, 0.2% to 1%, 0.5% to 1%, etc.

[0047] Any suitable chelating agent can be present in the pharmaceutical formulation. The chelating agent can be, for example, ethylenediaminetetraacetic acid (EDTA), amidoxime compounds (AOX), and / or dithiothreitol (DTT). The chelating agent can be present in the pharmaceutical formulation at any suitable concentration. The chelating agent can be present in the pharmaceutical formulation at a concentration of 10 μM or more, 20 μM or more, 30 μM or more, 40 μM or more, 50 μM or more, 60 μM or more, 70 μM or more, 80 μM or more, 90 μM or more, 100 μM or more, 120 μM or more, or 150 μM or more. Alternatively, or additionally, the chelating agent may be present in the pharmaceutical formulation at a concentration of 500 μM or less, 400 μM or less, 300 μM or less, 200 μM or less, 150 μM or less, 140 μM or less, 130 μM or less, 120 μM or less, 110 μM or less, 100 μM or less, 80 μM or less, 70 μM or less, 60 μM or less, or 50 μM or less. The chelating agent may be present in the pharmaceutical formulation at any concentration within a range bounded by any of the aforementioned endpoints. For example, the chelating agent may be present in the pharmaceutical formulation at a concentration of 10 μM to 500 μM, 10 μM to 200 μM, 10 μM to 150 μM, 10 μM to 100 μM, 50 μM to 500 μM, 50 μM to 200 μM, 50 μM to 150 μM, 50 μM to 100 μM, etc.

[0048] 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. The sugar(s) may be present in the pharmaceutical formulation at any suitable concentration. The sugar(s) may be present in the pharmaceutical formulation at concentrations of 0.1 mM or more, 0.2 mM or more, 0.4 mM or more, 0.6 mM or more, 0.8 mM or more, 1 mM or more, 1.2 mM or more, 1.4 mM or more, 1.6 mM or more, 1.8 mM or more, 2 mM or more, 3 mM or more, 4 mM or more, 5 mM or more, 6 mM or more, 7 mM or more, 8 mM or more, 9 mM or more, 10 mM or more, 20 mM or more, 3 mM or more, 40 mM or more, 50 mM or more, 60 mM or more, 70 mM or more, 80 mM or more, 90 mM or more, 10 ... The compound may be present at a concentration of 0 mM or more, 40 mM or more, 50 mM or more, 60 mM or more, 70 mM or more, 80 mM or more, 90 mM or more, or 100 mM or more, 200 mM or more, 300 mM or more, 400 mM or more, 500 mM or more, 600 mM or more, 700 mM or more, 800 mM or more, 900 mM or more, 1000 mM or more, or 1500 mM or more. Alternatively, or in addition, the sugar(s) are present in the pharmaceutical formulation at 2000 mM or less, 1500 mM or less, 1000 mM or less, 900 mM or less, 800 mM or less, 700 mM or less, 600 mM or less, 500 mM or less, 450 mM or less, 400 mM or less, 350 mM or less, 300 mM or less, 250 mM or less, 200 mM or less, 150 mM or less, 100 mM or less, 50 mM or less, 45 mM or less. , 40 mM or less, 35 mM or less, 30 mM or less, 25 mM or less, 20 mM or less, 10 mM or less, 9 mM or less, 8 mM or less, 7 mM or less, 6 mM or less, 5 mM or less, 4 mM or less, 3 mM or less, 2 mM or less, 1.8 mM or less, 1.6 mM or less, 1.4 mM or less, 1.2 mM or less, 1 mM or less, 0.8 mM or less, 0.6 mM or less, 0.4 mM or less, or 0.2 mM or less. The sugar(s) may be present in the pharmaceutical formulation at any concentration within a range bounded by any of the foregoing endpoints.For example, the sugar(s) may be present in the pharmaceutical formulation at concentrations of 0.1 mM to 2000 mM, 0.1 mM to 1500 mM, 0.1 mM to 1000 mM, 0.1 mM to 500 mM, 0.1 mM to 250 mM, 0.1 mM to 100 mM, 0.1 to 50 mM, 0.1 mM to 10 mM, 1 mM to 2000 mM, 1 mM to 1500 mM, 1 mM to 1000 mM, 1 mM to 500 mM, 1 mM to 250 mM, 1 mM to 100 mM, 1 mM to 50 mM, 1 mM to 10 mM It may be present at a concentration of 100 mM, 10 mM to 2000 mM, 10 mM to 1500 mM, 10 mM to 1000 mM, 10 mM to 500 mM, 10 mM to 250 mM, 10 mM to 100 mM, 10 mM to 50 mM, 100 mM to 2000 mM, 100 mM to 1500 mM, 100 mM to 1000 mM, 100 mM to 500 mM, 100 mM to 250 mM, 500 mM to 2000 mM, 500 mM to 1500 mM, 500 mM to 1000 mM, etc.

[0049] In other embodiments, the sugar(s) 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 sugar(s) may be present in the pharmaceutical formulation at a percentage concentration of 0.1% or more, 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more. Alternatively, or additionally, the sugar(s) may be present in the pharmaceutical formulation at a percentage concentration of 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less. The sugar(s) may be present in the pharmaceutical formulation at any percentage concentration within a range bounded by any of the aforementioned endpoints. For example, the sugar(s) may be present in the pharmaceutical formulation at percentage concentrations of 0.1% to 50%, 1% to 50%, 10% to 50%, 0.1% to 20%, 1% to 20%, 10% to 20%, 0.1% to 10%, 1% to 10%, etc.

[0050] Any suitable polyol can be present in the pharmaceutical formulation. The polyol can be, for example, sorbitol and / or mannitol. The polyol can be present in the pharmaceutical formulation at any suitable concentration. The polyol can be present in the pharmaceutical formulation at a concentration of 0.1 mM or more, 1 mM or more, 10 mM or more, 20 mM or more, 30 mM or more, 40 mM or more, 50 mM or more, 60 mM or more, 70 mM or more, 80 mM or more, 90 mM or more, 100 mM or more, 120 mM or more, 140 mM or more, 160 mM or more, 180 mM or more, 200 mM or more, 250 mM or more, 300 mM or more, 350 mM or more, 400 mM or more, 450 mM or more, or 500 mM or more. Alternatively, or additionally, the polyol can be present in the pharmaceutical formulation at a concentration of 1000 mM or less, 500 mM or less, 450 mM or less, 400 mM or less, 350 mM or less, 300 mM or less, 250 mM or less, 200 mM or less, 180 mM or less, 160 mM or less, 140 mM or less, 120 mM or less, 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, 10 mM or less, or 1 mM or less. The polyol can be present in the pharmaceutical formulation at any concentration within a range bounded by any of the foregoing endpoints. For example, the polyol may be present in the pharmaceutical formulation at a concentration of 0.1 mM to 1000 mM, 0.1 mM to 500 mM, 0.1 mM to 100 mM, 1 mM to 1000 mM, 1 mM to 500 mM, 1 mM to 100 mM, 100 mM to 1000 mM, 100 mM to 500 mM, etc.

[0051] In other embodiments, the polyol 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 polyol may be present in the pharmaceutical formulation at a percentage concentration of 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, or 45% or more. Alternatively, or additionally, the polyol may be present in the pharmaceutical formulation at a percentage concentration of 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. The polyol can be present in the pharmaceutical formulation at any percentage concentration within a range bounded by any of the aforementioned endpoints. For example, the polyol can be present in the pharmaceutical formulation at a percentage concentration of 0.1% to 50%, 1% to 50%, 5% to 50%, 10% to 50%, 15% to 50%, 0.1% to 25%, 1% to 25%, 5% to 25%, 10% to 25%, 15% to 25%, 0.1% to 15%, 1% to 15%, 5% to 15%, 10% to 15%, 0.1% to 10%, 1% to 10%, 5% to 10%, 0.1% to 5%, 1% to 5%, etc.

[0052] In one embodiment, the pharmaceutical formulation comprises an influenza virus of the invention, 0.5 M sucrose, 0.1 M or 0.5 M mannose, 0.3 M or 0.5 M trehalose, 50% SPG, and 0.05% polysorbate 20. In another embodiment, the pharmaceutical formulation comprises an influenza virus of the invention, 0.5 M sucrose, 0.3 M trehalose, and 0.05% polysorbate 20.

[0053] The at least one pharmaceutically acceptable carrier or excipient may be a component (e.g., a binder) that serves to bind the components of the pharmaceutical formulation. Binders may 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). The at least one pharmaceutically acceptable carrier or excipient may be a component (e.g., a bulking agent, diluent, and / or filler) that increases the bulk of the pharmaceutical formulation. Such bulking agents may include, but are not limited to, polysaccharides or their derivatives, sugars, and / or inorganic compounds. The pharmaceutically acceptable carrier or excipient may be a component (e.g., a flavoring agent, a sweetener, and / or a coloring agent) that enhances the taste and / or appearance of the pharmaceutical formulation. The pharmaceutically acceptable carrier or excipient may be a component (e.g., an adsorbent) that protects the pharmaceutical formulation by absorbing or adsorbing liquids or gases. Adsorbents include, but are not limited to, starch, calcium phosphate, and / or colloidal silicon dioxide. The pharmaceutically acceptable carrier or excipient can be an ingredient (e.g., a disintegrant) that promotes dissolution of the pharmaceutical formulation, such as starch, cellulose, and / or any other polymer known in the art, or a derivative thereof (e.g., cross-linked polyvinylpyrrolidone or sodium carboxymethylcellulose).

[0054] In some embodiments, the pharmaceutically acceptable carrier or excipient is a component (e.g., a glidant) that reduces interparticle adhesion and / or optimizes product flow during and during the manufacture of the pharmaceutical formulation. Examples of glidants include, but are not limited to, talc, colloidal silicon dioxide, and cornstarch. The pharmaceutically acceptable carrier or excipient may be a component (e.g., an anti-adherent) that reduces adhesion between components and, for example, punch surfaces during and during the manufacture of the pharmaceutical formulation, i.e., provides non-stick properties, such as a lubricant, particularly when the pharmaceutical formulation is formulated as an oral preparation. For example, an anti-adherent may include magnesium stearate. In other embodiments, the pharmaceutically acceptable carrier or excipient may be a component (e.g., a lubricant) that reduces aggregation of components during manufacturing and / or reduces friction, for example, between the surface of the pharmaceutical formulation (i.e., formulated as an oral preparation) and the die wall. In certain embodiments, both water-soluble and water-insoluble lubricants, such as magnesium stearate, stearic acid, vegetable oil, mineral oil, polyethylene glycol, and / or sodium lauryl sulfate, may be used. The pharmaceutically acceptable carrier or excipient may be a component that acts as a coating agent. Coating agents include, but are not limited to, gelatin and / or cellulose-based coating agents (e.g., hydroxypropylmethylcellulose).

[0055] Other suitable binders, flavoring agents, sweetening agents, coloring agents, disintegrants, glidants, anti-adherents, lubricants, and coating agents are well known in the art and can be readily identified.

