Influenza virus backbone

Abstract

To provide influenza virus exhibiting enhanced proliferation in Vero cells.SOLUTION: The invention provides an influenza virus that demonstrates enhanced growth in Vero cells. The influenza virus includes PB1, PB2, PA, NP, and NS gene segments encoding proteins having amino acid sequences with selected amino acids. Optionally, at least one of the PB1, PB2, and PA gene segments includes a cytosine to uracil promoter mutation at nucleotide position 4. The invention also provides a pharmaceutical formulation containing the influenza virus, as well as a method of eliciting an immune response in a mammal by administering the influenza virus to the mammal, and a method for generating the influenza virus.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Cross-reference of related applications This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 858,737, filed on 7 June 2019 (which is incorporated herein by reference in its entirety).

[0002] Incorporation by referencing electronically submitted properties A computer-readable nucleotide / amino acid sequence listing, submitted concurrently with this specification and identified as follows, is incorporated herein by reference in its entirety: one 73,600-byte ASCII (text) file named "749488SequenceListing_ST25.txt" created on 2 June 2020. [Background technology]

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

[0004] While vaccination is the best way to prevent influenza, influenza vaccines must be frequently re-prescribed because influenza viruses are subject to both continuous and discontinuous antigenic mutations. Furthermore, influenza viruses, especially influenza A, generally exhibit high mutation and evolutionary rates, meaning that influenza vaccine strains may not match circulating strains, resulting in minimal vaccine effectiveness. However, if the circulating influenza virus is a good match for the influenza vaccine, vaccination can reduce the risk of influenza-related illness by 40% to 60% of the total population. Therefore, researchers have studied vaccines that can induce cross-protective immunity across various influenza subtypes. One such example is a vaccine containing attenuated live influenza viruses that do not express the functional M2 protein (e.g., 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 lower risk of antigenic mutation in the virus. For example, M2SR vaccines can be grown in Vero cells that stably express the M2 protein. However, vaccine production in cell culture often results in undesirable yields. Furthermore, vaccine production in Vero cells is relatively inefficient because MDCK cells are generally more tolerant than Vero cells.

[0006] It has been shown that vaccine production is enhanced by modifications to the viral backbone, i.e., the six internal gene segments consisting of PB1, PB2, PA, NP, M, and NS. For example, the high-yielding vaccine backbone "PR8-HY," developed and described by 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 generalized and seasonal influenza vaccines in both cell culture and egg culture systems. However, these modifications described in the art have shown that, under particularly favorable manufacturing conditions, the viral production in Vero cells is enhanced. It was not effective in increasing viral replication. Therefore, it is necessary to enhance viral replication in Vero cells so that vaccines such as the M2SR vaccine can be produced more efficiently and effectively. [Overview of the project]

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

[0008] The present invention also provides a pharmaceutical preparation containing the influenza virus, a method for inducing an immune response in a mammal, which includes administering the influenza virus to the mammal, and a method for producing the influenza virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] FIG. 1A is a graph of virus titer (log TCID50 / ml) - time (days post - infection) showing the growth curve in Vero cells for the A / Massachusetts / 15 / 2013 M2SR virus (i.e., M2SR - MA15V virus) containing the Vero - adapted HA protein and including the UW - PR8 ("HG") and PR8 - HY ("HY") backbones. [Figure 1B] FIG. 1B is a graph of virus titer (TCID50 / ml) - time (days post - infection) showing the growth curve in Vero cells for the A / Brisbane / 10 / 2007 M2SR virus (i.e., Bris10 M2SR) including the HG and HY backbones. [Figure 1C] FIG. 1C is a graph of HA titer (HA / 50μl) - time (days post - infection) in Vero cells for the Bris10 M2SR - HG and Bris10 M2SR - HY viruses. [Figure 2] FIG. 2 is a graph of virus titer (log TCID50 / ml) - time (days post - infection) showing the growth curve in Vero cells for the FGHY1 - M2SR - MA15V and FGHY2 - M2SR - CA07 viruses compared with the growth curves of the HY - M2SR - MA15V and HY - M2SR - CA07 viruses. [Figure 3] FIG. 3 is a graph of virus titer (TCID50 / ml) - time (days post - infection) showing the growth curve in Vero cells for the FGHY1 - M2SR - MA15V and FGHY2 - M2SR - MA15V viruses compared with the growth curves of the HG - M2SR - MA15V and HY - M2SR - MA15V viruses. [Figure 4A] Figure 4A is a graph showing the viral titer (TCID50 / ml)-time (days after infection) of the FGHY1-M2SR-Bris10 and FGHY2-M2SR-Bris10 viruses in Vero cells, compared with the proliferation curves of the HY-M2SR-Bris10 and HG-M2SR-Bris10 viruses. [Figure 4B] Figure 4B is a graph showing the HA titer (HA / 50μl)-time (days after infection) in Vero cells for FGHY1-M2SR-Bris10 and FGHY2-M2SR-Bris10 viruses, compared to HY-M2SR-Bris10 and HG-M2SR-Bris10 viruses. [Figure 5A] Figure 5A is a graph of viral titer (TCID50 / ml)-time (days after infection) showing the proliferation curve in Vero cells for the A / Michigan / 45 / 2015 M2SR virus (i.e., FGHY1-M2SR-MI45V virus) containing the Vero-adapted HA protein and FGHY1 backbone, compared with the proliferation curves for FGHY1-M2SR-MI45, HY-M2SR-MI45, and HG-M2SR-MI45 viruses. [Figure 5B] Figure 5B is a graph of viral titer (log TCID50 / ml)-time (days after infection) showing the proliferation curve in Vero cells for the A / Hong Kong / 4801 / 2014 M2SR virus (i.e., FGHY1-M2SR-HK4801 virus) containing the FGHY1 backbone, compared with the proliferation curves for HY-M2SR-HK4801 and HG-M2SR-HK4801 viruses. [Figure 5C] Figure 5C is a graph of viral titer (log TCID50 / ml)-time (days after infection) showing the proliferation curve in Vero cells for the A / Vietnam / 1203 / 04 M2SR virus (i.e., FGHY1-M2SR-avVN1203 virus) containing the FGHY1 backbone, compared with the proliferation curves for HY-M2SR-avVN1203 and HG-M2SR-avVN1203 viruses. [Figure 6] Figure 6 is a graph showing the viral titers (log TCID50 / ml) of seasonal and generalized influenza M2SR viruses, including HG (i.e., UW-PR8), HY (i.e., PR8-HY), and FGHY1 backbone, in M2CK cells and M2VeroA cells. [Figure 7A] Figure 7A is a table showing HA mutations in Vero-adapted H1N1 viruses. [Figure 7B] Figure 7B is a table showing HA mutations in Vero-adapted H3N2 viruses. [Figure 7C] Figure 7C is a graph showing the proliferation curve of the A / Singapore / INFIMH-16-0019 / 2016 M2SR virus (i.e., the M2SR Sing2016 virus), including various Vero adaptive HA mutations, in terms of viral titer (log10 M2CK TCID50 / ml)-time (days after infection). [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 day 1 and day 2 post-infection for viruses containing HA and NA derived from A / Brisbane / 10 / 2007 (i.e., Bris10 virus) with various backbones (i.e., FGHY1, UW-PR8 ("HG"), and IVR-147). [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 day 1 and day 2 post-infection for viruses containing HA and NA derived from A / Singapore / INFIMH-16-0019 / 2016 (i.e., Sing2016 virus) with various backbones (i.e., FGHY1, UW-PR8 ("HG")) [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 various backbones (i.e., FGHY1, UW-PR8 ("HG"), and IVR-147) on post-infection day 1. [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 various backbones (i.e., FGHY1, UW-PR8 ("HG")) on day 1 post-infection. [Figure 10] Figure 10 is a graph showing the percentage change in body weight over time (days after inoculation) in mice inoculated with the FGHY1-M2SR mutant. The mean percentage body weight and the standard error of that mean are shown. [Figure 11] Figure 11 is a graph showing the anti-H3 HA ELISA IgG titers in the serum of ferrets immunized with 1 × 10⁸ TCID50 H3N2 FGHY1-M2SR, 1 × 10⁹ H3N2 FGHY1-M2SR, or SPG (control) on days 14, 30, and 49 of the study. [Figure 12] Figure 12 is a graph showing the anti-H3 HAI titers in the serum of ferrets immunized with 1 × 10⁸ TCID50 H3N2 FGHY1-M2SR, 1 × 10⁹ H3N2 FGHY1-M2SR, or SPG (control) on days 14, 30, and 49 of the study. [Figure 13] Figure 13 is a graph showing the anti-H3 PRNT50 titer in the serum of ferrets immunized with 1 × 10⁸ TCID50H3N2 FGHY1-M2SR, 1 × 10⁹ H3N2 FGHY1-M2SR, or SPG (control) on days 14, 30, and 49 of the study. [Figure 14A] Figure 14A is a graph showing the anti-H1 HA titer-time (days after immunization) in serum from mice immunized with monovalent H1N1 FGHY1-M2SR, monovalent H3N2 FGHY1-M2SR, divalent FGHY1-M2SR, trivalent FGHY1-M2SR+BM2SR-Vic, trivalent FGHY1-M2SR+BM2SR-Yam, tetravalent, or SPG (control). [Figure 14B]Figure 14B is a graph showing the anti-H3 HA titer-time (days after immunization) in serum from mice immunized with monovalent H1N1 FGHY1-M2SR, monovalent H3N2 FGHY1-M2SR, divalent 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 body weight over time (days after inoculation) of mice immunized with monovalent H1N1 FGHY1-M2SR, monovalent H3N2 FGHY1-M2SR, tetravalent FGHY1-M2SR, or SPG (control)-time (days after challenge) after inoculation with influenza A / California / 07 / 2009 (H1N1) virus. [Figure 16] Figure 16 is a graph showing the percentage change in body weight over time (days after vaccination) in 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 the anti-influenza A / H1 HA serum IgG ELISA titer-time (days after 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 the anti-influenza A / H3 HA serum IgG ELISA titer-time (number of days after 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 17C]Figure 17C is a graph showing the anti-influenza B / Yam HA serum IgG ELISA titer-time (days after 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 17D] Figure 17D is a graph showing the anti-influenza B / VicHA serum IgG ELISA titer-time (number of days after 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 18A] Figure 18A is a graph showing IgG ELISA titers and HA1 test antigens (A / H1, A / H3, B / Yam, or B / Vic) in tracheal-pulmonary lavage fluid from mice immunized with monovalent H3N2 FGHY1-M2SR, tetravalent FGHY1-M2SR, FluMist Quadrivalent, Fluzone Quadrivalent, Fluzone High Dose, or SPG (control). [Figure 18B] Figure 18B is a graph showing the anti-influenza HA1 IgA ELISA titer and HA1 test antigen (MI45, SingEgg, Phuket, or CO / 06) in tracheal-pulmonary lavage fluid from mice immunized with monovalent H3N2 FGHY1-M2SR, tetravalent FGHY1-M2SR, FluMist Quadrivalent, Fluzone Quadrivalent, Fluzone High Dose, or SPG (control). [Figure 19]Figure 19 is a graph showing the percentage change in body weight over time (days after vaccination) in mice immunized with H3N2 FGHY1-M2SR, tetravalent FGHY1-M2SR, FluMist Quadrivalent, Fluzone Quadrivalent, Fluzone High Dose, or SPG (control) after influenza A / H1N1 vaccination. [Modes for carrying out the invention]

[0010] Detailed description of the invention The influenza virus of the present invention may be any type of influenza virus. For example, the influenza virus may be any subtype of influenza A. In some embodiments, the influenza virus may be a generalized influenza A virus (e.g., H5N1). In other embodiments, the influenza virus may be a seasonal influenza A virus (e.g., H1N1 or H3N2). In some embodiments, the influenza virus may be a recombinant influenza virus. As used herein, a recombinant influenza virus (e.g., a reassembled influenza virus) is an influenza virus that contains genetic material (e.g., a gene segment) derived from a genetically different influenza virus (e.g., a heterogeneous gene segment). The influenza virus may also be an isolated influenza virus.

