Formulations of heat-stable vaccines

Abstract

This invention provides a stable pharmaceutical formulation comprising (a) a buffer, (b) a sugar, (c) a stabilizer, (d) one or more amino acids, and (e) at least one influenza virus backbone. This invention also provides a method for treating mammals in need of influenza vaccination by administering the pharmaceutical formulation of this invention. This invention further provides a method for inducing an immune response by administering the pharmaceutical formulation of this invention.

Description

[0001] By referencing materials incorporated into electronic submissions

[0002] The computer-readable nucleotide / amino acid sequence listing submitted with this article is incorporated herein by reference in its entirety and is identified as a 66,463-byte file named “772012.XML” created on October 10, 2024. Background Technology

[0003] Vaccines are an important tool for preventing illness caused by infectious diseases, which can infect millions of people worldwide. However, vaccines cannot be administered immediately after production, as they must be transported from the manufacturing center to the pharmacy or doctor's office where they are administered—a process that can be lengthy in some cases. During transport, vaccines may be stored in different locations for different periods, depending on their needs. Both transport and storage may involve varying temperature environments. These variations in time and storage can cause the vaccine to decompose and become less effective.

[0004] Therefore, it is still necessary to allow vaccine formulations to be stored at different temperatures for relatively long periods of time. Summary of the Invention

[0005] The present invention provides a stable pharmaceutical formulation comprising (a) a buffer, (b) a sugar, (c) a stabilizer, (d) one or more amino acids, and (e) at least one influenza virus backbone.

[0006] The present invention also provides a method for treating mammals in need of influenza vaccines by administering the pharmaceutical preparation of the present invention. The present invention further provides a method for inducing an immune response by administering the pharmaceutical preparation of the present invention. Attached Figure Description

[0007] Figure 1 This is a graph comparing the viral titers of drug formulations containing M2SR and BM2SR at 4°C with and without sucrose.

[0008] Figures 2A to 2C It contains the H1N1 M2SR skeleton ( Figure 2A ), Sing 2016 H3N2M2SR skeleton of influenza A ( Figure 2B ) and influenza B CO / O6 BM2SR skeleton ( Figure 2C A graph showing the comparison of viral titers at 4°C for different drug formulations.

[0009] Figure 3 This is a graph showing the comparison of viral titers over time between 2°C and 8°C for drug formulations containing rHSA and those without rHSA that contain the Bris10 H3N2 M2SR backbone.

[0010] Figures 4A to 4C It contains the H3N2 M2SR skeleton ( Figure 4A H1N1 M2SR skeleton ( Figure 4B ) and CA12 BM2SR skeleton ( Figure 4C A graph showing the comparison of viral titers of drug formulations at ambient temperature over time.

[0011] Figures 5A to 5F It contains sucrose phosphate and Sing 2016 H3N2 M2SR ( Figure 5A ), histidine and Sing2016 H3N2 M2SR ( Figure 5B ), sucrose phosphate and MT50 H1N1 M2SR ( Figure 5C ), histidine and MT50 H1N1 M2SR ( Figure 5D ), sucrose phosphate and CA / 12 (YL) BM2SR ( Figure 5E ), and histidine and CA / 12 (YL) BM2SR ( Figure 5F A graph showing the viral titer values ​​of the drug formulation after 0, 1, 8, or 16 freeze-thaw cycles.

[0012] Figures 6A to 6C It includes MT50 H1N1 M2SR with and without rHSA. Figure 6A Sing 2016H3N2 M2SR ( Figure 6B ) and CA / 12YL BM2SR ( Figure 6C A graph showing the comparison of viral titers at 4°C for different drug formulations.

[0013] Figures 7A to 7C It contains H3N2 M2SR ( Figure 7A H1N1 M2SR ( Figure 7B ) and CA12 BM2SR ( Figure 7C A graph showing the comparison of viral titers at -20°C for three different drug formulations.

[0014] Figure 8 This is a graph showing the viral titer of a drug formulation containing HEPES buffer at storage times between 2°C and 8°C.