[0056] The pharmaceutical formulation may further comprise a therapeutic agent (e.g., a chemotherapeutic agent or an anti-inflammatory agent). The pharmaceutical formulation may also comprise an agent that induces an immune response separate from the influenza virus. Such additional components other than the influenza virus of the present invention may be present in any suitable amount(s).

[0057] The additional component may be mixed with other components to form a pharmaceutical formulation before presentation to the immune system. The additional component may also be presented to the immune system separately from the pharmaceutical formulation. For example, the additional component and the pharmaceutical formulation may be presented to the immune system (e.g., administered to an organism) separately. If the additional component and the pharmaceutical formulation are administered separately, the additional component and the pharmaceutical formulation may be administered to the same site in the organism being immunized.

[0058] In one embodiment of the pharmaceutical formulation, the pharmaceutical formulation is a viral vaccine. The viral vaccine can 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 can be formulated as a monovalent vaccine, a bivalent vaccine, a trivalent vaccine, or a quadrivalent vaccine. For example, the vaccine can include multiple embodiments of the influenza viruses of the present invention. In some embodiments, the vaccine can further include at least one influenza virus different from the influenza viruses of the present invention.

[0059] The viral vaccine can be formulated into a composition for any suitable means of administration. For example, the viral vaccine can be formulated as an oral preparation (e.g., capsule, tablet, or oral film), a spray (e.g., nasal spray), or any composition suitable for intranasal or parenteral administration, e.g., intravenous, intramuscular, or subcutaneous administration, such as an aqueous or non-aqueous emulsion, solution, or suspension. Methods for eliciting an immune response

[0060] The invention provides a method for eliciting an immune response in a mammal, comprising administering to the mammal an influenza virus of the invention. In one embodiment, the influenza virus comprises PB1, PB2, PA, NP, and NS gene segments encoding proteins, i.e., PB1, PB2, PA, NP, and NS1 proteins, respectively, comprising selected amino acids, i.e., the influenza viruses of the invention described herein.

[0061] The mammal may be, for example, but is not limited to, a human or a primate.

[0062] In one embodiment of the invention, the influenza viruses of the invention are administered in a pharmaceutical formulation (e.g., a vaccine or other immunogenic composition) as described herein. The pharmaceutical formulation may be administered intranasally. In another embodiment, the pharmaceutical formulation is administered intramuscularly. The pharmaceutical formulation may also be administered subcutaneously or orally.

[0063] The dosage regimen for pharmaceutical preparations, e.g., viral vaccines, may depend on the age, weight, sex, and medical history of the mammal. For example, in one embodiment, a single dose of an attenuated viral vaccine for humans is administered in a dose of about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 or any range between two of the foregoing values. 50In some embodiments, the regimen for preventing or treating influenza virus comprises administering the pharmaceutical formulation as a single treatment. The pharmaceutical formulation can also be administered more than once, for example, the regimen can include a booster dose. For example, the booster dose of the pharmaceutical formulation can be administered over a period ranging from 7 days or more, e.g., 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 after the initial administration. Embodiment

[0064] The present invention provides the following embodiments:

[0065] (1) An influenza virus comprising PB1, PB2, PA, NP, and NS gene segments, wherein: (a) the PB1 gene segment encodes a PB1 protein having an amino acid sequence comprising selected amino acids, the selected amino acids comprising leucine at position 40 and tryptophan at position 180, and at least one of asparagine at position 464 or serine at position 607, and the PB1 gene segment optionally comprises a cytosine to uracil mutation in the promoter at the nucleotide at position 4; (b) the PB2 gene segment encodes a PB2 protein having an amino acid sequence comprising selected amino acids, the selected amino acids comprising valine at position 504, and optionally isoleucine at position 467 and valine at position 529, and the PB2 gene segment optionally comprises a cytosine to uracil mutation in the promoter at the nucleotide at position 4. and (c) the PA gene segment encodes a PA protein having an amino acid sequence comprising selected amino acids, the selected amino acids comprising lysine at position 401, and the PA gene segment optionally comprises a promoter cytosine to uracil mutation at nucleotide position 4; (d) the NP gene segment encodes an NP protein having an amino acid sequence comprising selected amino acids, the selected amino acids comprising leucine at position 116 and at least one of lysine at position 294 or arginine at position 311; and (e) the NS gene segment encodes an NS1 protein having an amino acid sequence comprising selected amino acids, the selected amino acids comprising proline at position 30 and lysine at position 118.

[0066] (2) An influenza virus of embodiment (1), wherein: (a) the PB1 gene segment encodes a PB1 protein having an amino acid sequence comprising selected amino acids, the selected amino acids being leucine at position 40, tryptophan at position 180, and asparagine at position 464, and the PB1 gene segment optionally comprises a cytosine to uracil mutation in the promoter at nucleotide position 4; (b) the PB2 gene segment encodes a PB2 protein having an amino acid sequence comprising selected amino acids, the selected amino acid being valine at position 504, and the PB2 gene segment optionally comprises a cytosine to uracil mutation in the promoter at nucleotide position 4; and (c) the NP gene segment encodes an NP protein having an amino acid sequence comprising selected amino acids, the selected amino acids being leucine at position 116 and lysine at position 294.

[0067] (3) The influenza virus of embodiment (1) or (2), wherein the PB1 gene segment has a nucleotide sequence represented by SEQ ID NO:2.

[0068] (4) The influenza virus of any of embodiments (1) to (3), wherein the NP gene segment has a nucleotide sequence represented by SEQ ID NO:1.

[0069] (5) The influenza virus of any one of embodiments (1) to (4), wherein the PB1 gene segment encodes a PB1 protein having the amino acid sequence of SEQ ID NO: 7.

[0070] (6) The influenza virus of any of embodiments (1) to (5), wherein the NP gene segment encodes an NP protein having the amino acid sequence of SEQ ID NO:6.

[0071] (7) The influenza virus of any of embodiments (1) to (6), wherein the selected amino acid is conserved in at least one of the PB1 and NP proteins after at least 10 serial passages in a Vero cell line.

[0072] (8) The influenza virus of any of embodiments (1) to (7), wherein the selected amino acid is conserved in at least one of the PB1 and NP proteins after at least 10 serial passages in a Vero cell line stably expressing the M2 ion channel protein of influenza A virus.

[0073] (9) The influenza virus of any of embodiments (1) to (8), wherein the influenza virus is an influenza A virus and the selected amino acid is conserved in at least one of the PB1 and NP proteins after at least 10 serial passages in a Vero cell line.

[0074] (10) The influenza virus of embodiment (1), wherein: (a) the PB1 gene segment encodes a PB1 protein having an amino acid sequence comprising selected amino acids, the selected amino acids being leucine at position 40, tryptophan at position 180, and serine at position 607, and the PB1 gene segment optionally comprises a cytosine to uracil mutation in the promoter at nucleotide position 4; (b) the PB2 gene segment encodes a PB2 protein having an amino acid sequence comprising selected amino acids, the selected amino acids being valine at position 504, isoleucine at position 467, and valine at position 529, and the PB2 gene segment optionally comprises a cytosine to uracil mutation in the promoter at nucleotide position 4; and (c) the NP gene segment encodes an NP protein having an amino acid sequence comprising selected amino acids, the selected amino acids being leucine at position 116 and arginine at position 311.

[0075] (11) The influenza virus of embodiment (1) or (10), wherein the PB1 gene segment has a nucleotide sequence represented by SEQ ID NO:4.

[0076] (12) The influenza virus of any of embodiments (1), (10), and (11), wherein the PB2 gene segment has a nucleotide sequence represented by SEQ ID NO:5.

[0077] (13) The influenza virus of any of embodiments (1) and (10) to (12), wherein the NP gene segment has a nucleotide sequence represented by SEQ ID NO: 3.

[0078] (14) The influenza virus of any one of embodiments (1) and (10) to (13), wherein the PB1 gene segment encodes a PB1 protein having the amino acid sequence of SEQ ID NO: 9.

[0079] (15) The influenza virus of any one of embodiments (1) and (10) to (14), wherein the PB2 gene segment encodes a PB2 protein having the amino acid sequence of SEQ ID NO: 10.

[0080] (16) The influenza virus of any one of embodiments (1) and (10) to (15), wherein the NP gene segment encodes an NP protein having the amino acid sequence of SEQ ID NO: 8.

[0081] (17) The influenza virus of any of embodiments (1) and (10) to (16), wherein the selected amino acid is conserved in at least one of the PB1, PB2, and NP proteins after at least 10 serial passages of the virus in a Vero cell line.

[0082] (18) The influenza virus of any of embodiments (1) and (10) to (17), wherein the selected amino acid is conserved in at least the PB1, PB2, and NP proteins after at least 10 serial passages in a Vero cell line stably expressing the M2 ion channel protein of influenza A virus.

[0083] (19) The influenza virus of any of embodiments (1) to (18), wherein at least one of the PB1, PB2, and PA gene segments comprises a promoter cytosine to uracil mutation at nucleotide position 4.

[0084] (20) The influenza virus according to any one of embodiments (1) to (19), wherein the influenza virus is a recombinant influenza virus.

[0085] (21) The influenza virus of any of embodiments (1) to (20), wherein the virus further comprises an NA gene segment and an HA gene segment.

[0086] (22) The influenza virus of embodiment (21), wherein the HA gene segment encodes an HA protein having an amino acid sequence containing at least one amino acid mutation in HA1.

[0087] (23) The influenza virus of embodiment (21) or (22), wherein the HA gene segment encodes an HA protein having an amino acid sequence comprising at least one amino acid mutation in HA2.

[0088] (24) An influenza virus according to embodiment (23), wherein at least one amino acid mutation in HA2 is asparagine at position 107.

[0089] (25) The influenza virus of any of embodiments (21) to (24), wherein the PB1, PB2, PA, NP, and NS gene segments are derived from a single influenza strain.

[0090] (26) The influenza virus of embodiment (25), wherein the HA gene segment is derived from an influenza strain different from the single influenza strain from which the PB1, PB2, PA, NP, and NS gene segments are derived.

[0091] (27) The influenza virus of embodiment (25) or (26), wherein the NA gene segment is derived from an influenza strain different from the single influenza strain from which the PB1, PB2, PA, NP, and NS gene segments are derived.

[0092] (28) The influenza virus of any one of embodiments (1) to (27), further comprising a mutant M gene segment.

[0093] (29) The influenza virus of embodiment (28), wherein the influenza virus does not encode a functional M2 protein.

[0094] (30) The influenza virus of any of embodiments (1) to (29), wherein the virus is capable of replicating in human cells.

[0095] (31) The influenza virus of any of embodiments (1) to (30), wherein the virus exhibits enhanced growth under identical conditions in Vero cells compared to an identical influenza virus except for the absence of the selected amino acid.

[0096] (32) A pharmaceutical preparation comprising the influenza virus of any one of embodiments (1) to (32).

[0097] (33) The pharmaceutical preparation of embodiment (32), wherein the pharmaceutical preparation is a vaccine.

[0098] (34) The pharmaceutical formulation of embodiment (33), wherein the vaccine is formulated as a monovalent vaccine.