[0011] As used herein, the term “gene segment” refers to a nucleotide sequence that codes for a viral protein. A gene segment may be represented by a cDNA (complementary DNA) sequence that codes for viral RNA (vRNA), i.e., sequence numbers 1-5, 11, 14, 16, and 18 that codes for a viral protein.

[0012] As used herein, the term “backbone” refers to the influenza gene segments encoding PB1, PB2, PA, NP, NS1 and / or NS2, as well as the M protein. 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 particular position in an amino acid sequence. In some embodiments, the selected amino acid is the result of a gene mutation into the 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 may encode a protein (i.e., PB1 protein) comprising 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 may optionally include a mutation in the promoter of the nucleotide at position 4, from cytosine to uracil.

[0015] The selected amino acids can be acquired by gene mutations into the 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 to the RNA-dependent RNA polymerase activity domain. Generally, aspartic acid at position 464 is highly conserved among influenza viruses isolated in egg and MDCK cells. Although the role of this amino acid is not specified, the amino acid change to asparagine(N) observed at this position may affect the conformation of the PB1 protein, potentially influencing its interaction with host cell factors and thus its polymerase activity in Vero cells. Furthermore, since histidine at position 465 of the PB1 protein interacts with glutamic acid at position 243 of the PA protein, the amino acid change 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 region and the PB2 binding region, which suggests 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 also encode a protein (i.e., the PB2 protein) containing at least one selected amino acid. In preferred embodiments, 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 in the promoter of the nucleotide at position 4. 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 (potion) 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 the host's capped RNA and utilizes the cap from the host RNA to create 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 of 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 acid protein) gene segment of the present invention may also encode a protein (i.e., a PA protein) containing at least one selected amino acid. In a preferred embodiment, the selected amino acid includes lysine at position 401. The PA gene segment may optionally contain a mutation in the promoter of the nucleotide at position 4, from cytosine to uracil.

[0018] The NP (nucleoprotein) gene segment of the present invention may also encode a protein (i.e., an NP protein) containing 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 position 294 of the NP protein And 311 are located within the main body of the NP protein, and they do not function as either nuclear localization signals or nuclear export signals.

[0019] The NS (non-structural) gene segment of the present invention may also encode a protein (i.e., NS1 and / or NS2 protein) comprising 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 includes a PB1 gene segment encoding a protein (i.e., PB1 protein) having selected amino acids at positions 40, 180, and 464, namely leucine at position 40, tryptophan at position 180, and asparagine at position 464. The PB1 gene segment may have the nucleotide sequence represented by SEQ ID NO: 2. The PB1 gene segment may encode a protein (i.e., PB1 protein) having the amino acid sequence of SEQ ID NO: 7. In another embodiment of the present invention, the influenza virus may include a PB2 gene segment encoding a protein (i.e., PB2 protein) having a selected amino acid at position 504, namely valine at position 504. The PB2 gene segment may have the nucleotide sequence represented by SEQ ID NO: 14. The PB2 gene segment may encode a protein (i.e., PB2 protein) having the amino acid sequence of SEQ ID NO: 15. The NP gene segment of this embodiment may encode a protein having selected amino acids at positions 116 and 294, i.e., leucine at position 116 and lysine at position 294 (i.e., the NP protein). The NP gene segment may have the 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 having 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 NS1 and / or NS2 proteins). The PA gene segment may have the 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 the nucleotide sequence represented by SEQ ID NO: 18. The NS gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 19 (i.e., the NS1 protein).The NS gene segment may 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 may also include a mutation in the promoter of the nucleotide at position 4, from cytosine to uracil.

[0021] In another embodiment of the present invention, the influenza virus includes a PB1 gene segment encoding a protein (i.e., PB1 protein) having selected amino acids at positions 40, 180, and 607, namely leucine at position 40, tryptophan at position 180, and serine at position 607. The PB1 gene segment may have the nucleotide sequence represented by SEQ ID NO: 4. The PB1 gene segment may encode a protein (i.e., PB1 protein) having the amino acid sequence of SEQ ID NO: 9. In another embodiment of the present invention, the influenza virus may include a PB2 gene segment encoding a protein (i.e., PB2 protein) having selected amino acids at positions 504, 467, and 529, namely valine at position 504, isoleucine at position 467, and valine at position 529. The PB2 gene segment may have the nucleotide sequence represented by SEQ ID NO: 5. The PB2 gene segment may encode a protein (i.e., PB2 protein) having the amino acid sequence of SEQ ID NO: 10. The NP gene segment of this embodiment has amino acids selected at positions 116 and 311, namely leucine at position 116 and alpha at position 311. The NP gene segment may encode a protein containing ginine (i.e., an NP protein). The NP gene segment may have the 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., an 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., PA protein and NS1 and / or NS2 protein). The PA gene segment may have the 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., a PA protein). The NS gene segment may have the nucleotide sequence represented by SEQ ID NO: 18. The NS gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 19 (i.e., an NS1 protein). The NS gene segment may 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 may also include a mutation in the promoter of the nucleotide at position 4, from cytosine to uracil.

[0022] The selected amino acids in the embodiment confer enhanced proliferation characteristics to the influenza virus, particularly in most of the backbone proteins, compared to an influenza virus that is identical to the influenza virus under the same conditions except for the absence of the selected amino acids. For example, the influenza virus of the present invention exhibits enhanced proliferation in Vero cells.

[0023] The influenza virus of the present invention may also include an M (membrane protein) gene segment. In one embodiment of the present invention, the M gene segment may be a mutant gene segment derived from influenza A, such that the virus lacks the expression of a functional M2 protein. Such a virus is referred to herein as the "M2SR" virus. The M2SR virus is a single-replication influenza virus. The M gene segment of the M2SR virus may be represented by Sequence ID No. 11. The M gene segment may encode a protein, e.g., a truncated M2 protein having the amino acid sequence of Sequence ID No. 12. The M2SR virus can be grown in Vero cells that stably express the M2 protein (i.e., M2VeroA cells) and become capable of multi-cycle replication. High yields in Vero cells are independent of mutations in the M gene segment. Therefore, the influenza virus of the present invention may include an M gene segment encoding a functional M2 protein. (B) Surface protein

[0024] In further embodiments of the present invention, the influenza virus comprises NA (neuraminidase) and HA (hemagglutinin) gene segments. In one embodiment of the present invention, the HA gene segment may encode an HA protein having an amino acid sequence comprising at least one selected amino acid (e.g., 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, at least one amino acid mutation in the HA2 subunit may be asparagine at position 107. Such a mutation may also contribute to enhanced viral replication 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 may be derived from an influenza strain different from the single influenza strain from which the PB1, PB2, PA, NP, and NS gene segments are derived. Similarly, the NA gene segment may be derived from an influenza strain different from the single influenza strain from which the PB1, PB2, PA, NP, and NS gene segments are derived. It may be derived from an influenza strain. Therefore, the influenza virus of the present invention may be a generalized influenza virus (e.g., H5N1, H7N9) or a seasonal influenza virus (e.g., H1N1, H3N2, influenza B). (C) Properties of the influenza virus

[0026] The influenza virus backbone of the present invention confers high proliferation characteristics to influenza viruses, particularly in Vero cells, regardless of the type of influenza virus (e.g., seasonal or generalized influenza virus). The influenza virus of the present invention exhibits high yields even in manufacturing processes using low MOI (e.g., 0.001). MOI refers to the average number of pathogens (e.g., viruses) per infectious target (e.g., cell). Lower MOIs are used when multiple infection cycles are required (e.g., in the production of viral vaccines). Current Good Manufacturing Practices (Good Manufacturing Practices) regulations, enforced by the FDA, generally require the use of the lowest possible MOI while still producing high viral yields. 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 of the backbone proteins, particularly the PB1, PB2, PA, NP, and NS1 proteins, are highly conserved even when grown at a low MOI. For example, in one embodiment of the present invention, the selected amino acids are conserved in at least one of the PB1, PB2, 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 consecutive passages in the Vero cell line. In one embodiment, the Vero cell line may include Vero cells that stably express 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 include Vero cells that stably express the BM2 ion channel protein of influenza B virus (i.e., BM2Vero cells). BM2 is considered to be a functional counterpart of influenza A virus M2. In such embodiments, the selected amino acids can be conserved even when the influenza virus is influenza A virus.

[0028] Genetically modified Vero cells (i.e., those expressing influenza M2 or BM2 proteins) behave like normal Vero cells and support replication of influenza A or B viruses comparable to that of normal Vero cells. The viral titer of M2SR virus in M2VeroA cells is comparable to that of a replicating influenza virus expressing functional M2 in an unmodified Vero cell line. Furthermore, the viral titer of BM2SR virus (i.e., an influenza virus containing a mutated M gene segment from influenza B, resulting in the absence of a functional BM2 protein) in BM2Vero cells is comparable to that of a replicating influenza virus expressing functional BM2 in an unmodified Vero cell line. Therefore, M2SR and BM2SR viruses behave like replicating influenza viruses in M2VeroA and BM2Vero cell lines, respectively.

[0029] In one embodiment of the present invention, the influenza virus can replicate in human cells. Method for producing influenza virus

[0030] A method for producing influenza viruses is also provided herein, and the produced influenza viruses are proteins having selected amino acids, i.e., disclosed herein. The present invention includes PB1, PB2, PA, NP, and NS gene segments that express the recombinant influenza virus of the present invention.

[0031] In one embodiment of the method of the present invention, a method for producing recombinant influenza virus comprises serial passage of a recombinant influenza virus (e.g., a first influenza virus) in Vero cells to produce a prepared influenza virus (e.g., a second influenza virus). The first influenza virus may include PB1, PB2, PA, NP, and NS gene segments expressing proteins, i.e., PB1, PB2, PA, NP, and NS1 proteins having selected amino acids, as described with respect to the influenza virus of the present invention. For example, the first influenza virus may include a PB1 gene segment encoding a protein (i.e., PB1 protein) containing leucine at position 40, tryptophan at position 180, aspartic acid at position 464, and proline at position 607. The PB2 gene segment of the first influenza virus may encode a protein (i.e., PB2 protein) containing methionine at position 467, valine at position 504, and isoleucine at position 529. The PA gene segment of the first influenza virus may encode a protein containing lysine at position 401 (i.e., the PA protein). The NP gene segment of the first influenza virus may encode a protein containing leucine at position 116, glutamic acid at position 294, and glutamine at position 311 (i.e., the NP protein). The NS gene segment of the first influenza virus may encode a protein containing proline at position 30 and lysine at position 118 (i.e., the NS1 protein). The PB1, PB2, and PA gene segments of the first influenza virus may optionally contain uracil at position 4. In one embodiment of the present invention, the second influenza virus (e.g., the produced influenza virus) is produced after at least four or at least five consecutive passages of the first influenza virus in Vero cells.