[0015] Figure 9 This is a graph comparing viral titers of drug formulations containing histidine buffer at storage times from 2°C to 8°C. Detailed Implementation

[0016] The stable pharmaceutical formulation of this application comprises (a) a buffer, (b) a sugar, (c) a stabilizer, (d) one or more amino acids, and (e) at least one influenza virus backbone.

[0017] Any suitable buffer may be present in the pharmaceutical formulation. In some embodiments, the buffer includes at least one of potassium phosphate, sodium phosphate, imidazole, histidine, citrate (e.g., sodium citrate), HEPES or phosphate-buffered saline (PBS), and Dulbecco phosphate-buffered saline (DPBS).

[0018] The buffer can be present in the pharmaceutical formulation at any suitable concentration. In some embodiments, the concentration of the buffer is from about 20 mM to about 150 mM, for example about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, or about 120 mM, or a concentration within the range defined by any two of the foregoing values.

[0019] Any suitable sugar may be present in the pharmaceutical formulation. In some embodiments, the pharmaceutical formulation contains at least one sugar, such as at least two, three, or four sugars. In some embodiments, the sugar includes at least one of sucrose, lactose, and trehalose.

[0020] Sugar can be present in pharmaceutical preparations at any suitable concentration. In some implementations, the sugar concentration is from about 1.0% w / v to about 15.0% w / v, for example, about 1.0% w / v, about 1.5% w / v, about 2.0% w / v, about 2.5% w / v, about 3.0% w / v, about 3.5% w / v, about 4.0% w / v, about 4.5% w / v, about 5.0% w / v, about 5.5% w / v, about 6.0% w / v, about 6.5% w / v, about 7.0% w / v, about 7.5% w / v, about 8.0% w / v, about 8.5% w / v, about 9.0% w / v, about 9.5% w / v, about 10.0% w / v, about 10.5% w / v, about 11.0% w / v, about 11.5% w / v, about 12.0% w / v, and about 12.5%. w / v, about 13.0% w / v, about 13.5% w / v, about 14.0% w / v, about 14.5% w / v or about 15.0% w / v, or a concentration within the range defined by any two of the foregoing values.

[0021] Any suitable stabilizer may be present in the pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises at least one stabilizer, such as at least two, at least three, or at least four stabilizers. In some embodiments, the stabilizer includes dextran 40K, dextran 70K, polyvinylpyrrolidone (PVP) 40K, PVP 70K, hydroxyethyl starch (HES), gelatin, polyethylene glycol (PEG) 4600, or recombinant human serum albumin (rHSA).

[0022] Stabilizers can be present in pharmaceutical formulations at any suitable concentration. In some embodiments, the concentration of the stabilizer is from about 0.01% w / v to about 3.0% w / v, for example, about 0.01% w / v, about 0.05% w / v, about 0.10% w / v, about 0.15% w / v, about 0.20% w / v, about 0.25% w / v, about 0.30% w / v, about 0.35% w / v, about 0.40% w / v, about 0.45% w / v, about 0.50% w / v, about 0.55% w / v, about 0.60% w / v, about 0.65% w / v, about 0.70% w / v, about 0.75% w / v, about 0.80% w / v, about 0.85% w / v, about 0.90% w / v, about 0.95% w / v, about 1.00% w / v, and about 1.05%. w / v, approximately 1.10% w / v, approximately 1.15% w / v, approximately 1.20% w / v, approximately 1.25% w / v, approximately 1.30% w / v, approximately 1.35% w / v, approximately 1.40% w / v, approximately 1.45% w / v, approximately 1.50% w / v, approximately 1.55% w / v, approximately 1.60% w / v, approximately 1.65% w / v, approximately 1.70% w / v, approximately 1.75% w / v, approximately 1.80% w / v, approximately 1.85% w / v, approximately 1.90% w / v, approximately 1.95%, approximately 2.00% w / v, approximately 2.05% w / v, approximately 2.10% w / v, approximately 2.15% w / v, approximately 2.20% w / v, approximately 2.25% w / v, approximately 2.30% w / v, about 2.35% w / v, about 2.40% w / v, about 2.45% w / v, about 2.50% w / v, about 2.55% w / v, about 2.60% w / v, about 2.65% w / v, about 2.70% w / v, about 2.75% w / v, about 2.80% w / v, about 2.85% w / v, about 2.90% w / v, about 2.95% w / v or about 3.0% w / v, or concentrations within the range defined by any two of the foregoing values.