[0099] (39) The pharmaceutical formulation of embodiment (33), wherein the vaccine is formulated as a bivalent vaccine.

[0100] (40) The pharmaceutical formulation of embodiment (33), wherein the vaccine is formulated as a trivalent vaccine.

[0101] (41) The pharmaceutical formulation of embodiment (33), wherein the vaccine is formulated as a tetravalent vaccine.

[0102] (42) A method for inducing an immune response in a mammal, comprising administering to the mammal an influenza virus of any of embodiments (1) to (31) or a pharmaceutical formulation of any of embodiments (32) to (41), thereby inducing an immune response in the mammal.

[0103] (43) The method of embodiment (42), wherein the mammal is a human.

[0104] (44) A method for producing an influenza virus according to any of embodiments (1) to (13), comprising serially passage of the influenza virus in a Vero cell line. [Example]

[0105] Example The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0106] Example 1 This example compares the growth of viruses with different backbones in Vero cells.

[0107] The high-yield PR8 ("PR8-HY") backbone described in Ping et al., Nature Communications, 6:8148 (2015), which contains mutated backbone gene segments derived from the influenza A A / Puerto Rico / 8 / 1934 ("PR8") strain, was used to generate M2SR viruses encoding HA and NA from two different influenza viruses representing the two influenza A subtypes present in seasonal vaccines: A / Massachusetts / 15 / 2013 (MA15; H1N1) and A / Brisbane / 10 / 2007 (Bris10, H3N2).

[0108] Specifically, cDNAs encoding the HA and NA gene segments from these viruses were co-transfected with cDNAs encoding the PR8-HY backbone gene segment and the M2SR M gene segment (SEQ ID NO: 11). The HA derived from MA15 was Vero-adapted (MA15V), as described in Example 8. Two viruses, HY-M2SR-MA15V and Bris10 M2SR-HY, were generated. Viruses were generated by standard virus rescue techniques and amplified in MDCK cells stably expressing M2 (i.e., M2CK cells), as described herein.

[0109] Influenza A virus RNA (vRNA) segments, including the PB1, PB2, PA, NP, and NS vRNA segments from the influenza PR8-HY backbone, and the M vRNA segment lacking the entire M2 open reading frame (ORF), as well as the HA and NA vRNA segments from influenza A / Brisbane / 10 / 2007 (Bris10, H3N2) or A / Massachusetts / 15 / 2013 (MA15V), were cloned into RNA polymerase I expression cassettes (Sarawar et al., Vaccine, 34:5090-5098 (2016) and Neumann et al., Proc. Natl. Acad. Sci. USA, 96:9345-9350 (1999)). The resulting plasmids were co-transfected into 293T cells with viral polymerase subunits and NP expression plasmids, and the released viruses were amplified in M2CK cells.

[0110] The growth of primary influenza viruses with the PR8-HY backbone and the high-growth ("HG") M2SR backbone (i.e., the "UW-PR8 backbone"), also described in Ping et al., Nature Communications, 6:8148 (2015), was compared in M2 Vero cells. To examine the growth of these viruses for comparison, M2 VeroA cell monolayers in 6 cm dishes were infected with each virus at an MOI of 0.001 using standard procedures. Infected cells were incubated at 35°C for 5 days. Aliquots were taken from the supernatant daily, and virus titers were calculated as TCID using the Reed & Muench method (Reed & Muench, Am. J. Hygiene, 27:493-497 (1938)). 50 was measured by assay.

[0111] The results are shown as growth curves in Figures 1A and 1B. These results demonstrate that neither HY-M2SR-MA15V nor Bris10 M2SR-HY grew better in M2VeroA cells than the UW-PR8 backbone, indicating that the PR8-HY backbone does not enhance virus growth in Vero cells.

[0112] Because H3N2 viruses (i.e., Bris10 M2SR-HY and Bris10 M2SR-HG) were observed to grow to higher titers than H1N1 viruses (i.e., HY-M2SR-MA15V and HG-M2SR-MA15), H3N2 virus supernatants were evaluated by hemagglutination (HA) assay to determine whether the PR8-HY and UW-PR8 backbones demonstrated differences in HA titers. As shown in Figure 1C, the HA titer of Bris10 M2SR-HY was lower than that of Bris10 M2SR-HG, further suggesting that the PR8-HY backbone does not enhance virus growth in Vero cells.

[0113] To confirm that HY-M2SR-MA15V and Bris10 M2SR-HY were not outliers but rather representative of other viruses with H1N1 and H3N2 subtypes, additional strains were used to generate M2SR viruses with PR8-HY and UW-PR8 backbones. These viruses were then tested for growth using the same methods described herein to verify growth of HY-M2SR-MA15V and Bris10 M2SR-HY viruses. Table 1 provides a summary of all strains tested.

[0114] [Table 1]

[0115] As can be seen from the results presented in Table 1, the PR8-HY backbone does not enhance virus growth in Vero cells compared to the UW-PR8 backbone for the H1N1, H3N2, and H5N1 virus strains. These results also demonstrate that the PR8-HY backbone is not a suitable backbone for influenza vaccine production.

[0116] Example 2 This example demonstrates the generation of viruses capable of enhanced growth in Vero cells. To generate these viruses, two M2SR viruses containing either the NA and Vero-adapted HA from A / Massachusetts / 15 / 2013 (i.e., MA15V M2SR virus) or the NA and HA from A / California / 07 / 2009 (i.e., CA07 M2SR virus) and a PR8-HY backbone (i.e., HY-M2SR-MA15V and HY-M2SR-CA07) were serially passaged in M2VeroA cells.

[0117] Virus was serially diluted 10-fold and adsorbed to M2 VeroA cells in TC-6 plates using standard influenza virus techniques. However, prior to virus infection, the cell culture medium was removed and cells were washed with PBS. After 60 minutes of adsorption at 35°C, virus growth medium containing trypsin / TPCK was added. Cultures were incubated at 35°C for 4–7 days. Culture supernatants were collected from wells containing the highest dilution that showed cytopathic effect and HA activity. Cytopathic effect (CPE) was determined by visual inspection of the monolayer at low magnification under a light microscope to detect rounding or other structural changes. HA activity was determined by a standard hemagglutination assay as described in the WHO Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza, 2011. Fifty microliters of a 0.5% suspension of turkey erythrocytes (Innovative Research, Novi, MN) was added to serial 2-fold dilutions of the culture supernatant, and hemagglutination was assessed after 30 minutes of incubation at room temperature. The reciprocal of the highest dilution of culture supernatant that agglutinated red blood cells was recorded as the HA titer for that sample.

[0118] The harvested supernatant was then serially diluted again and used to infect fresh M2 VeroA monolayers. The passage history is listed in Table 2.

[0119] [Table 2]

[0120] With increasing passage number, virus was recovered at higher dilutions, indicating that the virus grew to higher titers in Vero cells. Therefore, the process was stopped at passage 5, and growth curve studies were performed to assess whether the passage 5 (p5) virus had indeed grown to higher titers than the starting (p0) virus.

[0121] The p0 and (p5) viruses were evaluated in growth curves in M2 VeroA cells. The (p0) viruses were HY-M2SR-MA15V and HY-M2SR-CA07 viruses. The (p5) viruses were designated FGHY1-M2SR-MA15V and FGHY2-M2SR-CA07 viruses. FGHY1 and FGHY2 specifically refer to the backbone of the p5 virus.

[0122] To assess the growth curve, cell monolayers were infected at an MOI of 0.001 and incubated for 6 days in a CO2 incubator at 35°C. TCID 50 Aliquots were taken daily for virus titration using the assay. The resulting growth curves are shown in Figure 2. As indicated by the dashed line in Figure 2, passage 5 (p5) virus demonstrated higher virus titers and faster growth kinetics than the starting (p0) virus.

[0123] Example 3 This example identifies mutations that conferred increased growth characteristics associated with passage 5 viruses in M2 VeroA cells from Example 2 (i.e., FGHY1-M2SR-MA15V and FGHY2-M2SR-CA07).

[0124] HY-M2SR-MA15V and HY-M2SR-CA07 were serially passaged according to the method described in Example 2, and the entire viral genome sequences were determined for the passage 6 (p6) viruses, i.e., FGHY1-M2SR-MA15V and FGHY2-M2SR-CA07. Specifically, viral RNA was extracted from the supernatant of virus-infected cells using the Macherey-Nagel NUCLEOSPIN® RNA extraction kit. cDNA was generated using 12-base pair universal primers that amplify all eight segments of the viral genome in a multisegment reverse transcription reaction. Influenza genes were amplified using primers as described by Hoffmann et al., Arch. Virol., 146:2275-2289 (2001). Gene-specific primers were used to obtain bulk cDNA sequences. The resulting cDNA sequences were then aligned to the starting plasmid sequence, which served as a reference sequence, using a sequence comparison algorithm with program parameters specified to highlight non-identical residues. Table 3 shows the amino acid changes observed in each gene compared to the UW-PR8 and PR8-HY backbones.

[0125] As seen in Table 3, the backbones of the FGHY1-M2SR-MA15V and FGHY2-M2SR-CA07 viruses contain different mutations, and the FGHY1 and FGHY2 backbones are different. For example, FGHY1 contains a mutation at amino acid position 464 in the PB1 protein and at amino acid position 294 in the NP protein compared to the PR8-HY backbone, while FGHY2 contains a mutation at amino acid position 607 in the PB1 protein, at amino acids positions 467 and 529 in the PB2 protein, and at amino acid position 311 in the NP protein compared to the PR8-HY backbone.

[0126] [Table 3]

[0127] FGHY1-M2SR-MA15V and FGHY2-M2SR-CA07 further contained a Vero-adaptation mutation in HA2. Specifically, as described in Example 8, FGHY1-M2SR-MA15V contained a Vero-adaptation mutation at position 107 in HA2, changing threonine to asparagine. The amino acid sequence of Vero-adapted HA-MA15V is SEQ ID NO: 13. After serial passage, FGHY2-M2SR-CA07 developed a mutation at position 496 in HA, as shown in Table 5B.

[0128] To confirm that the amino acid changes observed in the FGHY1-M2SR-MA15V and FGHY2-M2SR-CA07 viruses confer high-yield properties, the FGHY1-M2SR-MA15V and FGHY2-M2SR-MA15V viruses were regenerated using the virus rescue technique described herein.

[0129] Specifically, individual backbone genes were cloned into pPolI plasmids using standard molecular techniques known in the art. These genes included the PB1 and NP genes of HY-M2SR-MA15V p6 (i.e., FGHY-PB1 and FGHY1-NP), identified by SEQ ID NOs:2 and 1, respectively, and the PB1, PB2, and NP genes of HY-M2SR-CA07 (i.e., FGHY2-PB1, FGHY2-PB2, and FGHY2-NP), identified by SEQ ID NOs:4, 5, and 3, respectively. M2SR viruses were then generated using a plasmid-based influenza virus rescue method similar to that described in Example 1. The viruses further contained HA-MA15V and NA-MA15.