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

[0033] The present invention provides a pharmaceutical formulation (e.g., a vaccine or other immunogenic composition) containing the influenza virus of the present 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” means 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] At least one pharmaceutically acceptable carrier or excipient may be any suitable pharmaceutically acceptable carrier or excipient, many of which are known in the art. Exemplary pharmaceutically acceptable carriers or excipients include components that maintain the pH of a pharmaceutical formulation (e.g., buffers), adjust tonicity (e.g., tonic modifiers such as inorganic salts), improve the stability and / or immunogenicity of proteins (e.g., viruses), improve mucosal adhesion, prevent protein aggregation, and / or preserve a pharmaceutical formulation (e.g., preservatives). For example, a pharmaceutically acceptable carrier or excipient may include at least one of inorganic salts, surfactants, amino acids, polymers or polymer compounds (e.g., proteins, polysaccharides, or hydrogels), chelating agents, sugars, polyols, and / or adjuvants (e.g., any substance that enhances a particular immune response) (many of which are known in the art). Because a particular carrier or excipient may serve more than one purpose in a formulation, the following embodiments are not limited to those described 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., 1×DPBS), histidine buffer, sodium citrate buffer, and sucrose phosphate glutamate (SPG) buffer. The PBS and / or DPBS preparation may, for example, contain sodium chloride, potassium chloride, monobasic potassium phosphate, and dibasic sodium phosphate, and may further contain calcium chloride and / or magnesium chloride. In some embodiments, the PBS and / or DPBS preparation contains 136.9 mM sodium chloride, 2.67 mM potassium chloride, 1.47 mM monobasic potassium phosphate, and 8.1 mM dibasic sodium phosphate, but any suitable PBS and / or DPBS preparation (many of which are known in the art) may be used as the buffer in the pharmaceutical formulation.

[0037] The buffer may be present in the pharmaceutical formulation at any preferred concentration. The buffer may be present in the pharmaceutical formulation at concentrations of 0.1 mM or higher, 1 mM or higher, 10 mM or higher, 20 mM or higher, 30 mM or higher, 40 mM or higher, 50 mM or higher, 60 mM or higher, 70 mM or higher, 80 mM or higher, 90 mM or higher, 100 mM or higher, 120 mM or higher, 140 mM or higher, 160 mM or higher, 180 mM or higher, 200 mM or higher, 250 mM or higher, 300 mM or higher, 350 mM or higher, 400 mM or higher, 450 mM or higher, or 500 mM or higher. Alternatively, or additionally, the buffer may be present in the pharmaceutical formulation at concentrations 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 the range where any of the aforementioned endpoints are the boundary. For example, the buffer may be present in the pharmaceutical formulation at concentrations of 0.1 mM to 1000 mM, 0.1 mM to 500 mM, 0. It can exist at concentrations such as 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, and 100 mM to 500 mM.

[0038] In further embodiments, the buffer may be present in the pharmaceutical formulation at a percentage concentration (e.g., volume / volume percentage (%v / v); weight / volume percentage (%w / v); or weight / weight percentage (%w / w)). The 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 the range where any of the aforementioned endpoints are boundaries. For example, buffer solutions may be present in pharmaceutical formulations at percentage concentrations such as 0.1%-60%, 1%-60%, 10%-60%, 0.1%-50%, 1%-50%, 10%-50%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-40%, and 40%-50%.

[0039] A buffer can maintain the pH of a pharmaceutical formulation at any suitable pH. For example, a buffer can maintain the pH of a 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, a buffer can maintain the pH of a 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. A buffer can maintain the pH of a pharmaceutical formulation within a range where any of the aforementioned endpoints is the boundary. For example, a buffer can maintain the pH of a pharmaceutical formulation at pH levels such as 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 tonic modifier may be present in the pharmaceutical formulation. In certain embodiments, one or more inorganic salts are present in the pharmaceutical formulation as tonic modifiers. The inorganic salt(s) may be at least one of sodium chloride (NaCl), magnesium sulfate (MgSO4), and magnesium chloride (MgCl2). The tonic modifier, e.g., inorganic salt(s), may be present in the pharmaceutical formulation in any suitable amount. Tension modifiers, e.g., inorganic salts (or more), may be present in pharmaceutical preparations at concentrations of 0.1 mM or higher, 0.2 mM or higher, 0.4 mM or higher, 0.6 mM or higher, 0.8 mM or higher, 1 mM or higher, 1.2 mM or higher, 1.4 mM or higher, 1.6 mM or higher, 1.8 mM or higher, 2 mM or higher, 3 mM or higher, 4 mM or higher, 5 mM or higher, 6 mM or higher, 7 mM or higher, 8 mM or higher, 9 mM or higher, 10 mM or higher, 20 mM or higher, 30 mM or higher, 40 mM or higher, 50 mM or higher, 100 mM or higher, 200 mM or higher, 300 mM or higher, 400 mM or higher, 500 mM or higher, 600 mM or higher, 700 mM or higher, 800 mM or higher, 900 mM or higher, 1000 mM or higher, or 1500 mM or higher. Alternatively, or additionally, tonicity modifiers, e.g., inorganic salts, may be added to pharmaceutical formulations at concentrations 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 They may be present at concentrations 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. Tension modifiers, e.g., inorganic salts (plural) may be present in pharmaceutical formulations at any concentration within the range where any of the aforementioned endpoints are boundaries. For example, tonicity modifiers, e.g., inorganic salts (multiple may be used), are used in pharmaceutical formulations 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, and 0.1 mM. M~250mM, 0.1mM~100mM, 0.1~50mM, 0.1mM~10mM, 1mM~2000mM, 1mM~1500mM, 1mM~1000mM, 1mM~5 00mM, 1mM~250mM, 1mM~100mM, 1mM~50mM, 1mM~10mM, 10mM~2000mM, 10mM~1500mM, 10mM~1000mM It can exist at concentrations such as 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, and 500 mM to 1000 mM.

[0041] In further embodiments, inorganic salts 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)). Tension modifiers, e.g., inorganic salts, 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, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more. Alternatively or additionally, tension modifiers, e.g., inorganic salts, 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. Tension modifiers, e.g., inorganic salts, may be present in pharmaceutical formulations at any percentage concentration within the range where any of the aforementioned endpoints are the boundary. For example, tension modifiers, e.g., inorganic salts, may be present in pharmaceutical formulations at percentage concentrations such as 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, surfactants 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)). Surfactants 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, surfactants may be present in the pharmaceutical formulation at percentage concentrations 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. Surfactants may be present in the pharmaceutical formulation at any percentage concentration within the range where any of the aforementioned endpoints are the boundary. For example, surfactants may be present in the pharmaceutical formulation at percentage concentrations such as 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 concentrations 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 concentrations 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. It is possible. Amino acids may be present in pharmaceutical formulations at any concentration within the range where any of the aforementioned endpoints are the boundary. For example, amino acids may be present in pharmaceutical formulations at concentrations such as 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, amino acids are present in the pharmaceutical formulation at a percentage concentration (e.g., volume / volume percentage (%v / v); weight / volume percentage (%w / v); or weight / weight percentage (%w / w)). Amino acids 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, amino acids 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. Amino acids(s) may be present in a pharmaceutical preparation at any percentage concentration within a range where any of the aforementioned endpoints are the boundary. For example, amino acids(s) may be present in a pharmaceutical preparation at percentage concentrations such as 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 may be present in the pharmaceutical formulation. The polymer or polymer compound may be, for example, a protein, polysaccharide, hydrogel, or any other suitable polymer or polymer compound, many of which are known in the art. For example, the polymer or polymer compound may be recombinant human serum albumin (rHSA), serum albumin (SA), gelatin, hydroxyethyl starch (HES), chitosan, dextran (DEX70K, DEX40K), and polyvinylpyrrolidone (PVP40K).

[0046] Polymers or polymer compounds may be present in a pharmaceutical formulation in any suitable amount. Polymers or polymer compounds may be present in a pharmaceutical formulation at a percentage concentration (e.g., volume / volume percentage (%v / v); weight / volume percentage (%w / v); or weight / weight percentage (%w / w)). Polymers or polymer compounds may be present in a 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, polymers or polymer compounds 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. Polymers or polymer compounds may be present in the pharmaceutical formulation at any percentage concentration within the range where any of the aforementioned endpoints are the boundary. For example, polymers or polymer compounds may be present in the pharmaceutical formulation at percentage concentrations such as 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 may be present in the pharmaceutical formulation. The chelating agent may be, for example, ethylenediaminetetraacetic acid (EDTA), amidoxime compounds (AOX), and / or dithiothreitol (DTT). The chelating agent may be present in the pharmaceutical formulation at any suitable concentration. The chelating agent may be present in the pharmaceutical formulation at concentrations of 10 μM or higher, 20 μM or higher, 30 μM or higher, 40 μM or higher, 50 μM or higher, 60 μM or higher, 70 μM or higher, 80 μM or higher, 90 μM or higher, 100 μM or higher, 120 μM or higher, or 150 μM or higher. Alternatively, Alternatively, chelating agents may be present in pharmaceutical formulations at concentrations 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. Chelating agents may be present in pharmaceutical formulations at any concentration within the range where any of the aforementioned endpoints are the boundary. For example, chelating agents may be present in pharmaceutical formulations at concentrations such as 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 higher, 0.2 mM or higher, 0.4 mM or higher, 0.6 mM or higher, 0.8 mM or higher, 1 mM or higher, 1.2 mM or higher, 1.4 mM or higher, 1.6 mM or higher, 1.8 mM or higher, 2 mM or higher, 3 mM or higher, 4 mM or higher, 5 mM or higher, 6 mM or higher, 7 mM or higher, 8 mM or higher, 9 mM or higher, 10 mM or higher, 20 mM or higher, 3 It may be present at concentrations of 0 mM or higher, 40 mM or higher, 50 mM or higher, 60 mM or higher, 70 mM or higher, 80 mM or higher, 90 mM or higher, or 100 mM or higher, 200 mM or higher, 300 mM or higher, 400 mM or higher, 500 mM or higher, 600 mM or higher, 700 mM or higher, 800 mM or higher, 900 mM or higher, 1000 mM or higher, or 1500 mM or higher. Alternatively, or additionally, sugars may be present in pharmaceutical preparations at concentrations 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, and 45 mM or less. It may be present at concentrations of 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. Sugars (multiple sugars) may be present in the pharmaceutical preparation at any concentration within the range where any of the aforementioned endpoints are the boundary.For example, sugars (multiple concentrations possible) are present in pharmaceutical preparations in concentrations of 0.1mM-2000mM, 0.1mM-1500mM, 0.1mM-1000mM, 0.1mM-500mM, 0.1mM-250mM, 0.1mM-100mM, 0.1mM-50mM, 0.1mM-10mM, 1mM-2000mM, 1mM-1500mM, 1mM-1000mM, 1mM-500mM, 1mM-250mM, 1mM-100mM, 1mM-50mM, and 1mM-10mM. It can exist at concentrations such as M, 10mM-2000mM, 10mM-1500mM, 10mM-1000mM, 10mM-500mM, 10mM-250mM, 10mM-100mM, 10mM-50mM, 100mM-2000mM, 100mM-1500mM, 100mM-1000mM, 100mM-500mM, 100mM-250mM, 500mM-2000mM, 500mM-1500mM, and 500mM-1000mM.

[0049] In other embodiments, sugars may be present in the pharmaceutical formulation at a percentage concentration (e.g., volume / volume percentage (%v / v); weight / volume percentage (%w / v); or weight / weight percentage (%w / w)). Sugars 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, or 40% or more. Alternatively or additionally, sugars 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. Sugars may be present in the pharmaceutical formulation at any percentage concentration within the range where any of the aforementioned endpoints are the boundary. For example, sugars (multiple sugars are possible) are present in pharmaceutical preparations at concentrations of 0.1% to 50%, 1% to 50%, 10% to 50%, 0.1% to 20%, 1% to 20%, 10% to 20%, and 0.1%. It can exist at percentage concentrations such as 10%, 1% to 10%, etc.