[0023] Any suitable amino acid may be present in the pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises one or more amino acids, such as two or more amino acids, three or more amino acids, or four or more amino acids. In some embodiments, one or more amino acids are selected from glutamic acid, histidine, glycine, and arginine.

[0024] One or more amino acids may be present in a pharmaceutical preparation at any suitable concentration. In some embodiments, the concentration of one or more amino acids is from about 0.05% w / v to about 2.0% w / v, for example, about 0.05% w / v, about 0.10% w / v, about 0.15% w / v, about 0.20% w / v, about 0.25% w / v, about 0.30% w / v, about 0.35% w / v, about 0.40% w / v, about 0.45% w / v, about 0.50% w / v, about 0.55% w / v, about 0.60% w / v, about 0.65% w / v, about 0.70% w / v, about 0.75% w / v, about 0.80% w / v, about 0.85% w / v, about 0.90% w / v, about 0.95% w / v, about 1.0% w / v, about 1.05% w / v, or about 1.10%. w / v, about 1.15% w / v, about 1.20% w / v, about 1.25% w / v, about 1.30% w / v, about 1.35% w / v, about 1.40% w / v, about 1.45% w / v, about 1.50% w / v, about 1.55% w / v, about 1.60% w / v, about 1.65% w / v, about 1.70% w / v, about 1.75% w / v, about 1.80% w / v, about 1.85% w / v, about 1.90% w / v, about 1.95% or about 2.0%, or concentrations within the range defined by any two of the foregoing values.

[0025] Any suitable influenza virus backbone can be present in the pharmaceutical formulation. As used herein, the term "backbone" refers to an influenza gene segment encoding the PB1, PB2, PA, NP, NS1, and / or NS2 proteins. The backbone may also include gene segments encoding the M protein. The backbone may also include gene segments encoding NA (neuraminidase) and HA (hemagglutinin) gene segments. The gene segments of the present invention encode proteins having selected amino acids. Based on the core proteins of influenza viruses, there are four types of influenza viruses (i.e., influenza A, B, C, and D), and seasonal epidemics are most commonly caused by circulating influenza A and influenza B viruses. In some embodiments, the pharmaceutical formulation comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight influenza virus backbones. In some embodiments, the at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight influenza virus backbones are independently selected from influenza A and influenza B virus backbones. For example, in an embodiment where the pharmaceutical formulation comprises one influenza virus backbone, the influenza virus backbone is either an influenza A virus backbone or an influenza B virus backbone. In an embodiment where the pharmaceutical formulation comprises two influenza virus backbones, the two influenza virus backbones can be two influenza A virus backbones, two influenza B virus backbones, or one influenza A virus backbone and one influenza B virus backbone. In an embodiment where the pharmaceutical formulation comprises three influenza virus backbones, the three influenza virus backbones can be three influenza A virus backbones, two influenza A virus backbones and one influenza B virus backbone, two influenza B virus backbones and one influenza A virus backbone, or three influenza B virus backbones. In an embodiment where the pharmaceutical formulation comprises four influenza virus backbones, the four influenza virus backbones can be four influenza A virus backbones, three influenza A virus backbones and one influenza B virus backbone, two influenza A virus backbones and two influenza B virus backbones, one influenza A virus backbone and three influenza B virus backbones, or four influenza B virus backbones. In a preferred embodiment, the pharmaceutical formulation comprises two influenza A virus backbones and two influenza B virus backbones.