[0130] The generated M2SR viruses (i.e., FGHY1-M2SR-MA15V and FGHY2-M2SR-MA15V) were then evaluated for growth kinetics in M2VeroA cells. Standard M2SR backbone (UW-PR8) and PR8-HY viruses (i.e., HG-M2SR-MA15V and HY-M2SR-MA15V, respectively) were used as standards for comparison. M2VeroA cells grown in 6-cm dishes were infected at a multiplicity of infection (MOI) of 0.001. Virus growth medium containing trypsin / TPCK (1 μg / mL) was added, and the cells were incubated at 35°C for 7 days. Aliquots were taken daily and stored at -80°C until virus titer determination.

[0131] The resulting growth curves are shown in Figure 3. As shown in Figure 3, FGHY1-M2SR-MA15V and FGHY2-M2SR-MA15V grew faster than HG-M2SR-MA15V and HY-M2SR-MA15V. FGHY1-M2SR-MA15V and FGHY2-M2SR-MA15V also reached peak titers earlier than HG-M2SR-MA15V and HY-M2SR-MA15V, reaching a plateau 2–3 days earlier. These results indicate that the amino acid mutations found in the passaged viruses (i.e., viruses with FGHY1 and FGHY2 backbones) confer high-yield characteristics and promote infection at low multiplicities of infection in M2 VeroA cells, which are highly desirable characteristics for vaccine production.

[0132] Example 4 This example demonstrates that mutations in the PB1 and NP proteins for FGHY1 and the PB1, PB2, and NP proteins for FGHY2 are responsible for conferring high growth properties to influenza viruses, regardless of HA and NA subtypes. Thus, this example demonstrates that the FGHY1 and FGHY2 backbones can be updated with various influenza HAs and NAs for the production of seasonal and pandemic influenza vaccines.

[0133] M2SR viruses carrying seasonal influenza H3N2 HA and NA (A / Brisbane / 10 / 2007) were generated using standard influenza virus rescue techniques described herein. Four M2SR viruses, FGHY1-M2SR-Bris10 and FGHY2-M2SR-Bris10, were generated, along with comparative controls, HY-M2SR-Bris10 (PR8-HY backbone) and HG-M2SR-Bris10 (UW-PR8 backbone). All viruses expressed H3N2 HA and NA proteins. The HA and NA proteins did not contain Vero-adaptive mutations.

[0134] Viral growth was assessed in M2 VeroA cells infected at an MOI of 0.001. Aliquots were collected daily and the virus titer was calculated in TCID 50 The HA production was measured by hemagglutination assay. Supernatants were also evaluated by hemagglutination assay to assess HA production. The resulting growth curves for virus titer and HA titer are shown in Figures 4A and 4B, respectively. As shown in Figure 4A, both FGHY1-M2SR-Bris10 and FGHY2-M2SR-Bris10 grew faster and to higher virus titers than HY-M2SR-Bris10 and HG-M2SR-Bris10. Furthermore, as shown in Figure 4B, both FGHY1-M2SR-Bris10 and FGHY2-M2SR-Bris10 demonstrated rapid HA titer kinetics compared to the other two viruses.

[0135] These results demonstrate that viruses containing the FGHY1 and FGHY2 backbones grow faster than viruses containing either the UW-PR8 or PR8-HY backbones, regardless of the subtype of the HA and NA surface proteins. The FGHY1 and FGHY2 backbones allow viruses with seasonal influenza A virus HA and NA to grow faster in Vero cells for both infectious virus production and HA production (i.e., live or inactivated vaccines). The FGHY1 and FGHY2 backbones also allow viruses with pandemic HA and NA to grow faster in Vero cells.

[0136] We generated M2SR viruses containing the FGHY1 backbone and the HA and NA of multiple influenza A subtypes, namely, seasonal (H1N1, H3N2) and pandemic (H5N1) viruses. Growth studies were performed in M2VeroA cells as previously described at an MOI of 0.001, comparing M2SR viruses containing UW-PR8, PR8-HY, and FGHY1 backbones. The resulting growth curves are shown in Figures 5A, 5B, and 5C for H1N1 (MI45), H3N2 (HK4801), and H5N1 (avVN1203) viruses, respectively.

[0137] As is evident from the data presented, the FGHY1 backbone exhibited faster growth kinetics and higher titers, further demonstrating that the FGHY1 backbone offers a growth advantage, making it suitable for use in vaccine production.

[0138] Figure 5A further shows a comparison between the FGHY1-M2SR-MI45 virus and the FGHY1-M2SR-MI45V virus, which further contains a Vero adaptation mutation at position 107 in HA2, changing threonine to asparagine. Thus, the results shown in Figure 5A also demonstrate that Vero adaptation mutations in the HA protein can further provide enhanced growth effects of the backbones described herein.

[0139] Example 5 This example demonstrates that while viruses containing the UW-PR8 and HY-PR8 backbones grow in MDCK cells, they show weaker growth in Vero cells, whereas viruses containing the FGHY1 backbone show higher yields in Vero cells.

[0140] Figure 6 shows the viral titers of M2SR viruses (i.e., Bris10 (H3N2), HK4801 (H3N2), MA15V (H1N1), and avVN1203 (H5N1)) containing various backbones (i.e., UW-PR8, HY-PR8, and FGHY1) grown in M2CK and M2VeroA cells for 4 days. Viruses were generated using standard influenza virus rescue techniques and evaluated using methods similar to those detailed in Examples 1-4. The data presented in Figure 6 demonstrate that the FGHY1 backbone specifically supports the growth of seasonal and pandemic influenza subtype viruses in Vero cells compared to MDCK cells.

[0141] Similarly, the data presented in Table 4 demonstrate that FGHY1 enhances the growth of multiple subtype viruses in Vero cells, resulting in titers that more closely resemble those produced in MDCK cells.

[0142] [Table 4]

[0143] These results indicate that mutations in the FGHY1 backbone overcome host restriction in Vero cells, allowing Vero cells to behave more like MDCK cells with respect to vaccine production.

[0144] Example 6A This example evaluates the genetic stability of the FGHY1 and FGHY2 backbones.

[0145] The HY-M2SR-MA15 and HY-M2SR-CA07 viruses, generated by the virus rescue technique described herein, were passaged twice in M2CK cells and then 20 and 9 times in M2VeroA cells, respectively. At each passage, serial 10-fold dilutions of tissue culture supernatant collected from the previous passage, ranging from 1:10 to 1:10,000,000, were used to infect fresh monolayers of M2VeroA cells. After infection, the culture supernatants were tested for CPE and HA titers. Supernatants from the highest dilutions that showed CPE and HA titers were collected anywhere between days 3 and 7 postinfection. FGHY1-M2SR-MA15 and FGHY2-M2SR-CA07 were generated at passage 4 in Vero cells.

[0146] At the end of the passages, the nucleotide sequence of the entire viral genome was determined and compared to the starting sequence from passage 4 in Vero cells (i.e., FGHY1-M2SR-MA15 and FGHY2-M2SR-CA07). The results are shown in Tables 5A and 5B.

[0147] [Table 5-1]

[0148] [Table 5-2]

[0149] As can be seen from the results shown in Table 5A, after 16 additional passages in M2VeroA cells, HY-M2SR-MA15 retained the same amino acid changes in the PB1 and NP proteins. The NP protein acquired one additional mutation at amino acid position 50, a serine to asparagine mutation. As can be seen from the results shown in Table 5B, HY-M2SR-CA07 also retained the same mutations in the PB1, PB2, and NP proteins as the starting virus after five additional passages in M2VeroA cells. These results demonstrate that the FGHY1-M2SR and FGHY2-M2SR backbones are stable in M2VeroA cells and are therefore suitable for use as vaccine backbones.

[0150] Example 6B To further demonstrate the stability of the FGHY1 backbone, influenza A / Hong Kong / 4801 / 2014 FGHY1 M2SR (H3N2) was serially passaged in BM2Vero cells. BM2Vero cells are Vero cells expressing the influenza BM2 protein, an ion channel protein of influenza B serotypes. Strong selective pressure against type A viruses interacting with the BM2 protein was expected. However, after 13 passages, the viral genome was sequenced, and surprisingly, only silent amino acid mutations were observed. The results, shown in Table 6, further demonstrate the stability of the FGHY1 backbone.

[0151] [Table 6]

[0152] Example 7 This example demonstrates that the M2SR-FGHY1 virus can replicate in human cells and that the enhanced growth in Vero cells is not species-specific.

[0153] The original M2SR backbone (UW-PR8) has been tested in human subjects (ClinicalTrials.gov Identifier: NCT02822105) and shown to elicit an immune response, indicating that it is functional in human cells. Therefore, the original backbone was used as a standard of comparison to determine whether M2SR-FGHY1 could replicate in human cells and thus serve as a viable clinical candidate.

[0154] The two viruses tested encoded the HA and NA of the H3N2 influenza virus A / Hong Kong / 4801 / 2014 (HK4801). The two viruses, M2SR-Original-HK4801 (lot number B17A12YH1) and M2SR-FGHY1-HK4801 (lot number B17A23YH1), were tested for infection in A549 cells (ATCC CCL-185), a human lung epithelial cell line, in 96-well plates using the infected-cell NP enzyme-linked immunosorbent assay (ELISA) protocol as described in the WHO Influenza Manual.

[0155] At day 2 post-infection, cells were stained for intracellular influenza nucleoprotein (NP) with anti-NP monoclonal antibody and processed as ELISA to determine the TCID 50 To normalize A549 titers to M2CK titers, a standard TCID 50 Assay was carried out and virus titer was obtained for each virus.Table 7 shows the virus titer for each virus in each cell line and the ratio between two cell lines.The ratio is similar for the two viruses, which indicates that M2SR-FGHY1-HK4801 grows in human cell lines similar to M2SR-Original-HK4801.

[0156] [Table 7]

[0157] Example 8 This example demonstrates that passage of wild-type virus in Vero cells can allow for better virus growth by acquiring mutations in the HA gene segment (eg, the HA2 region of H1N1pdm).

[0158] Influenza A / Massachusetts / 15 / 2013 (H1N1) (MA15) (e.g., parent virus) was obtained from International Reagent Resource (IRR, catalog number FR-1319, lot number 62525202), passaged in Vero cells, and amplified in MDCK cells. Viral RNA in tissue culture supernatant from passage 8 was extracted, and the nucleotide and corresponding amino acid sequences of the HA and NA segments were determined. The HA sequence from passage 8 contained one amino acid change from the parent virus, at amino acid position 451 (SEQ ID NO: 13) (i.e., position 107 in HA2). Specifically, a threonine was changed to an asparagine. Two separate, independent passages from independent wells yielded identical adaptive mutations.

[0159] This Vero-adapted HA2 mutation from MA15 (MA15V) (SEQ ID NO: 13) is similar to the mutations described in Example 4 for the Vero-adapted HA from MI45 (MI45V). As can be seen in Figure 5A, such mutations enhance virus growth in Vero cells.