[0050] Any suitable polyol may be present in the pharmaceutical formulation. The polyol may be, for example, sorbitol and / or mannitol. The polyol may be present in the pharmaceutical formulation at any suitable concentration. The polyol may be present in the pharmaceutical formulation at concentrations of 0.1 mM or higher, 1 mM or higher, 10 mM or higher, 20 mM or higher, 30 mM or higher, 40 mM or higher, 50 mM or higher, 60 mM or higher, 70 mM or higher, 80 mM or higher, 90 mM or higher, 100 mM or higher, 120 mM or higher, 140 mM or higher, 160 mM or higher, 180 mM or higher, 200 mM or higher, 250 mM or higher, 300 mM or higher, 350 mM or higher, 400 mM or higher, 450 mM or higher, or 500 mM or higher. Alternatively, or additionally, polyols may be present in pharmaceutical formulations at concentrations 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. Polyols may be present in pharmaceutical formulations at any concentration within the range where any of the aforementioned endpoints are boundaries. For example, polyols can be present in pharmaceutical formulations at concentrations such as 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, and 100 mM to 500 mM.

[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, or 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. Polyols may be present in pharmaceutical formulations at any percentage concentration within the range where any of the aforementioned endpoints are the boundary. For example, polyols may be present in pharmaceutical formulations at percentage concentrations such as 0.1%~50%, 1%~50%, 5%~50%, 10%~50%, 15%~50%, 0.1%~25%, 1%~25%, 5%~25%, 10%~25%, 15%~25%, 0.1%~15%, 1%~15%, 5%~15%, 10%~15%, 0.1%~10%, 1%~10%, 5%~10%, 0.1%~5%, and 1%~5%.

[0052] In one embodiment, the pharmaceutical formulation comprises the influenza virus of the present 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 the influenza virus of the present invention, 0.5 M sucrose, 0.3 M trehalose, and 0.05% polysorbate 20.

[0053] At least one pharmaceutically acceptable carrier or excipient may be an ingredient that helps to bind components of a pharmaceutical formulation (e.g., a binder). Examples of binders include, but are not limited to, proteins (e.g., gelatin), polymers (e.g., polyethylene glycol, polyvinylpyrrolidone), and / or polysaccharides or their derivatives (e.g., starch and cellulose). At least one pharmaceutically acceptable carrier or excipient may be an ingredient that increases the bulk of a pharmaceutical formulation (e.g., a bulking agent, diluent, and / or filler). Examples of such bulking agents include, but are not limited to, polysaccharides or their derivatives, sugars, and / or inorganic compounds. pharmaceutically acceptable carriers or excipients may enhance the taste and / or appearance of a pharmaceutical formulation. These may be components that absorb or adsorb liquids or gases (e.g., flavoring agents, sweeteners, and / or coloring agents). pharmaceutically acceptable carriers or excipients may be components that protect pharmaceutical formulations from moisture by absorbing or adsorbing liquids or gases (e.g., adsorbents). Examples of adsorbents include, but are not limited to, starch, calcium phosphate, and / or colloidal silicon dioxide. pharmaceutically acceptable carriers or excipients may be components that promote the dissolution of pharmaceutical formulations (e.g., disintegrants), such as starch, cellulose, and / or any other polymer known in the art, or derivatives thereof (e.g., cross-linked polyvinylpyrrolidone or sodium carboxymethylcellulose).

[0054] In some embodiments, pharmaceutically acceptable carriers or excipients are components (e.g., flow enhancers) that reduce interparticle adhesion and / or optimize the flow of the product during and after the manufacture of the pharmaceutical formulation. Examples of flow enhancers include, but are not limited to, talc, colloidal silicon dioxide, and corn starch. In particular, when the pharmaceutical formulation is formulated as an oral preparation, pharmaceutically acceptable carriers or excipients may be components (e.g., anti-adhesion agents) that reduce adhesion between components and, for example, the perforator surface during and after the manufacture of the pharmaceutical formulation, i.e., lubricants. Anti-adhesion agents may include, for example, magnesium stearate. In other embodiments, pharmaceutically acceptable carriers or excipients may be components (e.g., lubricants) that reduce aggregation of components during manufacture and / or reduce friction between, for example, the surface of the pharmaceutical formulation (i.e., formulated as an oral preparation) and the die wall. Depending on the specific embodiment, both water-soluble and water-insoluble lubricants may be used, such as magnesium stearate, stearic acid, vegetable oil, mineral oil, polyethylene glycol, and / or sodium lauryl sulfate. pharmaceutically acceptable carriers or excipients may be components acting as coating agents. Examples of coating agents include, but are not limited to, gelatin and / or cellulose-based coating agents (e.g., hydroxypropyl methylcellulose).

[0055] Other suitable binders, flavoring agents, sweeteners, coloring agents, disintegrants, flow promoters, anti-adhesion agents, lubricants, and coating agents are well known in the art and can be easily identified.

[0056] The pharmaceutical formulation may further include therapeutic agents (e.g., chemotherapeutic agents or anti-inflammatory agents). The pharmaceutical formulation may also include agents that induce an immune response in addition to the influenza virus. Such further components other than the influenza virus of the present invention may be present in any preferred amount(s).

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

[0058] In one embodiment of the pharmaceutical formulation, the pharmaceutical formulation is a viral vaccine. The viral vaccine may be a live attenuated viral vaccine or an inactivated viral vaccine (e.g., a whole virus vaccine, a split viral vaccine, or a subunit vaccine). The viral vaccine may be formulated as a monovalent, bivalent, trivalent, or quaternary vaccine. For example, the vaccine may comprise multiple embodiments of the influenza virus of the present invention. In some embodiments, the vaccine may further comprise at least one influenza virus different from the influenza virus of the present invention.

[0059] Viral vaccines can be formulated into compositions for any preferred means of administration. For example, viral vaccines can be formulated into oral preparations (e.g., capsules, tablets, or oral films), sprays (e.g., nasal sprays), or aqueous or non-aqueous emulsions, solutions, or suspensions suitable for intranasal administration, or parenteral administration, e.g., intravenous, intramuscular, or subcutaneous administration. It can be formulated as a composition of the same name. Methods to induce an immune response

[0060] The present invention provides a method of inducing an immune response in a mammal, which includes administering an influenza virus of the present invention to the mammal. In one embodiment, the influenza virus includes proteins containing selected amino acids, that is, PB1 gene segment, PB2 gene segment, PA gene segment, NP gene segment, and NS gene segment encoding PB1 protein, PB2 protein, PA protein, NP protein, and NS1 protein respectively, that is, the influenza virus of the present invention described herein.

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

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

[0063] The dosing schedule of a pharmaceutical preparation, e.g., a viral vaccine, can depend on the age, weight, sex, and medical history of the mammal. For example, in one embodiment, the single dose of a live attenuated viral vaccine for humans is 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 aforementioned values of the particle forming unit (PFU), focus forming unit (FFU), or TCID of the influenza virus of the present invention 50It may contain. In some embodiments, the plan for preventing or treating influenza virus includes administering the pharmaceutical formulation as a single treatment. The pharmaceutical formulation may be administered in more than one dose, for example, the treatment plan may include additional immunization doses. For example, additional immunization doses of the pharmaceutical formulation may be administered over a period of time 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 dose. 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 containing 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 mutation of cytosine to uracil in the promoter of the nucleotide at position 4; (b) the PB2 gene segment encodes a PB2 protein having an amino acid sequence containing selected amino acids, the selected amino acids comprising valine at position 504, (c) The PB2 gene segment optionally includes isoleucine at position 467 and valine at position 529, and optionally includes a cytosine to uracil mutation in the promoter of the nucleotide at position 4; (d) The PA gene segment encodes a PA protein having an amino acid sequence containing the selected amino acid, the selected amino acid containing lysine at position 401, and the PA gene segment optionally includes a cytosine to uracil mutation in the promoter of the nucleotide at position 4; (d) The NP gene segment encodes an NP protein having an amino acid sequence containing the selected amino acid, the selected amino acid containing leucine at position 116 and lysine at position 294 (e) an influenza virus comprising at least one of n or arginine at position 311; and an NS gene segment encoding an NS1 protein having an amino acid sequence comprising the selected amino acids, wherein the selected amino acids comprise proline at position 30 and lysine at position 118.

[0066] (2)(a) The influenza virus of Embodiment (1), wherein the PB1 gene segment encodes a PB1 protein having an amino acid sequence containing 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 includes a mutation from cytosine to uracil in the promoter of the nucleotide at position 4; (b) The PB2 gene segment encodes a PB2 protein having an amino acid sequence containing selected amino acids, the selected amino acid being valine at position 504, and the PB2 gene segment optionally includes a mutation from cytosine to uracil in the promoter of the nucleotide at position 4; (c) The NP gene segment encodes an NP protein having an amino acid sequence containing 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 the nucleotide sequence represented by SEQ ID NO: 2.

[0068] (4) An influenza virus of any of embodiments (1) to (3), wherein the NP gene segment has the nucleotide sequence represented by Sequence ID No. 1.

[0069] (5) Any influenza virus 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) Any influenza virus 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) An influenza virus of any of embodiments (1) to (6), wherein the selected amino acids are conserved in at least one of the PB1 and NP proteins after at least 10 consecutive passages in the Vero cell line.

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

[0073] (9) An 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 consecutive passages in the Vero cell line.

[0074] (10)(a) The PB1 gene segment encodes a PB1 protein having an amino acid sequence containing 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 contains a mutation from cytosine to uracil in the promoter of the nucleotide at position 4; (b) The PB2 gene segment encodes a PB2 protein having an amino acid sequence containing 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 contains a mutation from cytosine to uracil in the promoter of the nucleotide at position 4; and (c) NP The influenza virus of embodiment (1), wherein the gene segment encodes an NP protein having an amino acid sequence containing 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 the nucleotide sequence represented by SEQ ID NO: 4.

[0076] (12) An influenza virus of any of embodiments (1), (10), and (11), wherein the PB2 gene segment has the nucleotide sequence represented by Sequence ID No. 5.

[0077] (13) An influenza virus of any embodiment (1) and (10) to (12), wherein the NP gene segment has the nucleotide sequence represented by Sequence ID No. 3.

[0078] (14) An influenza virus of any embodiment (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) An influenza virus of any embodiment (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) An influenza virus of any embodiment (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) An influenza virus of any embodiment (1) and (10) to (16), wherein the selected amino acid is conserved in at least one of the PB1, PB2, and NP proteins of the virus after at least 10 consecutive passages in the Vero cell line.

[0082] (18) An influenza virus of any embodiment (1) and (10) to (17), wherein the selected amino acids are conserved in at least PB1, PB2, and NP proteins after at least 10 consecutive passages in a Vero cell line that stably expresses the M2 ion channel protein of influenza A virus.

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

[0084] (20) An influenza virus of any embodiment (1) to (19), wherein the influenza virus is a recombinant influenza virus.

[0085] (21) Any influenza virus 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 containing at least one amino acid mutation in HA2.

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

[0089] (25) An 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) An 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) Any influenza virus of embodiment (1) to (27), further comprising a mutant M gene segment.

[0093] (29) An influenza virus of embodiment (28) in which the influenza virus does not encode a functional M2 protein.

[0094] (30) Any influenza virus of embodiment (1) to (29) that can replicate in human cells.

[0095] (31) Any influenza virus of Embodiments (1) to (30), wherein the virus is identical to an influenza virus except that it lacks selected amino acids, and replicates more readily in Vero cells under the same conditions.

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

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

[0098] (34) A pharmaceutical product of embodiment (33) in which the vaccine is formulated as a monovalent vaccine.

[0099] (39) A pharmaceutical formulation of embodiment (33) in which the vaccine is formulated as a bivalent vaccine.

[0100] (40) A pharmaceutical product of embodiment (33) in which the vaccine is formulated as a trivalent vaccine.

[0101] (41) A pharmaceutical product of embodiment (33) in which the vaccine is formulated as a quadrivalent vaccine.

[0102] (42) A method for inducing an immune response in a mammal, comprising administering to the mammal an influenza virus of any embodiment (1) to (31) or a pharmaceutical preparation of any embodiment (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 generating influenza virus according to any of embodiments (1) to (13), comprising continuously passage the influenza virus in a Vero cell line. [Examples]

[0105] Examples The following embodiments further illustrate the present invention, but should not be interpreted as limiting its scope.

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

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

[0108] Specifically, cDNA encoding the HA and NA gene segments derived from these viruses was transfected together with cDNA encoding the PR8-HY backbone gene segment and the M2SR M gene segment (SEQ ID NO: 11). As described in Example 8, the HA derived from MA15 was Vero-compatible (MA15V). Two viruses, HY-M2SR-MA15V and Bris10 M2SR-HY, were produced. The viruses were produced using standard virus rescue techniques as described herein and amplified in MDCK cells that stably express M2 (i.e., M2CK cells).

[0109] Influenza A virus RNA (vRNA) segments, specifically the PB1, PB2, PA, NP, and NS vRNA segments derived from the influenza PR8-HY backbone, as well as the M vRNA segment lacking the entire M2 open reading frame (ORF), and the HA and NA vRNA segments of 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 transfected into 293T cells along with viral polymerase subunits and NP expression plasmids, and the viruses released into the supernatant were amplified in M2CK cells.

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

[0111] The results are shown as proliferation curves in Figures 1A and 1B. These results demonstrate that neither HY-M2SR-MA15V nor Bris10 M2SR-HY proliferated better in M2VeroA cells than the UW-PR8 backbone. This indicates that the PR8-HY backbone does not enhance viral proliferation in Vero cells.

[0112] Since H3N2 viruses (i.e., Bris10 M2SR-HY and Bris10 M2SR-HG) were observed to proliferate to higher titers than H1N1 viruses (i.e., HY-M2SR-MA15V and HG-M2SR-MA15), the supernatant of H3N2 viruses was evaluated by hemagglutination (HA) assay to determine whether a difference in HA titer was demonstrated by the PR8-HY and UW-PR8 backbones. 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 viral proliferation in Vero cells.

[0113] To confirm that HY-M2SR-MA15V and Bris10 M2SR-HY are not outliers but rather representative of other viruses possessing H1N1 and H3N2 subtypes, M2SR viruses with PR8-HY and UW-PR8 backbones were generated using additional strains. These viruses were then tested for replication using the same method described herein for verifying the replication of HY-M2SR-MA15V and Bris10 M2SR-HY viruses. Table 1 summarizes all the strains tested.

[0114] [Table 1]

[0115] As is clear from the results presented in Table 1, the PR8-HY backbone does not enhance viral replication in Vero cells compared to the UW-PR8 backbone for H1N1, H3N2, and H5N1 virus strains. These results also demonstrate that the PR8-HY backbone is not a suitable backbone for the manufacture of influenza vaccines.

[0116] Example 2 This example illustrates the creation of viruses that can enhance proliferation in Vero cells. To create these viruses, either NA and Vero-adapted HA derived from A / Massachusetts / 15 / 2013 (i.e., MA15V M2SR virus) or NA and HA derived from A / California / 07 / 2009 (i.e., CA07 M2SR virus) are included, along with a PR8-HY backbone (i.e., HY-M2SR-M Two M2SR viruses, including A15V and HY-M2SR-CA07, were serially passaged in M2VeroA cells.

[0117] The virus was serially diluted 10-fold and adsorbed onto M2 VeroA cells in TC-6 plates using standard influenza virus techniques. However, prior to viral infection, the cell culture medium was removed and the cells were washed with PBS. After adsorption at 35°C for 60 minutes, viral growth medium containing trypsin / TPCK was added. The cultures were incubated at 35°C for 4–7 days. The culture supernatant was collected from the wells of the maximum dilution that showed cytopathic effect and HA activity. Cytopathic effect (CPE) was determined by visual inspection of a monolayer at low magnification with a light microscope to detect curling and other structural changes. HA activity was measured according to the WHO Manual for the Laboratory Diagnosis and The titer was determined by a standard hemagglutination assay as described in virological surveillance of influenza (2011). 50 μL of a 0.5% suspension of turkey erythrocytes (Innovative Research, Novi, MN) was added to stepwise 2-fold dilutions of the culture supernatant, and hemagglutination was evaluated after incubation at room temperature for 30 minutes. The reciprocal of the maximum dilution of the culture supernatant that agglutinated the erythrocytes was recorded as the HA titer for that sample.

[0118] Next, the recovered supernatant was serially diluted again and used to infect fresh M2VeroA monolayers. The subculturing history is shown in Table 2.

[0119] [Table 2]

[0120] As the passage number increased, the virus was recovered at higher dilutions. This indicated that the virus proliferated to higher titers in Vero cells. Therefore, the process was stopped at passage 5, and proliferation curve studies were used to evaluate whether the passage 5 (p5) virus actually proliferated to a higher titer than the initiation (p0) virus.

[0121] p0 and (p5) viruses were evaluated using proliferation curves in M2VeroA cells. The (p0) viruses were identified as HY-M2SR-MA15V and HY-M2SR-CA07 viruses. The (p5) viruses were named FGHY1-M2SR-MA15V and FGHY2-M2SR-CA07 viruses. FGHY1 and FGHY2 specifically refer to the backbone of the p5 virus.

[0122] To evaluate the growth curve, cell monolayers were infected with an MOI of 0.001 and incubated in a 35°C CO2 incubator for 6 days. TCID 50 Aliquotes were collected daily for viral titer measurement using an assay. The resulting proliferation curves are shown in Figure 2. As shown by the dashed line in Figure 2, passage 5 (p5) virus demonstrated higher viral titers and faster proliferation dynamics than the initiation (p0) virus.

[0123] Example 3 This example identifies mutations that resulted in increased proliferation characteristics associated with passage 5 viruses (i.e., FGHY1-M2SR-MA15V and FGHY2-M2SR-CA07) in the M2VeroA cells of Example 2.

[0124] HY-M2SR-MA15V and HY-M2SR-CA07 were successively passaged according to the method of Example 2, and the complete viral genome sequences were determined for 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, and cDNA was constructed using 12-base pair universal primers that amplified all eight segments of the viral genome by multi-segment reverse transcription. The influenza gene was then amplified using primers as described in Hoffmann et al., Arch. Virol., 146:2275-2289 (2001). Bulk cDNA sequences were obtained using gene-specific primers. The obtained cDNA sequences were then aligned to start plasmid sequences that functioned as reference sequences using a sequence comparison algorithm with programmed parameters specified to emphasize non-identical residues. Table 3 shows the amino acid changes observed in each gene compared to the UW-PR8 backbone and the PR8-HY backbone.

[0125] As shown 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 distinct. For example, FGHY1 contains mutations at the 464th amino acid in the PB1 protein and at the 294th amino acid in the NP protein compared to the PR8-HY backbone, while FGHY2 contains mutations at the 607th amino acid in the PB1 protein, at the 467th and 529th amino acids in the PB2 protein, and at the 311th amino acid in the NP protein compared to the PR8-HY backbone.

[0126] [Table 3]

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

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

[0129] Specifically, individual backbone genes were cloned into pPolI plasmids using standard molecular techniques known in the art. These genes include 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. Next, M2SR viruses were produced using a plasmid-based influenza virus rescue method similar to that described in Example 1. These viruses further contained HA-MA15V and NA-MA15.

[0130] Next, the proliferation dynamics of the constructed M2SR viruses (i.e., FGHY1-M2SR-MA15V and FGHY2-M2SR-MA15V) in M2VeroA cells were evaluated. For comparison, the standard M2SR backbone (UW-PR8) and PR8- HY viruses (i.e., HG-M2SR-MA15V and HY-M2SR-MA15V, respectively) were used. M2VeroA cells grown in a 6cm dish were infected with a multiple of infection (MOI) of 0.001. A viral growth medium containing trypsin / TPCK (1 μg / mL) was added, and the cells were incubated at 35°C for 7 days. Aliquots were collected daily and stored at -80°C until viral titer measurement.

[0131] The obtained proliferation curves are shown in Figure 3. As shown in Figure 3, FGHY1-M2SR-MA15V and FGHY2-M2SR-MA15V proliferated faster than HG-M2SR-MA15V and HY-M2SR-MA15V. FGHY1-M2SR-MA15V and FGHY2-M2SR-MA15V also reached their peak titers earlier than HG-M2SR-MA15V and HY-M2SR-MA15V, and plateaued 2-3 days earlier. These results indicate that amino acid mutations observed in the passaged viruses (i.e., viruses with FGHY1 and FGHY2 backbones) confer high yield characteristics and promote infection with low infection multiplicity in M2VeroA cells (a highly desirable characteristic in vaccine production).

[0132] Example 4 This example demonstrates that mutations in the PB1 and NP proteins of FGHY1, and in the PB1, PB2, and NP proteins of FGHY2, contribute to the conferral of high proliferation characteristics of influenza viruses, regardless of HA and NA subtypes. Therefore, this example shows that the FGHY1 and FGHY2 backbones can be updated with various influenza HA and NA for the production of seasonal and generalized influenza vaccines.

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

[0134] Viral proliferation was evaluated in M2 VeroA cells infected with an MOI of 0.001. Aliquots were collected daily, and viral titers were measured using TCID. 50 The titers were measured by assay. To assess HA production, the supernatant was also evaluated by hemagglutination assay. The resulting proliferation curves for viral 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 proliferated faster and to higher viral 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 dynamics compared to the other two viruses.

[0135] These results demonstrate that viruses containing the FAGHY1 and FGHY2 backbones proliferate 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 enable seasonal influenza A viruses with HA and NA to proliferate faster in Vero cells, both for the production of infectious viruses and HA (i.e., live vaccines or inactivated vaccines). The FGHY1 and FGHY2 backbones also enable viruses with generalized HA and NA to proliferate faster in Vero cells.

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

[0137] As is evident from the presented data, the FGHY1 backbone exhibited faster growth kinetics and higher titer. This further demonstrates that the FGHY1 backbone offers growth advantages and is therefore 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, specifically a virus containing an additional Vero adaptive mutation at position 107 in HA2, in which threonine is replaced with asparagine. Thus, the results shown in Figure 5A demonstrate that the Vero adaptive mutation in the HA protein may further enhance the proliferative effect of the backbone described herein.

[0139] Example 5 This example demonstrates that viruses containing UW-PR8 and HY-PR8 backbones proliferate in MDCK cells, while their proliferation is weaker in Vero cells. On the other hand, viruses containing the FGHY1 backbone show a higher yield in Vero cells.

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

[0141] Similarly, the data presented in Table 4 demonstrate that FGHY1 enhances the proliferation of multiple viral subtypes in Vero cells, resulting in titers closer to those produced in MDCK cells.

[0142] [Table 4]

[0143] These results suggest that mutations in the FGHY1 backbone can overcome host limitations in Vero cells, allowing Vero cells to behave more like MDCK cells in relation to vaccine production.

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

[0145] HY-M2SR-MA15 and HY-M2SR-CA07 viruses, prepared using the virus rescue technique described herein, were passaged twice in M2CK cells, and then 20 and 9 times, respectively, in M2VeroA cells. For each passage, the tissue culture supernatant recovered from the previous passage was used, progressively diluted 10-fold from 1:10 to 1:10,000,000, to infect fresh monolayers of M2VeroA cells. After infection, the culture supernatant was validated for CPE and HA titers. The supernatant was collected from the dilution showing the highest CPE and HA titers at some point between day 3 and day 7 post-infection. FGHY1-M2SR-MA15 and FGHY2-M2SR-CA07 were prepared in Vero cells at passage 4.