[0160] Other amino acid changes in the HA (e.g., HA2) of Vero-passaged influenza HA (e.g., H1N1pdm HA) have been shown to stabilize the HA protein at lower pH, further enhancing viral infection of Vero cells. See, e.g., Cotter, Jin, and Chen, PLoS Pathog, 10(1):e1003831 (2014); Maurer-Stroh et al., PLoS Curr, 2:RRN1162 (2010); and Russier et al., PNAS, 113(6):1636-1641 (2016). Thus, the HA mutations described herein may also affect the stability of the HA protein at lower pH.

[0161] Figure 7A is a table showing mutations in the amino acid sequence of the HA protein in H1N1 viruses that emerged during Vero adaptation. Specifically, Table 7A shows the Vero-adapted mutations from MA15V (2013 Mass 15) and M145V (2015 MI45) described herein. The table in Figure 7A also shows the Vero-adapted HAs from influenza viruses that were passaged six times in Vero cells: A / Slovenia / 2903 / 2015 (2015 Slovenia), A / Lisboa / 32 / 2015 (2015 Lisboa), A / Scotland / P2 / 2015 (2015 Scotland), and A / Montana / 50 / 2016 (2016 Montana 50).

[0162] Figure 7B is a table showing mutations in the nucleic acid sequence and the resulting mutations in the amino acid sequence of a Vero-adapted HA derived from an H3N2 virus (i.e., A / Singapore / INFIMH-16-0019 / 2016). As can be seen from the table in Figure 7B, the possible amino acid mutations for each HA shown therein are as follows: T176K, L210P, T219I, S221P, D241G, P243H, K407E, D435N, E459K, and / or R472G. The "clinical" HA correlates with the wild-type HA. The HA designations V1 through V8 correlate with the sequences of the various HAs obtained by various passages. The shaded boxes indicate the nucleotide changes that result in the amino acid mutations. The Origin column lists the passage history of the starting virus.

[0163] Figure 7C shows the growth curves in M2VeroA cells of M2SR Sing2016 viruses containing various HA mutations (i.e., M2SR Sing2016 V5, M2SR Sing2016 V5 R2, and M2SR Sing2016 V6) as shown in Figure 7B. These viruses also contained the FGHY1 backbone. M2VeroA cell monolayers were infected with each virus at an MOI of 0.005. Infectious supernatants were harvested at the indicated time points, and virus titers were calculated as TCID in M2CK cells. 50 As determined by the assay, M2SR Sing2016 with the most Vero-adapted mutations (i.e., M2SR Sing2016 V6) showed the highest growth.

[0164] Example 9 This example demonstrates that the FGHY1-M2SR virus is attenuated in vivo.

[0165] Seven-week-old BALB / c, female mice were immunized intranasally with one of the following virus mutants: H1N1 FGHY1-M2SR, H3N2 FGHY1-M2SR (both of which contained the mutant M segment set forth in SEQ ID NO: 11). These mutants were administered at 1x10 per mouse. 6 TCID 50A control group of mice received SPG. Mice were observed for 14 days after immunization for any changes in weight and symptoms of infection.

[0166] No clinical symptoms of infection or weight loss were observed over a 14-day period in mice immunized with the FGHY1-M2SR mutant or SPG control. Figure 10 shows the weight change rate of mice after immunization. Furthermore, weight changes between groups were comparable over a 14-day period. These results indicate that the FGHY1-M2SR virus is attenuated and not pathogenic in mice.

[0167] Example 10 Although backbones that confer a growth advantage in cells of a certain species might be expected to exhibit host restriction and not replicate and / or produce antigens in the target host, this example demonstrates that antigen production of the FGHY1-M2SR virus is not restricted in human cell lines.

[0168] The following human cell lines were tested: A549 (ATCC No. CCL-185) human lung carcinoma; Calu-3 (ATCC No. HTB-55) human lung adenocarcinoma; and MRC-5 (ATCC No. CCL-171) human lung fibroblasts. These cell lines are derived from the human respiratory tract and represent target substrates for influenza vaccine viruses. Control cell lines used for influenza virus propagation were MDCK (Sigma No. 84121903) cells and Vero (ATCC No. CCL-81) cells.

[0169] One day before infection, cells were seeded in 60 mm dishes and immunized at an approximate MOI of 0.5 with the following viruses: primary, FGHY1, and IVR-147, a replicating vaccine reassortant virus from the CDC containing backbone gene segments from A / Puerto Rico / 8 / 1934, all of which expressed HA and NA from A / Brisbane / 10 / 2007. A subset of cells was infected with viruses containing primary and FGHY1, which expressed HA and NA from A / Singapore / INFIMH-16-0019 / 2016. The culture medium was supplemented with 10% FCS for 1 or 2 days post-viral infection at 35°C. Cells in the culture medium and on the culture surface were harvested and fixed with 10% buffered formalin. Cells were permeabilized in BD Cytofix / Cytoperm solution (BD, Cat. No. 554714), and influenza NP protein was stained with FITC-labeled mouse anti-influenza A NP monoclonal antibody (D67J, Invitrogen, Cat. No. MA1-7322). Fluorescein intensity was measured with a BD LSRII and analyzed with FlowJo software.

[0170] The single cell population was gated by forward scatter height (FSC-H) versus forward scatter area (FSC-A), and FITC-positive and -negative cells were separated by side scatter area (SSC-A) versus FITC. FITC-positive cells were considered to be virus-infected cells, and the intensity of FITC reflected the amount of NP expression in the cells.

[0171] The resulting NP expression level data are shown in Figures 8A, 8B, 9A, and 9B.

[0172] Figures 8A and 8B show that the FGHY1 virus expresses similar levels of NP in various cell lines compared to the IVR-147 and HG viruses.

[0173] Figures 9A and 9B show that the FGHY1-M2SR virus has a similar or higher proportion of cells expressing high NP levels in human cell lines compared to other viruses. These results demonstrate that the FGHY1 virus can infect human cell lines and produce influenza antigens. Therefore, the FGHY1-M2SR virus is expected to induce immunity when administered to human subjects.

[0174] Example 11 This example demonstrates that the FGHY1-M2SR vaccine elicits an antibody response in vivo that increases upon repeated administration without toxicity to the host. summary

[0175] To demonstrate that the FGHY1-M2SR vaccine virus induces an immune response to the components without causing toxicity to the host, 15 male and 15 female ferrets were infected with 1 × 10 8 TCID 50 (low dose) or 1x10 9 TCID 50 Ferrets were immunized intranasally with the FGHY1-M2SR vaccine at a dose level of 100 mg / kg (high dose). A third group of ferrets was mock-immunized intranasally with SPG as a placebo control. A three-dose vaccination schedule was utilized for each treatment group. Ferrets received a primary immunization (study day 1) and two booster immunizations 13 and 27 days later (study days 14 and 28). After each immunization, ferrets were observed for mortality for seven days, and weight, temperature, and clinical signs were measured daily. Blood was collected from all surviving ferrets before the study and on study days 14, 16, 30, and 49 to assess clinical pathology. Serum samples were collected before the study and on study days 14, 30, and 49, and antibody levels over time were assessed by ELISA, hemagglutination inhibition (HAI) assay, and virus neutralization (VN) assay. Necropsies, including examination of the exterior of the body, all orifices, skull, thoracic and peritoneal cavities, and their contents, were performed on five males and five females per group on study days 3, 30, and 49. B. Materials and Methods

[0176] Ferrets were immunized with the vaccine virus, 1x10 8 TCID 50 dose or 1x10 9 TCID 50 Ferrets were immunized intranasally with three doses of H3N2FGHY1-M2SR vaccine at either 0.05 mg / nostril or 0.05 mg / nostril. Vials of frozen vaccine virus stock were thawed at room temperature for at least 10 minutes and then stored refrigerated or on wet ice until use. Ferrets were anesthetized with ketamine / xylazine and administered one dose of virus intranasally in a volume of 500 μL (250 μL per nostril).

[0177] The H3N2 FGHY1-M2SR vaccine virus, which encodes the HA and NA genes of influenza A / Singapore / INFIMH-16-0019 / 2016 (H3N2), is a recombinant influenza A virus that does not express a functional M2 protein. The M gene segment of influenza A virus is represented by SEQ ID NO: 11.

[0178] Experimental Design. Ninety ferrets (Triple F Farms, Sayre, PA), 45 males and 45 females, aged 16–22 weeks at study initiation, were utilized in this study. All animal procedures were performed in an animal biosafety level 2 facility according to protocols approved by the IIT Institute Animal Care and Use Committee. Prior to immunization, ferrets were monitored for 4 days to establish baseline body temperature. Temperature measurements were recorded daily via a transponder (BioMedic data systems, Seaford, DE) implanted subcutaneously in each ferret. Prior to study initiation, blood samples were collected and serologically tested for influenza antibodies. Preimmune serum samples were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) to remove nonspecific inhibitors, then serially diluted and tested against defined amounts of influenza A / Michigan / 45 / 2015 (H1N1), A / Singapore / INFIMH-16-0019 / 2016 (H3N2), B / Phuket / 3073 / 2013 (Yamagata lineage), and B / Colorado / 06 / 2017 (Victoria lineage) viruses, mixed with 0.5% turkey red blood cells. Antibody titers were defined as the lowest serum dilution causing inhibition of hemagglutination. Only ferrets with HAI titers below 40 were considered seronegative and used in this study. Study animals were randomized and divided into three groups (15 male and 15 female ferrets per group).

[0179] To evaluate vaccine efficacy and toxicity, ferrets were administered 1 x 10 8 TCID 50 3 doses or 1 x 10 9 TCID 50All ferrets were immunized intranasally with three doses of H3N2 FGHY1-M2SR. A control group was mock-immunized intranasally with SPG on study days 1, 14, and 28. Ferrets' temperature, weight, and clinical signs were monitored daily for 7 days after immunization. Blood was collected from all surviving ferrets on study days -5, 14, 16, 30, and 49 for clinical pathology evaluation. Serum samples were collected on study days -5, 14, 30, and 49 and kept at approximately 70°C until antibody titer determination by ELISA, virus neutralization assay, and HAI assay. All study animals were euthanized and necropsied on scheduled days (days 3, 30, or 49, five males and five females per group). Necropsy consisted of examination of the external body surface, all orifices, and the cranial, thoracic, and peritoneal cavities and their contents. Tissues were collected, fixed, and evaluated histopathologically by a board-certified veterinary pathologist. C. Results

[0180] Morbidity / Mortality and Clinical Observations: All ferrets survived until their scheduled sacrifice date. The most common clinical sign observed in the group receiving H3N2FGHY1-M2SR was diarrhea; however, animals in the SPG control group also exhibited diarrhea. The activity level scores of all ferrets were "0" (alert and playful) for all time points measured between days 1 and 49, except for one male and one female from the SPG control group, which received a score of "1" (alert, but playful only when provoked) on day 20.

[0181] Body Weight and Body Weight Change: Several groups had statistically significant differences in mean body weight change compared to SPG controls, but these differences appeared randomly distributed and were not significant.

[0182] Body Temperature: Several groups experienced statistically significant increases and decreases in mean body temperature compared to the SPG control group, but these differences appeared randomly distributed and were not significant.

[0183] Enzyme-linked immunosorbent assay (ELISA): Anti-HA IgG antibody titers from serum samples were determined by ELISA. ELISA plates were coated with recombinant HA protein from A / Singapore / INFIMH-16-0019 / 2016 (H3N2) (Immune Technology Corp., New York, NY), blocked with skim milk, and then loaded with samples. Ferret IgG antibodies were detected using horseradish peroxidase-conjugated goat anti-ferret IgG antibody (SeraCare Life Sciences, Milford, MA) and 1-Step™ Ultra TMB-ELISA (Thermo Fisher Scientific Inc., Maltham, MA) substrate.

[0184] Anti-H3 HA ELISA IgG titers obtained in the serum data are shown in Figure 11. Ferrets in each immunized group showed a significant increase in anti-H3 HA antibodies in the serum, whereas antibody levels in animals administered SPG alone were unchanged from baseline. Anti-H3 HA antibody titers were higher in the immunized groups than in the SPG control group two weeks after the first dose. The mean antibody titer per immunized group further increased after the first and second doses of vaccine.

[0185] Hemagglutination Inhibition (HAI) Assay: To verify the functional activity of antibodies detected by ELISA, serum samples were analyzed by HAI assay. To eliminate nonspecific hemagglutination inhibitors, serum samples were treated with RDE. RDE was reconstituted according to the manufacturer's instructions. Serum was diluted 1:3 in RDE and incubated in a water bath at 37°C ± 2°C for 18–20 hours. After adding an equal volume of 2.5% (v / v) sodium citrate, the samples were incubated in a water bath at 56°C ± 2°C for 30±5 minutes. A solution consisting of 0.85% NaCl was added to each sample until the final serum dilution after RDE treatment was 1:10. The samples were then further diluted 2-fold (1:10 to 1:1280) in PBS and incubated with 4 hemagglutination units of influenza A / Singapore / INFIMH-16-0019 / 2016 (H3N2) virus. After incubation, 0.5% avian red blood cells were added to each sample and incubated for 30±5 minutes, then the presence or absence of hemagglutination was scored.

[0186] Anti-H3 HAI titers obtained in serum data are shown in Figure 12. 9 TCID 50 ) group received a low dose (1x10 8 TCID 50 The SPG (control) group did not develop HAI titers. 9 TCID 50 With the exception of one male in the study, all H3N2 FGHY1-M2SR-immunized ferrets demonstrated HAI titers of 80 or higher against the test virus. The CDC states that a serum HAI antibody titer of 40 is associated with at least a 50% reduction in the risk of influenza infection or disease in a population. Therefore, these results suggest that the FGHY1-M2SR virus can induce a protective immune response.

[0187] Virus Neutralization Assay: Prestudy and treatment-phase serum samples (from study days 3, 14, 30, and 49) were tested against A / Singapore / INFIMH-16-0019 / 2016 (H3N2) influenza virus in a virus neutralization assay. Serum samples were inactivated at 56°C for 30 minutes. The serum was then serially diluted two-fold and incubated with standardized virus (concentrations of 80–140 PFU) for 60 minutes at 37±2°C and 5.0±1% CO2. One hundred microliters (100 μL) of each serum-virus mixture was then transferred to each well of a 96-well plate containing a monolayer of MDCK cells. The plate (containing the sample) was then incubated for 18–22 hours at 37±2°C in 5.0±1% CO2. After incubation, cells were fixed with paraformaldehyde and stained with an anti-influenza A nucleoprotein monoclonal antibody pool (1 part MAB8257: 1 part MAB8258 (Millipore; Billerica, MA)), followed by peroxidase-conjugated goat anti-mouse IgG. Spots were developed using TrueBlue Peroxidase Substrate (Kirkegaard and Perry Laboratories; Gaithersburg, MD). Plaques were visualized and counted using an ELISPOT device (AID GmbH, Strassberg, Germany). The 50% plaque reduction neutralization titer (PRNT) was defined as the neutralization titer of 50% of the plaques. 50 ) was calculated based on a back titration of control plaques by counting plaques and recording the titer as the reciprocal of the final serum dilution showing a 50% reduction in viral plaque count of the input control.

[0188] Anti-H3 PRNT50 titers obtained in the serum data are shown in Figure 13. All ferrets in the SPG group tested negative for the duration of the study.

[0189]

number

[0190] It remained as it was. 1x10 8 TCID 50 Ferrets immunized with H3N2 FGHY1-M2SR at a dose of 1 x 10 showed VN titers of 1916, 1838, and 1970 geometric mean titers (GMTs) on days 14, 30, and 49, respectively. 9 Ferrets immunized with H3N2 FGHY1-M2SR at a TCID50 dose showed GMT VN titers of 4434, 5572, and 6400 on days 14, 30, and 49, respectively. Sera were diluted up to 1:6400, resulting in a PRNT of >6400. 50 Titer not specifically determined. 1x10 in triplicate 9 TCID 50 All ferrets immunized with H3N2FGHY1-M2SR had a PRNT of over 6400. 50 The titer was shown.

[0191] Clinical Pathology: For all surviving ferrets, blood samples for analysis of clinical chemistry, hematology, and coagulation parameters were collected from the jugular vein or vena cava before the study and on days 3, 14, 16, 30, and 49. Animals were fasted for 4-6 hours before blood collection. EDTA was used as an anticoagulant for hematology samples, and sodium citrate was used for coagulation samples. Samples for clinical chemistry were collected without anticoagulant. Urine samples were collected directly from each ferret's bladder at necropsy.

[0192] No treatment-related or toxicologically significant findings were noted in any of the clinical chemistry or hematology parameters evaluated during the study. The increase in fibrinogen observed in the vaccine-treated group was considered an "expected inflammatory response" following treatment with the immunogenic agent. Fibrinogen returned to control levels after 14 and 21-day recovery periods, suggesting that the effect was acute and reversible. The decrease in prothrombin time (PT) was reversible after discontinuation of medication. Therefore, this effect was considered to be of little toxicological importance.

[0193] Gross necropsy and histopathology: Gross necropsy and histopathology were performed on 5 males and 5 females per group on study days 3, 30, and 49. 8 TCID 50 Intranasal immunization of ferrets with H3N2 FGHY1-M2SR at a dose of 1x10 resulted in no macroscopic findings, but microscopic findings (mixed cell infiltrates) were observed in the lungs on days 3 and 30. 9 At the TCID50 dose, macroscopic findings were observed in the lungs (pigmentation, dark or mottled) and microscopic findings (mixed cell infiltrates) were observed in the lungs on days 3 and 30. After a 3-week recovery, no test article-related macroscopic lesions were observed on study day 49.

[0194] Thus, this example demonstrates that intranasal immunity to the H3N2 FGHY1-M2SR vaccine virus is not transmitted to vaccinated hosts and is not associated with any vaccine-related adverse events (e.g., elevated body temperature, weight loss, or clinical signs). These results demonstrate that the H3N2 FGHY1-M2SR virus elicits a protective immune response against a homologous test virus after a single dose, which can be further boosted with repeated administration and is useful as an intranasal influenza vaccine.

[0195] Example 12 This example demonstrates that the FGHY1-M2SR virus formulated in a multivalent vaccine elicits an antibody response against influenza A virus.

[0196] Influenza A H1N1 or H3N2 FGHY1-M2SR viruses induce antibody responses when formulated as monovalent, bivalent, trivalent, or tetravalent vaccines.

[0197] Seven-week-old BALB / c female mice (N = 8) were intranasally immunized with monovalent H1N1 FGHY1-M2SR, monovalent H3N2 FGHY1-M2SR, bivalent H1N1 and H3N2 FGHY1-M2MR, trivalent H1N1 and H3N2 FGHY1-M2SR and BM2SR Victoria or Yamagata, or tetravalent H1N1 and H3N2 FGHY1-M2SR and BM2SR Victoria and Yamagata vaccines. Control mice were mock immunized with SPG. Twenty-eight days after vaccination, mice were intranasally immunized with a booster consisting of the same vaccine administered for the primary immunization. Serum samples were collected on days 7, 14, and 21 after the primary immunization and on days 35, 42, and 49 after the booster (day 28). Anti-H1 HA and anti-H3 HA serum IgG antibody titers from serum samples were determined by ELISA.

[0198] The resulting anti-H1 HA data is shown in Figure 14A. The resulting anti-H3 HA data is shown in Figure 14B. The results showed that all vaccines were able to elevate anti-influenza virus antibodies above the SPG control, and these increases were comparable across vaccine formulations. These results demonstrate that there is no interference between the monovalent components when formulated into a multivalent vaccine.

[0199] Example 13 This example demonstrates that intranasally administered monovalent or tetravalent FGHY1-M2SR vaccines protect mice against lethal influenza A virus not contained in the vaccine.

[0200] BALB / c female mice described in Example 12 were challenged with a lethal dose of influenza A / California / 07 / 2009 (H1N1) virus (>10 mice 50% lethal dose (MLD)) 70 days after the first immunization (6 weeks after the booster immunization). 50All mice immunized with the monovalent H1N1 (influenza A / Montana / 50 / 2016) or H3N2 (influenza A / Singapore / INFIMH-16-0019 / 2016) FGHY1-M2SR and tetravalent FGHY1-M2SR and BM2SR vaccines survived inoculation. Mice immunized with the monovalent H1N1 FGHY1-M2SR and tetravalent FGHY1-M2SR and BM2SR vaccines remained healthy without weight loss. The weight loss data are shown in Figure 15. Mice immunized with the monovalent H3N2 FGHY1-M2SR vaccine temporarily lost weight but fully recovered. Control mice mock-immunized with SPG alone lost weight and died of infection within 5 days after inoculation. Three days after inoculation, lungs were harvested from three mice per group, and viral load was measured by plaque assay in MDCK cells. As shown in Table 8, virus titers in the lungs of mice immunized with the monovalent H1N1 FGHY1-M2SR and tetravalent vaccines were below the limit of detection (less than 76 plaque-forming units (PFU) per lung). Viral loads were detected in mice immunized with H3N2 FGHY1-M2SR (6.60 log PFU / g). However, the average virus titer of the three mice was approximately 1 log lower than that of naive control SPG mice (7.52 log PFU / g). These results indicate that the M2SR monovalent and tetravalent vaccines confer cross-protection and limit the replication of the inoculum virus independent of any of the vaccine components.

[0201] [Table 8]

[0202] Example 14 This example demonstrates that the tetravalent FGHY1-M2SR vaccine provides a favorable safety profile compared to the licensed intranasal influenza vaccine and superior protection against influenza viruses not contained in the licensed intramuscular inactivated influenza vaccine. The antigenically shifted monovalent H3N2 FGHY1-M2SR vaccine provides equivalent protection to the antigenically matched licensed vaccine in protecting mice against lethal influenza A virus.