[0146] At the end of each passage, the entire viral genome was sequenced and compared with the start sequences 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 is clear from the results shown in Table 5A, after 16 further passages in M2VeroA cells, HY-M2SR-MA15 retained the same amino acid changes in the PB1 and NP proteins. The NP protein acquired a further mutation of 1 at the 50th amino acid position, i.e., a mutation from serine to asparagine. As is clear from the results shown in Table 5B, HY-M 2SR-CA07 also retained the same mutations as the initiating virus in PB1, PB2, and NP proteins after five further passages in M2VeroA cells. These results demonstrate that the FGHY1-M2SR and FGHY2-M2SR backbones are stable in M2VeroA cells and 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 that express the influenza BM2 protein, i.e., the ion channel protein of influenza B serotype. 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 illustrate 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 proliferation in Vero cells is not species-specific.

[0153] The original M2SR backbone (UW-PR8) was tested in human subjects (ClinicalTrials.gov Identifier:NCT02822105) and demonstrated that it induced an immune response. This indicates that it is functional in human cells. Therefore, the original backbone was used as a benchmark to determine whether M2SR-FGHY1 can replicate in human cells and, consequently, whether it is 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 human lung epithelial cell line A549 cells (ATCC CCL-185) using the infected cell NP enzyme-linked immunosorbent assay (ELISA) protocol described in the WHO Influenza Manual in 96-well plates.

[0155] On the second day after infection, cells were tested for anti-NPs (anti-influenza nucleoproteins) in the influenza nucleoprotein (NP) within the cells. Stain with monoclonal antibody, process as ELISA, and obtain TCID. 50 The titer was determined. To normalize the A549 titer to the M2CK titer, standard TCID was used in M2CK cells. 50 Assays were performed to obtain viral titers for each virus. Table 7 shows the viral titers for each virus in each cell line and the ratios between the two cell lines. The ratios for the two viruses were similar, indicating that M2SR-FGHY1-HK4801 proliferates in human cell lines, similar to M2SR-Original-HK4801.

[0156] [Table 7]

[0157] Example 8 This example demonstrates that better viral replication can be achieved by acquiring mutations in the HA gene segment (e.g., the HA2 region of H1N1pdm) through passage of wild-type viruses in Vero cells.

[0158] Influenza A / Massachusetts / 15 / 2013(H1N1)(MA15) (e.g., parent virus) was obtained from the International Reagent Resource (IRR, catalog number FR-1319, lot number 62525202), passaged in Vero cells, and amplified in MDCK cells. Viral RNA was extracted from the tissue culture supernatant of passage 8, and the nucleotides 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 position 451 (sequence number 13) (i.e., position 107 in HA2). Specifically, threonine was changed to asparagine. Two separate, independent passages from independent wells yielded the same adaptive mutation.

[0159] This Vero-adaptive HA2 mutation from MA15 (MA15V) (SEQ ID NO: 13) is similar to the mutation described in Example 4 for Vero-adaptive HA derived from MI45 (MI45V). As is evident from Figure 5A, such a mutation promotes viral replication in Vero cells.

[0160] Other amino acid changes in the HA (e.g., HA2) of influenza HA (e.g., H1N1pdm HA) passaged in Vero have been shown to stabilize the HA protein at lower pH levels, further promoting viral infection of Vero cells. See, for example, 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). Therefore, the HA mutations described herein may also affect the stability of the HA protein at lower pH levels.

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

[0162] Figure 7B is a table showing nucleic acid sequence mutations and resulting amino acid sequence mutations of Vero-adapted HAs derived from the H3N2 virus (i.e., A / Singapore / INFIMH-16-0019 / 2016). As is clear 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. "Clinical" HAs are correlated with wild-type HAs. HA names V1 to V8 are correlated with the sequences of various HAs obtained through various passages. Shaded boxes indicate nucleotide mutations that result in amino acid mutations. The Origin column describes the passage history of the initiating virus.

[0163] Figure 7C shows the proliferation curves in M2VeroA cells for 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 supernatant was collected at the indicated time, and viral titers were measured using TCID in M2CK cells. 50This was determined by assay. MSRSing2016 (i.e., M2SR Sing2016 V6), which has the most Vero-adapted mutation, showed the highest growth rate.

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

[0165] Seven-week-old BALB / c, female mice were intranasally immunized with one of the following viral variants: H1N1 FGHY1-M2SR and H3N2 FGHY1-M2SR (both containing the variant M segment described in SEQ ID NO: 11). These variants were administered at a rate of 1 x 10⁶ per mouse. 6 TCID 50 The drug was administered at the specified dose. The control group of mice was given SPG. For 14 days after immunization, the mice were observed for any changes in body weight and symptoms of infection.

[0166] In mice immunized with the FGHY1-M2SR mutant or SPG control, no clinical symptoms of infection or weight loss were observed over a 14-day period. Figure 10 shows the percentage change in body weight of immunized mice. Furthermore, the change in body weight between the groups was similar over the 14-day period. These results indicate that the FGHY1-M2SR virus is attenuated and non-pathogenic in mice.

[0167] Example 10 A backbone that confers advantages to proliferation in cells of a certain origin may exhibit host limitation, potentially preventing the replication and / or production of the antigen in the target host. However, this embodiment demonstrates that antigen production of the FGHY1-M2SR virus is not limited in human cell lines.

[0168] The following human cell lines were tested: A549 (ATCC number CCL-185) human lung cancer; Calu-3 (ATCC number HTB-55) human lung adenocarcinoma; and MRC-5 (ATCC number CCL-171) human lung fibroblasts. These cell lines are derived from the human airway and serve as a target substrate for the influenza vaccine virus. The control cell lines used for influenza virus replication were MDCK (Sigma number 84121903) cells and Vero (ATCC number CCL-81) cells.

[0169] One day prior to infection, cells were seeded in 60 mm dishes and immunized with IVR-147, a replicating vaccine reassortment virus from the CDC, containing primary UW-PR8 ("HG") backbone, FGHY1, and a backbone gene segment derived from A / Puerto Rico / 8 / 1934, all with an approximate MOI of 0.5. All of these viruses expressed HA and NA derived from A / Brisbane / 10 / 2007. A subset of cells was infected with viruses including primary and FGHY1 expressing HA and NA derived from A / Singapore / INFIMH-16-0019 / 2016. 10% FCS was added to the culture medium for 1 or 2 days post-infection at 35°C. Cells were harvested from the culture medium and on the culture surface and fixed with 10% buffered formalin. Cells were permeabilized in BD Cytofix / Cytoperm solution (BD, catalog number 554714), and influenza NP proteins were stained with FITC-labeled mouse anti-influenza A NP monoclonal antibody (D67J, Invitrogen, catalog number MA1-7322). Fluorescein intensity was measured using BD LSRII and analyzed with FlowJo software.

[0170] Single-cell populations were gated by forward scattering height (FSC-H) versus forward scattering area (FSC-A), and FITC-positive and FITC-negative cells were separated by lateral scattering area (SSC-A) versus FITC. FITC-positive cells were considered to be virus-infected cells, and the strength of the FITC intensity reflected the amount of NP expression in the cells.

[0171] The obtained 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 IVR-147 and HG viruses.

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

[0174] Example 11 This embodiment demonstrates that the FGHY1-M2SR vaccine induces an in vivo antibody response that increases with repeated administration, without toxicity to the host. summary

[0175] To demonstrate that the FGHY1-M2SR vaccine virus induces an immune response to its components without causing toxicity in the host, 15 male and 15 female ferrets were subjected to a 1 × 10⁻¹⁶ experiment. 8 TCID 50 (Low dose) or 1x10 9 TCID 50Intranasal immunization was performed with FGHY1-M2SR vaccine at a high dose level. A third group of ferrets was intranasally immunized with SPG as a placebo control. A three-dose vaccination regimen was used for each treatment group. Ferrets received primary immunization (day 1 of the study) and two booster immunizations at 13 and 27 days (days 14 and 28 of the study). After each immunization, ferrets were observed for 7 days for mortality, and body weight, temperature, and clinical symptoms were measured daily. To assess clinicopathology, blood was collected from all surviving ferrets before the study and on days 14, 16, 30, and 49 of the study. Serum samples were collected before the study and on days 14, 30, and 49 of the study. The specimens were collected on day 1, and antibody levels over time were evaluated by ELISA, hemagglutination inhibition (HAI) assay, and viral neutralization (VN) assay. Necropsies, including examination of the external body, all orifices, skull, thoracic cavity, peritoneal cavity, and their contents, were performed on 5 males and 5 females per group on days 3, 30, and 49 of the study. B. Materials and Methods

[0176] Vaccine virus immunity. Ferret, 1x10 8 TCID 50 Dosage or 1x10 9 TCID 50 Three doses of H3N2FGHY1-M2SR vaccine were administered intranasally at one of the following doses. The vials of frozen vaccine virus stock were thawed at room temperature for at least 10 minutes, and then stored in the refrigerator or on moist ice until use. Ferrets were anesthetized with ketamine / xylazine, and one dose of virus was administered intranasally at 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 the influenza A virus is represented by SEQ ID NO: 11.

[0178] Experimental Design. At the start of the study, 90 ferrets (Triple F Farms, Sayre PA), aged 16–22 weeks, consisting of 45 males and 45 females, were used. All animal procedures were performed in a biosafety level 2 facility for animals, following protocols approved by the Animal Care and Use Committee of the IIT Laboratory. Prior to immunization, the ferrets were monitored for 4 days to determine their baseline body temperature. Temperature measurements were recorded daily via a subcutaneously implanted transponder (BioMedic data systems, Seaford, DE) in each ferret. Blood samples were collected and serologically tested for influenza antibodies before the start of the study. Preimmunized serum samples were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) to remove nonspecific inhibitors, then serially diluted and tested for 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, and mixed with 0.5% turkey erythrocytes. Antibody titer was defined by the lowest serum dilution that caused inhibition of hemagglutination. Only ferrets with HAI titers less than 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 the efficacy and toxicity of the vaccine, ferrets were given 1 x 10⁶ doses on days 1, 14, and 28 of the study. 8 TCID 50 Dosage: 3 doses or 1 x 10 9 TCID 50Ferrets were intranasally immunized with three doses of H3N2 FGHY1-M2SR. The control group was intranasally immunized with SPG on days 1, 14, and 28 of the study. Ferrets' body temperature, weight, and clinical symptoms were monitored daily for 7 days post-immunization. Blood was collected from all surviving ferrets on days 5, 14, 16, 30, and 49 of the study for clinicopathological evaluation. Serum samples were collected on days 5, 14, 30, and 49 of the study and kept at approximately 70°C until antibody titer measurement by ELISA, viral neutralization assay, and HAI assay. All study animals were euthanized on the scheduled day (days 3, 30, or 49, 5 males and 5 females per group) and necropsy was performed. Necropsy consisted of examination of the external body, all orifices, and the skull, thoracic cavity, peritoneal cavity, and their contents. The tissue was collected, fixed, and evaluated histopathologically by a qualified veterinary pathologist. C. Results

[0180] Morphology / Mortality and Clinical Observations: All ferrets survived until their scheduled slaughter days. 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 score for all ferrets was "0" (alert and playful) at all time points measured between days 1 and 49. However, one male and one female from the SPG control group were given a score of "1" (alert, but playful only when stimulated) on day 20.