[0203] Seven-week-old BALB / c female mice (N=13) were immunized with one of the following vaccines: monovalent H3N2 FGHY1-M2SR, tetravalent FGHY-M2SR, FluMist® Quadrivalent (AstraZeneca, Wilmington, DE), Fluzone® Quadrivalent (Sanofi, Bridgewater, NJ), or Fluzone® High Dose (Sanofi). The strain composition for each vaccine is shown in Table 9. FGHY1-M2SR and FluMist were administered intranasally, and both Fluzone vaccines were administered intramuscularly. A control group of mice was mock-immunized intranasally with SPG. Mice were observed for any changes in body weight for 14 days post-immunization.

[0204] [Table 9]

[0205] The resulting weight loss data are shown in Figure 16. Mice immunized with monovalent H3N2 FGHY1-M2SR, tetravalent FGHY1-M2SR, Fluzone Quadrivalent, Fluzone High Dose, or SPG control did not experience weight loss over 14 days. However, mice immunized with FluMist lost an average of 7% body weight on day 3 post-vaccination and took 14 days to recover. These data indicate that the FGHY1-M2SR vaccine has a superior safety profile to the licensed live-attenuated influenza vaccine, FluMist.

[0206] Twenty-eight days after the primary immunization, mice were immunized with a booster immunization. The booster immunization consisted of the same vaccine as the mice were immunized with in the primary immunization. Serum samples were collected weekly after the primary and boost immunizations, and pooled serum IgG titers against each vaccine component were determined by ELISA. The resulting anti-influenza A / H1 HA serum (serium) IgG ELISA titer data are shown in Figure 17A. The resulting anti-influenza A / H3 HA serum (serium) IgG ELISA titer data are shown in Figure 17B. The resulting anti-influenza B / Yam HA serum IgG ELISA titer data are shown in Figure 17C. The resulting anti-influenza B / Vic HA serum (serium) IgG ELISA titer data are shown in Figure 17D. The results show that all vaccines except the monovalent H3N2 FGHY1-M2SR were able to increase serum IgG titers against influenza A H1 and H3 HA compared to the SPG control, and these increases were comparable across vaccine formulations. Fluzone Quadrivalent and Fluzone High Dose induced lower serum IgG titers against influenza B HA antigen compared with the live vaccine. The monovalent H3N2FGHY1-M2SR vaccine, as expected, induced an increase in serum IgG only against the H3 HA antigen.

[0207] Tracheal-lung lavage fluids were obtained from four mice per group on day 49 after the prime immunization (day 21 after the boost), and IgG and IgA titers were determined by ELISA to assess mucosal immune responses. The resulting IgG titer data are shown in Figure 18A, and the resulting IgA titer data are shown in Figure 18B. Tetravalent FGHY1-M2SR and FluMist elicited both IgG and IgA titers against all tested antigens. Mice immunized with monovalent H3N2FGHY1-M2SR exhibited IgG and IgA titers against the H3 HA antigen, but not against the H1 or influenza B HA antigens. IgG titers against all four antigens were elevated in groups immunized with Fluzone Quadrivalent and Fluzone High Dose vaccines, but IgA antibody titers against any antigen were not detected. These data indicate that the FGHY1-M2SR vaccine induces mucosal immune responses comparable to those of the licensed live-attenuated influenza vaccine, FluMist, whereas the licensed intramuscular influenza vaccine does not induce any mucosal immune responses.

[0208] Six weeks after the booster immunization, mice were challenged with a lethal dose of influenza A / California / 07 / 2009 (H1N1pdm). Mock-immunized mice with the SPG control became infected and died by day 5 post-inoculation, whereas all vaccine recipients survived. Figure 19 shows the weight changes following inoculation. As seen in Table 10, the tetravalent FGHY1-M2SR and FluMist groups did not lose any weight, and no inoculum virus was detected in the lungs or nasal turbinates on day 3 post-inoculation. This suggests that these intranasal vaccines provide virucidal immunity against lethal infection with drift-mutated influenza viruses. Mice in the monovalent H3N2 FGHY1-M2SR (heterosubtypic to the inoculum virus), Fluzone Quadrivalent, and Fluzone High Dose groups lost an average of more than 15% weight on day 3 or 4 post-inoculation and then recovered by day 21 post-inoculation. Infectious virus was detected in the lungs and nasal turbinates of mice vaccinated with monovalent H3N2 FGHY1-M2SR and Fluzone Quadrivalent. Mice immunized with Fluzone High Dose vaccine showed weight loss comparable to that of mice immunized with monovalent H3N2 FGHY1-M2SR and Fluzone Quadrivalent vaccines, but infectious virus was not recovered from the lungs or nasal turbinates. These data demonstrate that intranasally administered monovalent heterosubtypic H3N2 FGHY1-M2SR (i.e., lacking the H1N1 component) vaccine protects mice as well as intramuscularly administered licensed inactivated vaccines containing the H1N1 component.

[0209] [Table 10]

[0210] Example 15 This example demonstrates that production of FGHY1-M2SR virus is scalable in M2VeroA cells, reducing host cell DNA levels to levels recommended by WHO and FDA guidelines for vaccines produced in cell substrates.

[0211] M2VeroA cells (Vero cells stably expressing influenza A M2 protein) were cultured in OptiVero medium (InVitria, Aurora, CO) in a humidified incubator at 37°C in a 5% CO atmosphere. Approximately 242 million cells in CellSTACK®-5 Chambers (CS5; Corning, Corning, NY)—three or four per lot—were infected in OptiVero medium with H3N2FGHY1-M2SR virus encoding the HA and NA genes of influenza A / Singapore / INFIMH-16-0019 / 2016I (H3N2) at a multiplicity of infection (MOI) of 0.01. After 2–3 days at 35°C and 5% CO, when the HA titer of the supernatant was at least 32 HAU / 50 μL, the culture medium was harvested and cells and cell debris were removed by low-speed centrifugation. The supernatant was further clarified and sterilized by vacuum filtration through a 0.2 μM pore PES membrane. The clarified supernatant was then treated with nonspecific RNA and DNA nuclease (Benzonase, 5 units / mL) at 37°C for 2 hours to digest / hydrolyze residual cellular RNA and DNA. The Benzonase-digested material was then purified by filtration at 235 cm. 2Purification was performed by tangential flow filtration (TFF) using a 300 kD MWCO Modified Polyethersulfone (mPES) MidiKros® hollow fiber filter module (Repligen, Waltham, MA). The material was concentrated 10-20 times. Contaminating host cell proteins (HCPs) and residual DNA fragments were then removed by diafiltration using at least 15 column volumes of SPG. The purified virus in SPG buffer was further concentrated 10-100 times by ultracentrifugation at 25,000 rpm through a 25% sucrose PBS cushion. The resulting virus pellet was resuspended in SPG, aliquoted, and then flash-frozen in liquid nitrogen before storage at -80°C.

[0212] The sequence homology of the enriched and purified H3N2 FGHY1-M2SR gene was compared with that of the reference sequence of the H3N2 FGHY1-M2SR virus seed stock. Viral RNA extracted from the purified virus was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR), and cDNA was generated and subjected to Sanger sequencing. Analysis of the open reading frames (ORFs) encoded by the eight viral segments showed that all segments shared 100% nucleotide sequence identity with the reference.

[0213] The infectious titer of concentrated and purified H3N2 FGHY1-M2SR virus was measured as the 50% tissue culture infectious dose (TCID ) in at least three independent assays using M2CK cells (MDCK cells stably expressing influenza A M2). 50 ) assay. In the procedure, serial dilutions of vaccine samples were applied to replicate M2CK cells in 96-well plates and cultured for 4 days at 35°C in a 5% CO2 atmosphere. Four days after inoculation, cell monolayers were visually inspected and scored for CPE. Virus titers were calculated using the Reed-Münch method and expressed as TCID 50The results were expressed as 1000 / mL. HA activity was also tested in an aliquot of each well supernatant to verify the virus titer determined by CPE. As shown in Table 11, after concentration and purification, the H3N2 FGHY1-M2SR virus consistently reached 1000 / mL. 9.8 TCID 50 reached high titers exceeding / mL.

[0214] [Table 11]

[0215] To confirm that the H3N2 FGHY1-M2SR vaccine virus maintained its replication-deficient phenotype after concentration and purification, the presence of any replicating virus was assessed by three serial passages of the test article in MDCK cells, which are permissive for wild-type influenza virus but not for M2SR virus. For the first round of infection, serial dilutions of the test virus were inoculated onto cell monolayers. The infected cells were then cultured at 35°C in a 5% CO2 atmosphere for 4 days. The culture supernatant from the infected cells was then transferred to a fresh MDCK monolayer and incubated at 35°C in a 5% CO2 atmosphere for 4 days. For the third and final passage, the culture medium from this infected cell was transferred to another fresh MDCK monolayer and incubated at 35°C in a 5% CO2 atmosphere for an additional 4 days. MDCK cells were observed for CPE after every 4 days of incubation at 35°C, and HA activity in the culture supernatant was measured to confirm the presence of progeny virus particles. For each round of infection, the replication-deficient reference virus, Bris10 M2SR, was tested as a positive control. The negative control inoculum was medium alone. Results showed that the control performed as expected, with no infectious progeny detected after inoculation of any of the four test articles into normal cells. Thus, the H3N2 FGHY1-M2SR vaccine virus preparation was demonstrated to be replication-incompetent and unable to replicate.

[0216] The fragment size of residual host cell DNA in the concentrated and purified FHGY1-M2SR was measured by capillary electrophoresis on a 2100 BioAnalyzer instrument (Agilent, Santa Clara, CA). Total sample DNA was extracted using Axygen® magnetic silica beads (Corning, Corning, NY). DNA purified from the samples was loaded onto a DNA High Sensitivity Chip (Agilent) and subjected to automated capillary electrophoresis. Using the 2100 Expert software, data was analyzed to detect fragments, obtain fragment concentrations, and obtain relative percentages of total DNA. The size of the extracted residual DNA was measured in base pairs (bp). Results from quadruplicate runs of three lots of concentrated and purified FGHY1-M2SR demonstrated low or no fluorescent signal intensity on the 2100 Expert, indicating that they contained insufficient residual DNA for accurate size determination.

[0217] A commercially available ELISA kit (Cygnus Technologies, part number F500, Southport, NC) was used to quantify the Vero host HCPs present in concentrated, purified H3N2 FGHY1-M2SR vaccine lots. The resulting HCP presence data are shown in Table 12. The antibodies in the kit were generated and affinity-purified using Vero lysates and have been shown to detect HCPs from many commercially available Vero cell lines used to generate viral products. Therefore, this kit can be used as a tool to monitor the levels of Vero HCP contaminants. The kit was used according to the manufacturer's assay protocol. Multiple dilutions of each sample were prepared in sample diluent (Cygnus catalog number I028) and analyzed (in duplicate) to confirm that the samples exhibited dilutional linearity within the range of the standard provided with the kit (i.e., no high-dose hook effect). A four-parameter logistic regression was used to generate a standard curve and interpolate sample values.

[0218] [Table 12]

[0219] The average amount of HCP in the tested vaccine preparations was 1.4 ± 1.0 μg / mL. These values ​​are consistent with the levels of HCP previously detected in clinical trial material (Bris10 M2SR, lot number 15100251). HCP in the three vaccine preparations was reduced by 99.98% to 99.99%.

[0220] Sterility of the vaccine preparations was verified by procedures based on WHO standards for pharmaceutical preparations. The vaccine preparations were inoculated under aseptic conditions into three liquid media: Luria-Bertani broth (LB), tryptic soy broth (TSB), and thioglycollate medium (TGM). Cultures were grown at 37°C (LB and TSB) and ambient temperature (TGM). Cultures were grown for 14 days and then visually inspected for microbial growth. All preparations tested were negative for microbial growth under all growth conditions.

[0221] Osmolality values ​​for H3N2 FGHY1-M2SR vaccine lots were measured using a Fiske Model 210 Micro-Osmometer (Fiske Associates, Norwood, MA). The resulting osmolality data are shown in Table 13. A three-point calibration (50, 850, and 2000 mOsm / kg) was performed prior to each day's use, and a five-point linearity check (100, 500, 900, 1500, and 2000 mOsm) was evaluated weekly to confirm that these criteria were within the expected values ​​specified by the manufacturer. Calibration and measurements were performed according to the Fiske Model 210 Micro-Osmometer user's guide. All vaccine samples tested were found to have osmolality measurements within the range of 604–616 mOsm / kg, similar to the 595–626 mOsm / kg detected in the SPG vehicle. These values ​​are consistent with those obtained for clinical trial material (Bris10 M2SR, lot number 15100251).

[0222] [Table 13]

[0223] The H3N2 FGHY1-M2SR vaccine lot was manufactured in M2 VeroA cells using a process similar to that of the intended clinical material and formulated in the same SPG buffer as the intended clinical material. Characterization studies showed that the purification process successfully removed host cell impurities (DNA and proteins) and simulated the purity of the clinical material, while maintaining high infectious titers and preserving the genome sequence and replication-deficient phenotype of the vaccine virus.

[0224] All references cited herein, including publications, patent applications, and patents, are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and to the same extent as if each reference were set forth in its entirety herein.

[0225] With regard to describing the present invention (particularly with regard to the claims that follow), use of the terms "a," "an," "the," and "at least one" and similar referents should be construed to cover both the singular and the plural, unless otherwise stated herein or clearly contradicted by context. The use of the term "at least one" after a list of one or more items (e.g., "at least one of A and B") should be construed to mean any one item (A or B) selected from the listed items or any combination of two or more of the listed items (A and B), unless otherwise stated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise stated. Recitation of ranges of values ​​herein is intended solely to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary phrases (e.g., "such as") provided herein is intended only to better illustrate the invention and does not impose limitations on the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0226] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors anticipate that those of ordinary skill in the art will employ such variations as appropriate, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. 1. An influenza virus comprising PB1, PB2, PA, NP, and NS gene segments, (a) the PB1 gene segment encodes a PB1 protein having the amino acid sequence of SEQ ID NO:7, the PB2 gene segment encodes a PB2 protein having the amino acid sequence of SEQ ID NO:15, the PA gene segment encodes a PA protein having the amino acid sequence of SEQ ID NO:17, the NP gene segment encodes an NP protein having the amino acid sequence of SEQ ID NO:6, and the NS gene segment encodes an NS1 protein having the amino acid sequence of SEQ ID NO:19; or (b) the PB1 gene segment encodes a PB1 protein having the amino acid sequence of SEQ ID NO:9, the PB2 gene segment encodes a PB2 protein having the amino acid sequence of SEQ ID NO:10, the PA gene segment encodes a PA protein having the amino acid sequence of SEQ ID NO:17, the NP gene segment encodes a NP protein having the amino acid sequence of SEQ ID NO:8, and the NS gene segment encodes an NS1 protein having the amino acid sequence of SEQ ID NO:

19. Influenza virus.

2. The PB1 gene segment encodes a PB1 protein having the amino acid sequence of SEQ ID NO:7, the PB2 gene segment encodes a PB2 protein having the amino acid sequence of SEQ ID NO:15, the PA gene segment encodes a PA protein having the amino acid sequence of SEQ ID NO:17, the NP gene segment encodes a NP protein having the amino acid sequence of SEQ ID NO:6, and the NS gene segment encodes an NS1 protein having the amino acid sequence of SEQ ID NO:

19. The influenza virus of claim 1.

3. The PB1 gene segment has the nucleotide sequence of SEQ ID NO:

2. The influenza virus of claim 1 or 2.

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

1.

5. The influenza virus of any one of claims 1 to 4, wherein the PB2 gene segment has the nucleotide sequence of SEQ ID NO:

14.

6. An influenza virus according to any one of claims 1 to 5, wherein (a) leucine at position 40, tryptophan at position 180, and asparagine at position 464 in the PB1 protein, and / or (b) leucine at position 116 and lysine at position 294 in the NP protein are conserved in at least one of the PB1 and NP proteins after at least 10 serial passages in a Vero cell line.

7. An influenza virus according to any one of claims 1 to 6, wherein (a) the leucine at position 40, the tryptophan at position 180, and the asparagine at position 464 in the PB1 protein, and / or (b) the leucine at position 116 and the lysine at position 294 in the NP protein are conserved in at least one of the PB1 and NP proteins after at least 10 serial passages in a Vero cell line stably expressing the M2 ion channel protein of influenza A virus.

8. 7. The influenza virus of any one of claims 1 to 6, wherein the influenza virus is an influenza A virus, and (a) the leucine at position 40, the tryptophan at position 180, and the asparagine at position 464 in the PB1 protein, and / or (b) the leucine at position 116 and the lysine at position 294 in the NP protein are conserved in at least one of the PB1 and NP proteins after at least 10 serial passages in a Vero cell line stably expressing the BM2 ion channel protein of influenza B virus.

9. The PB1 gene segment encodes a PB1 protein having the amino acid sequence of SEQ ID NO:9, the PB2 gene segment encodes a PB2 protein having the amino acid sequence of SEQ ID NO:10, the PA gene segment encodes a PA protein having the amino acid sequence of SEQ ID NO:17, the NP gene segment encodes a NP protein having the amino acid sequence of SEQ ID NO:8, and the NS gene segment encodes an NS1 protein having the amino acid sequence of SEQ ID NO:

19. The influenza virus of claim 1.

10. 10. The influenza virus of claim 1 or 9, wherein the PB1 gene segment has the nucleotide sequence of SEQ ID NO:

4.

11. 11. The influenza virus of any one of claims 1, 9, and 10, wherein the PB2 gene segment has the nucleotide sequence of SEQ ID NO:

5.

12. 12. The influenza virus of any one of claims 1 and 9 to 11, wherein the NP gene segment has the nucleotide sequence represented by SEQ ID NO:

3.

13. An influenza virus according to any one of claims 1 and 9 to 12, wherein (a) leucine at position 40, tryptophan at position 180, and serine at position 607 in the PB1 protein, (b) valine at position 504, isoleucine at position 467, and valine at position 529 in the PB2 protein, and / or (c) leucine at position 116 and arginine at position 311 in the NP protein are conserved in at least one of the PB1, PB2, and NP proteins after at least 10 serial passages of the virus in a Vero cell line.

14. An influenza virus according to any one of claims 1 and 9 to 13, wherein (a) leucine at position 40, tryptophan at position 180, and serine at position 607 in the PB1 protein, (b) valine at position 504, isoleucine at position 467, and valine at position 529 in the PB2 protein, and / or (c) leucine at position 116 and arginine at position 311 in the NP protein are conserved in at least the PB1, PB2, and NP proteins after at least 10 serial passages in a Vero cell line stably expressing the M2 ion channel protein of influenza A virus.

15. 15. The influenza virus of any one of claims 1 to 14, wherein at least one of the PB1, PB2, and PA gene segments comprises a promoter cytosine to uracil mutation at nucleotide position 4.

16. The influenza virus of any one of claims 1 to 15, wherein the influenza virus is a recombinant influenza virus.

17. 17. The influenza virus of any one of claims 1 to 16, wherein the virus further comprises an NA gene segment and an HA gene segment.

18. 18. The influenza virus of claim 17, wherein the HA gene segment encodes an HA protein having an amino acid sequence that includes at least one amino acid mutation in HA1.

19. 19. The influenza virus of claim 17 or 18, wherein the HA gene segment encodes an HA protein having an amino acid sequence that includes at least one amino acid mutation in HA2.

20. 20. The influenza virus of claim 19, wherein the at least one amino acid mutation in HA2 is an asparagine at position 107.

21. 21. The influenza virus of any one of claims 17 to 20, wherein the PB1, PB2, PA, NP, and NS gene segments are derived from a single influenza strain.

22. 22. The influenza virus of claim 21, wherein the HA gene segment is derived from an influenza strain that is different from the single influenza strain from which the PB1, PB2, PA, NP, and NS gene segments are derived.

23. 23. The influenza virus of claim 21 or 22, wherein the NA gene segment is derived from an influenza strain different from the single influenza strain from which the PB1, PB2, PA, NP, and NS gene segments are derived.

24. 24. The influenza virus of any one of claims 1 to 23, further comprising a mutated M gene segment.

25. 25. The influenza virus of claim 24, wherein the influenza virus does not encode a functional M2 protein.

26. 26. The influenza virus of any one of claims 1 to 25, wherein the virus is capable of replicating in human cells.

27. 9. The influenza virus according to any one of claims 1 to 8, wherein the virus exhibits enhanced growth in Vero cells under the same conditions as an influenza virus identical to the virus except for the absence of leucine at position 40, tryptophan at position 180 in the PB1 protein, and asparagine at position 464, leucine at position 116, and lysine at position 294 in the NP protein; or 15. The influenza virus of claims 1 and 9 to 14, wherein the virus exhibits enhanced growth in Vero cells under the same conditions as an influenza virus identical to the virus except for the absence of leucine at position 40, tryptophan at position 180, and serine at position 607 in the PB1 protein, valine at position 504, isoleucine at position 467, and valine at position 529 in the PB2 protein, and leucine at position 116 and arginine at position 311 in the NP protein.

28. A pharmaceutical preparation comprising the influenza virus of any one of claims 1 to 27.

29. 29. The pharmaceutical preparation of claim 28, wherein the pharmaceutical preparation is a vaccine.

30. 30. The pharmaceutical preparation of claim 29, wherein the vaccine is formulated as a monovalent vaccine.

31. 30. The pharmaceutical preparation of claim 29, wherein the vaccine is formulated as a bivalent vaccine.

32. 30. The pharmaceutical preparation of claim 29, wherein the vaccine is formulated as a trivalent vaccine.

33. 30. The pharmaceutical preparation of claim 29, wherein the vaccine is formulated as a tetravalent vaccine.

34. An influenza virus according to any one of claims 1 to 27 or a pharmaceutical formulation according to any one of claims 28 to 33 for use in inducing an immune response against influenza virus in a mammal.

35. 35. The influenza virus of claim 34, wherein the mammal is a human.

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

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