[0181] Body weight and weight change: In some groups, there were statistically significant differences in mean weight change compared to the SPG control group, but these differences appeared to be randomly distributed and were not significant.

[0182] Body temperature: In some groups, statistically significant increases and decreases in mean body temperature were observed compared to the SPG control group. However, these differences appeared to be 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 identified as A / Singapore / INFIMH-16-0019 / 2016(H3N2)(Immune Technology). The sample was coated with recombinant HA protein from (Corporation, New York, NY), blocked with skim milk, and then coated with ferret IgG antibody (SeraCare Life). Detection was performed using the 1-Step® Ultra TMB-ELISA (Thermo Fisher Scientific Inc., Maltham, MA) substrate (Sciences, Milford, MA) and 1-Step® Ultra TMB-ELISA (Thermo Fisher Scientific Inc., Maltham, MA) substrates.

[0184] Figure 11 shows the anti-H3 HA ELISA IgG titers obtained from serum data. Ferrets in each immunized group showed a significant increase in serum anti-H3 HA antibodies, while antibody levels in animals administered only SPG did not change from baseline. Anti-H3 HA antibody titers were higher in the immunized group than in the SPG control group two weeks after the first dose. The mean antibody titer per immunized group increased further after the first and second doses of the vaccine.

[0185] Hemagglutination Inhibition (HAI) Assay: To demonstrate 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 37°C ± 2°C water bath for 18–20 hours. After adding an equal volume of 2.5% (v / v) sodium citrate, the samples were incubated in a 56±2°C water bath 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 in PBS (from 1:10 to 1:1280) and incubated with four 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] Figure 12 shows the anti-H3 HAI titers obtained from serum data. High dose (1x10 9 TCID 50 The ) group received a low dose (1x10 8 TCID 50 The SPG (control) group tended to show higher HAI titers than the control group. The SPG (control) group did not produce HAI titers. 1x1 0 9 TCID 50 With the exception of one male, 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] Viral neutralization assay: Serum samples from the pre-study and treatment phases (from days 3, 14, 30, and 49 of the study) were tested for influenza A / Singapore / INFIMH-16-0019 / 2016(H3N2) virus in a viral neutralization assay. Serum samples were inactivated at 56°C for 30 minutes. The serum was then serially diluted 2-fold and incubated with standardized virus (concentration of 80–140 PFU) at 37±2°C and 5.0±1% CO2 for 60 minutes. 100 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 at 37±2°C in 5.0±1% CO2 for 18–22 hours. 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 stained using TrueBlue Peroxidase Substrate (Kirkegaard and Perry Laboratories; Gaithersburg, MD). Plaques were visualized and counted using an ELISPOT instrument (AID GmbH, Strassberg, Germany). 50% plaque reduction neutralizing titer (PRNT) was calculated. 50 The titer was calculated by counting plaques based on back titration of control plaques, recording the titer as the reciprocal of the final serum dilution, and demonstrating a 50% reduction in the number of viral plaques in the input control.

[0188] Figure 13 shows the anti-H3 PRNT50 titers obtained from serum data. All ferrets in the SPG group tested negative throughout the study period.

[0189]

number

[0190] It remained as it was. 1x10 8 TCID 50 Ferrets immunized with H3N2 FGHY1-M2SR at the specified dose showed VN titers of 1916, 1838, and 1970 geometric mean titers (GMT) on days 14, 30, and 49, respectively. 9 Ferrets immunized with H3N2 FGHY1-M2SR at a dose of TCID50 showed GMT VN titers of 4434, 5572, and 6400 on days 14, 30, and 49, respectively. Since serum was diluted up to 1:6400, PRNT values ​​greater than 6400 were not recorded. 50 Titer was not specifically measured. 3 doses of 1x10 9 TCID 50 All ferrets immunized with H3N2FGHY1-M2SR had over 6400 PRNTs. 50 It showed potency.

[0191] Clinical Pathology: Blood samples for clinical chemistry, hematology, and coagulation parameter analysis were collected from all living ferrets 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 hematological samples, and sodium citrate was used for coagulation samples. Clinical chemistry samples were collected without anticoagulant. Urine samples were collected directly from the bladder of each ferret at necropsy.

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

[0193] Gross autopsy and histopathology: Gross autopsy and histopathology were performed on 5 males and 5 females per group on days 3, 30, and 49 of the study. 1x10 8 TCID 50 Intranasal immunization of ferrets with H3N2 FGHY1-M2SR at the specified dose yielded no macroscopic findings, but microscopic findings (mixed cell infiltration) were observed in the lungs on days 3 and 30. 1x10 9 At the TCID50 dose, macroscopic findings (pigmentation, darkening, or mottling) were observed in the lungs, and microscopic findings (mixed cell infiltration) were observed on days 3 and 30. After 3 weeks of recovery, no macroscopic lesions related to the test substance were observed on day 49 of the study.

[0194] Therefore, this embodiment demonstrates that intranasal immunity to the H3N2 FGHY1-M2SR vaccine virus is not transmitted to the vaccinated host and is not associated with any vaccine-related adverse events (e.g., elevated body temperature, weight loss, or clinical signs). These results indicate that the H3N2 FGHY1-M2SR virus induces a protective immune response against homologous test viruses after a single dose. This can be further enhanced with repeated doses and is useful as an intranasal influenza vaccine.

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

[0196] Influenza A H1N1 or H3N2 FGHY1-M2SR viruses induce an antibody response 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 vaccines, or quadrivalent H1N1 and H3N2 FGHY1-M2SR and BM2SR Victoria or Yamagata vaccines. Control mice were simulated with SPG. Twenty-eight days after vaccination, mice were intranasally immunized with a booster immunization 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 immunization (day 28). Anti-H1 HA and anti-H3 HA serum IgG antibody titers were determined from serum samples by ELISA.

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

[0199] Example 13 This example demonstrates that a monovalent or tetravalent FGHY1-M2SR vaccine administered intranasally protects mice from lethal influenza A viruses not contained in the vaccine. This indicates.

[0200] BALB / c female mice described in Example 12 were immunized with a lethal dose of influenza A / California / 07 / 2009(H1N1) virus (>10 mice, 50% lethal dose (MLD)) 70 days after the initial immunization (6 weeks after the booster immunization). 50Mice were immunized with the following vaccines: monovalent H1N1 (influenza A / Montana / 50 / 2016) or H3N2 (influenza A / Singapore / INFIMH-16-0019 / 2016) FGHY1-M2SR and quadrivalent FGHY1-M2SR and BM2SR vaccines. All mice immunized with monovalent H1N1 FGHY1-M2SR and quadrivalent FGHY1-M2SR and BM2SR vaccines remained healthy without weight loss. The weight loss data obtained is shown in Figure 15. Mice immunized with monovalent H3N2 FGHY1-M2SR experienced temporary weight loss but recovered completely. Control mice simulated with SPG alone experienced weight loss and died from infection within 5 days after inoculation. Three days after inoculation, lungs were obtained from three mice per group, and viral load was measured by plaque assay in MDCK cells. As shown in Table 8, monovalent H1N1 Viral titers in the lungs of mice immunized with FGHY1-M2SR and the quadrivalent vaccine were below the detection limit (less than 76 plaque-forming units (PFUs) per lung). Viral load was detected in mice immunized with H3N2 FGHY1-M2SR (6.60 log PFU / g). However, the average viral 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 monovalent and quadrivalent M2SR vaccines confer cross-protection and limit the replication of inoculated viruses that do not match any vaccine component.

[0201] [Table 8]

[0202] Example 14 This embodiment demonstrates that the tetravalent FGHY1-M2SR provides a favorable safety profile compared to approved intranasal influenza vaccines and offers superior protection against influenza viruses not contained in approved intramuscular inactivated influenza vaccines. The monovalent H3N2 FGHY1-M2SR vaccine with discontinuous antigenic mutations provides comparable protection to an antigenically matched approved vaccine in mice against lethal influenza A virus.

[0203] 7-week-old BALB / c female mice (N=13) were given the following vaccine: monovalent H3N2 Mice were immunized with one of the following: FGHY1-M2SR, tetravalent FGHY-M2SR, FluMist® Quadrivalent (AstraZeneca, Wilmington, DE), Fluzone® Quadrivalent (Sanofi, Bridgewater, NJ), or Fluzone® High Dose (Sanofi). The strain components for each vaccine are shown in Table 9. FGHY1-M2SR and FluMist were administered intranasally, while both Fluzone vaccines were administered intramuscularly. A control group of mice was simulated by intranasal immunization with SPG. Mice were observed for any changes in body weight for 14 days post-immunization.

[0204] [Table 9]

[0205] The obtained weight loss data is 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% of their body weight on day 3 after vaccination and took 14 days to recover. These data demonstrate that the FGHY1-M2SR vaccine has a superior safety profile compared to FluMist, an approved live attenuated influenza vaccine.

[0206] Mice were immunized with a booster immunization 28 days after primary immunization. The booster immunization consisted of the same vaccine used to immunize the mice in primary immunization. Serum samples were collected weekly after primary and booster immunization, and pooled serum IgG titers for each vaccine component were determined by ELISA. The obtained anti-influenza A / H1 HA serum IgG ELISA titer data is shown in Figure 17A. The obtained anti-influenza A / H3 HA serum IgG ELISA titer data is shown in Figure 17B. The obtained anti-influenza B / Yam HA serum IgG ELISA titer data is shown in Figure 17C. The obtained anti-influenza B / Vic HA serum IgG ELISA titer data is 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 all vaccine formulations. Fluzone Quadrivalent and Fluzone High Dose are more effective against influenza B HA antigen compared to live vaccines. This induced lower serum IgG titers. The monovalent H3N2FGHY1-M2SR vaccine, as expected, caused an increase in serum IgG only against the H3 HA antigen.

[0207] Tracheal-pulmonary lavage fluid was obtained from four mice per group 49 days after initial immunization (21 days after booster shot), and IgG and IgA titers were determined by ELISA to evaluate the mucosal immune response. The obtained IgG titer data are shown in Figure 18A, and the obtained IgA titer data are shown in Figure 18B. Quadrivalent FGHY1-M2SR and FluMist induced both IgG and IgA titers in response to all test antigens. Mice immunized with monovalent H3N2FGHY1-M2SR showed IgG and IgA titers in response to the H3 HA antigen, but not in response to the H1 or B influenza HA antigen. IgG titers for all four antigens were elevated in the groups immunized with Fluzone Quadrivalent and Fluzone High Dose vaccines, but no IgA antibody titers were detected for any of the antigens. These data demonstrate that the FGHY1-M2SR vaccine induces a mucosal immune response comparable to that of the approved live attenuated influenza vaccine, FluMist, whereas approved intramuscular influenza vaccines do not induce any mucosal immune response.

[0208] Six weeks after booster immunization, mice were inoculated with a lethal dose of influenza A / California / 07 / 2009 (H1N1pdm). Mice immunized with SPG controls became infected and died by day 5 post-inoculation, but all vaccinated mice survived. Post-inoculation weight changes are shown in Figure 19. As seen in Table 10, the quadrivalent FGHY1-M2SR and FluMist groups showed no weight loss, and no inoculated virus was detected in the lungs or nasal turbinates on day 3 post-inoculation. This suggests that these intranasal vaccines provide antiviral immunity against lethal infection by serial mutant influenza viruses. Mice in the monovalent H3N2 FGHY1-M2SR (heterozygous subtype to the inoculated virus), Fluzone Quadrivalent, and Fluzone High Dose groups experienced a mean weight loss of more than 15% on day 3 or 4 post-inoculation, followed by recovery by day 21 post-inoculation. Infectious viruses were detected in the lungs and nasal turbinates of mice immunized with monovalent H3N2 FGHY1-M2SR and Fluzone Quadrivalent. Mice immunized with Fluzone High Dose vaccine showed similar weight loss to mice immunized with monovalent H3N2 FGHY1-M2SR and Fluzone Quadrivalent vaccines, but infectious viruses were not recovered from the lungs or nasal turbinates. These data demonstrate that intranasally administered monovalent heterozygous H3N2 FGHY1-M2SR (i.e., lacking the H1N1 component) vaccine protects mice as well as intramuscularly administered approved inactivated vaccines containing the H1N1 component.

[0209] [Table 10]

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

[0211] M2VeroA cells (Vero cells that stably express influenza A M2 protein) were cultured in OptiVero medium (InVitria, Aurora, CO) in a humidified incubator at 37°C and a 5% CO2 atmosphere. Approximately 242 million cells in CellSTACK®-5 Chamber (CS5; Corning, Corning, NY), with 3 or 4 CellSTACKs 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 multiple of infection (MOI) of 0.01. After 2-3 days at 35°C and 5% CO2, when the HA titer of the supernatant was at least 32 HAU / 50 μL, the culture medium was collected and cells and cell residue were removed by slow centrifugation. The supernatant was further clarified and sterilized by suction filtration through a 0.2 μM pore PES membrane. Next, the clarified supernatant was 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 subjected to a 235 cm³ solution. 2The virus was purified using a tangential flow filter (TFF) with a 300kD MWCO Modified Polyethersulfone (mPES)MidiKros® hollow fiber filter module (Repligen, Waltham, MA). The substance was concentrated 10 to 20 times. Next, contaminating host cell proteins (HCPs) and residual DNA fragments were removed by dialysis filtration using SPG with at least 15 column volumes. The purified virus in SPG buffer was further concentrated 10 to 100 times by ultracentrifugation at 25,000 rpm using a 25% sucrose PBS cushion. The resulting virus pellet was resuspended in SPG, divided into equal portions, flash-frozen in liquid nitrogen, and then stored at -80°C.

[0212] The sequence homology of the concentrated 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-PC). The samples were subjected to R) to produce cDNA, which was then subjected to Sanger sequencing. Analysis of open reading frames (ORFs) encoded by the eight viral segments showed that all segments had 100% nucleotide sequence identity with respect to the reference.

[0213] The infectivity titer of concentrated and purified H3N2 FGHY1-M2SR virus was determined by the 50% tissue culture infectious dose (TCID) measured at least three times independently using M2CK cells (MDCK cells that stably express influenza A M2). 50 The titer was determined by assay. In the procedure, serial dilutions of the vaccine sample were applied to replicated M2CK cells in a 96-well plate and cultured at 35°C in a 5% CO2 atmosphere for 4 days. Four days after inoculation, the cell monolayer was visually inspected and scored for CPE. The viral titer was calculated using the Reed-Münch method and determined by TCID. 50The values ​​were expressed as / mL. Furthermore, HA activity was tested in aliquots of the supernatant in each well to verify the viral titer determined by CPE. As shown in Table 11, after concentration and purification, the H3N2 FGHY1-M2SR virus consistently showed a titer of 10. 9.8 TCID 50 It reached a high titer exceeding / mL.

[0214] [Table 11]

[0215] To confirm that the H3N2 FGHY1-M2SR vaccine virus maintains the replication-deficient phenotype after concentration and purification, the presence of any replicating virus was evaluated by three consecutive passages of the test sample in MDCK cells that tolerate wild-type influenza virus but not M2SR virus. For the first infection, the test virus was serially diluted and inoculated into a cell monolayer. The infected cells were then cultured at 35°C in a 5% CO2 atmosphere for 4 days. The culture supernatant of the infected cells was then transferred to a fresh MDCK monolayer and incubated at 35°C in a 5% CO2 atmosphere for 4 days. For the third and final passages, the culture medium of these infected cells was transferred to another fresh MDCK monolayer and incubated at 35°C in a 5% CO2 atmosphere for a further 4 days. MDCK cells were observed for CPE after each 4-day incubation at 35°C, and the presence of progeny virus particles was confirmed by measuring HA activity in the culture supernatant. For each infection, the replication-deficient reference virus, Bris10 M2SR, was tested as a positive control. The negative control inoculum consisted solely of culture medium. The results demonstrated that the control functioned as expected, and no infectious progeny were detected after inoculation of any of the four test preparations into normal cells. Therefore, the H3N2 FGHY1-M2SR vaccine virus preparation was demonstrated to be incapable of replication and lacking replication ability.

[0216] The fragment size of residual host cell DNA in 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). The DNA purified from the sample was loaded onto a DNA High Sensitivity Chip (Agilent) and subjected to automated capillary electrophoresis. Using 2100 Expert software, the data was analyzed to detect fragments, obtain fragment concentrations and the relative percentage of total DNA, and measure the size of the extracted residual DNA in base pairs (bp). Results obtained from four replicate measurements of 3 lots of concentrated and purified FGHY1-M2SR demonstrated that there was no or very low fluorescence signal intensity in 2100 Expert. This indicated that they contained residual DNA insufficient for accurate sizing. The commercially available ELISA kit (Cygnus Technologies part number F500, Southport, NC) was used to quantify Vero host HCP present in concentrated and purified H3N2 FGHY1-M2SR vaccine lots. The resulting HCP data is shown in Table 12. The antibodies in the kit were produced and affinity purified using Vero lysates and have been shown to detect HCP from many commercially available Vero cell lines used in the production of viral products. Thus, this kit can be used as a tool to monitor the level of Vero HCP contaminants. The kit was used according to the manufacturer's assay protocol. Multiple dilutions of each sample were prepared with sample diluent (Cygnus catalog number I028) and analyzed (in duplicate) to confirm that the samples showed dilution linearity within the range of the standards supplied with the kit (i.e., no high-dose hook effect). A four-parameter logistic regression was used to generate a standard curve and used to interpolate the values of the samples.

[0217] <0...901>

[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 detected previously in clinical trial materials (Bris10 M2SR, lot number 15100251). The HCP in the 3 vaccine preparations decreased by 99.98% - 99.99%.

[0220] The sterility of the vaccine preparations was verified by procedures based on WHO specifications for pharmaceutical preparations. The vaccine preparations were inoculated under aseptic conditions into 3 types of liquid media: Luria-Bertani broth (LB), tryptic soy broth (TSB), and thioglycollate medium (TGM). The cultures were incubated at 37°C (LB and TSB) and ambient temperature (TGM). The cultures were grown for 14 days and then visually examined for microbial growth. All the tested preparations were negative for microbial growth under all growth conditions.

[0221] The osmotic pressure values of the H3N2 FGHY1-M2SR vaccine lots were measured using a Fiske Model 210 Micro-Osmometer (Fiske Associates, Norwood, MA). The obtained osmotic pressure data are shown in Table 13. Calibration at 3 points (50, 850, and 2000 mOsm / kg) was performed daily before use, and a 5-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 measurement were performed according to the user guide of the Fiske Model 210 Micro-Osmometer. All the tested vaccine samples were found to have osmotic pressure measurements in the range of 604 - 616 mOsm / kg, similar to the osmotic pressure measurement values of 595 - 626 mOsm / kg detected in the SPG vehicle. These values are consistent with those obtained for the clinical trial materials (Bris10 M2SR, lot number 15100251).

[0222] [Table 13]

[0223] The H3N2 FGHY1-M2SR vaccine batch was manufactured in M2VeroA cells using a process similar to that of the target clinical material, and formulated in the same SPG buffer as the target clinical material. Characterization tests showed that the purification process successfully removed host cell impurities (DNA and proteins), simulating the purity of the clinical material, while maintaining a high infectivity titer and preserving the vaccine virus's genome sequence and replication deficiency phenotype.

[0224] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as each reference is individually and specifically indicated to be incorporated by reference, and to the same extent as they are incorporated herein in whole.

[0225] With regard to the description of the present invention (in particular with regard to the following claims), the use of the terms “a,” “an,” “the,” and “at least one,” and similar references, should be interpreted as covering both singular and plural forms, unless otherwise specifically noted herein or clearly inconsistent with the context. The use of the term “at least one” after the enumeration of one or more items (e.g., “at least one of A and B”) should be interpreted as meaning one item (A or B) selected from the enumerated items, or any combination of two or more items (A and B), unless otherwise specifically noted herein or clearly inconsistent with the context. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., “including but not limited to”) unless otherwise specifically noted herein. Unless otherwise specifically noted herein, descriptions of ranges of values ​​are intended only as an abbreviation to refer individually to each individual value that falls within that range, and each individual value is incorporated herein as if it were described individually herein. All methods described herein may be carried out in any preferred order, unless otherwise specified herein or otherwise clearly inconsistent with the context. The use of any and all examples or illustrative terms provided herein (e.g., "such as") is intended solely to better illustrate the invention and does not impose any limitation on the scope of the invention unless otherwise claimed. All terms herein should not be construed as indicating any unclaimed element as essential to the practice of the invention.

[0226] Preferred embodiments of the present invention, including the best mode known to the inventors for carrying out the invention, are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art by reading the above description. The inventors anticipate that those skilled in the art will use such variations as appropriate, and they intend that the present invention may be carried out in ways different from those specifically described herein. Accordingly, the present invention may be permitted by applicable law. As such, this includes all modifications and equivalents of the subject matter described in the claims attached herein. Furthermore, any combination of the above elements in all possible variations thereof is encompassed by the present invention unless otherwise specifically noted herein or is particularly obviously inconsistent with the context.

Claims

1. A pharmaceutical preparation comprising a first influenza virus and a second influenza virus, The first influenza virus and the second influenza virus are different, The first influenza virus and the second influenza virus include a PB1 gene segment encoding a PB1 protein having the amino acid sequence of SEQ ID NO: 7, a PB2 gene segment encoding a PB2 protein having the amino acid sequence of SEQ ID NO: 15, a PA gene segment encoding a PA protein having the amino acid sequence of SEQ ID NO: 17, an NP gene segment encoding an NP protein having the amino acid sequence of SEQ ID NO: 6, and an NS1 gene segment encoding an NS1 protein having the amino acid sequence of SEQ ID NO:

19. Pharmaceutical preparations.

2. The pharmaceutical formulation according to claim 1, wherein the first influenza virus and the second influenza virus further comprise an NA gene segment and an HA gene segment.

3. The pharmaceutical formulation according to claim 2, wherein the first influenza virus comprises an NA gene segment different from the NA gene segment of the second influenza virus.

4. The pharmaceutical formulation according to claim 2, wherein the first influenza virus comprises an HA gene segment different from the HA gene segment of the second influenza virus.

5. The pharmaceutical formulation according to claim 2, wherein the first influenza virus comprises an HA gene segment and an NA gene segment that are different from the HA gene segment and NA gene segment of the second influenza virus.

6. The pharmaceutical formulation according to claim 5, wherein the first influenza virus comprises an H3 HA gene segment and an N2 NA gene segment, and the second influenza virus comprises an H1 HA gene segment and an N1 NA gene segment.

7. The pharmaceutical preparation according to any one of claims 1 to 6, wherein the pharmaceutical preparation further comprises at least one type of influenza B virus.

8. The pharmaceutical preparation according to claim 7, wherein the pharmaceutical preparation contains two types of influenza B viruses, and the two types of influenza B viruses are not identical.

9. The pharmaceutical preparation according to any one of claims 1 to 8, wherein the pharmaceutical preparation is a vaccine.

10. A pharmaceutical formulation according to any one of claims 1 to 9, for use in inducing an immune response to influenza virus in mammals.

11. The pharmaceutical preparation according to claim 10, wherein the mammal is a human.

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