[0026] Influenza A skeletal protein

[0027] The PB1 (polymerase basic protein 1) gene fragment of the backbone can encode a protein containing at least one selected amino acid, namely the PB1 protein. In a preferred embodiment, the selected amino acid contains leucine at position 40 and tryptophan at position 180. The selected amino acid of the PB1 protein also contains at least one of asparagine at position 464 or serine at position 607. The PB1 gene fragment may optionally contain a cytosine-to-uracil promoter mutation at position 4.

[0028] The selected amino acid can be obtained by genetically mutagenesis of the parental PB1 sequence, which is, for example, a sequence identical to the PB1 amino acid sequence except for the position corresponding to the selected amino acid. Amino acid position 464 of the PB1 protein is located in the palm region of the influenza PB1 protein and is linked to the RNA-dependent RNA polymerase activity domain. Normally, aspartic acid at position 464 is highly conserved in influenza viruses isolated from oocytes and MDCK cells. Although the role of this amino acid has not been identified, observed amino acid changes at this position, such as asparagine (N), may affect the conformation of the PB1 protein and may affect its interaction with host cytokines, and thus affect influenza polymerase activity in Vero cells. Furthermore, influenza RNA polymerase is a heterotrimer composed of PA, PB1, and PB2 subunits. Histidine at position 465 of the PB1 protein interacts with glutamate at position 243 of the PA protein, and amino acid changes 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, suggesting that it may alter the interaction between PB1 and PB2, thereby affecting polymerase activity in Vero cells.

[0029] The PB2 (polymerase basic protein 2) gene fragment can encode a protein containing at least one selected amino acid, namely the PB2 protein. In a preferred embodiment, the selected amino acid contains valine at position 504 and optionally isoleucine at position 467 and valine at position 529. The PB2 gene fragment may optionally contain a cytosine-to-uracil promoter mutation at position 4. Amino acids at positions 467 and 529 of the PB2 protein are located in the PB2-C portion. Specifically, amino acid at position 467 is located in the cap-binding region of the PB2 protein, and 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 capped RNA and utilizes the cap from the host RNA to produce influenza mRNA. This process is called "cap-snatching." Furthermore, amino acid position 627 of PB2 is known to be a key determinant of host range and viral pathogenicity. Therefore, changes in amino acids near the cap-binding region can affect the efficiency of viral mRNA synthesis.

[0030] The PA (polymerase acid protein) gene fragment can also encode a protein containing at least one selected amino acid, i.e., a PA protein. In a preferred embodiment, the selected amino acid contains a lysine residue at position 401. The PA gene fragment may optionally contain a cytosine-to-uracil promoter mutation at position 4.

[0031] The NP (nuclear protein) gene fragment can encode a protein containing at least one selected amino acid, i.e., an NP protein. In a preferred embodiment, the selected amino acid contains at least one of leucine at position 116 and lysine at position 294 or arginine at position 311. The amino acids at positions 294 and 311 of the NP protein are located within the NP protein body, and therefore do not serve as nuclear localization signals or nuclear export signals.

[0032] The NS (non-structural) gene fragment can encode a protein containing at least one selected amino acid, namely the NS1 and / or NS2 protein. In a preferred embodiment, the selected amino acid contains proline at position 30 (NS1 protein) and lysine at position 118 (NS1 protein).

[0033] In one embodiment, the influenza virus backbone includes a PB1 gene fragment encoding a protein having selected amino acids (i.e., leucine at position 40, tryptophan at position 180, and asparagine at position 464), namely the PB1 protein. The PB1 gene fragment may have the nucleotide sequence shown in SEQ ID NO: 2. The PB1 gene fragment may encode a protein having the amino acid sequence shown in SEQ ID NO: 7, namely the PB1 protein. In another embodiment, the influenza virus backbone may include a PB2 gene fragment encoding a protein having selected amino acid (i.e., valine at position 504), namely the PB2 protein. The PB2 gene fragment may have the nucleotide sequence shown in SEQ ID NO: 13. The PB2 gene fragment may encode a protein having the amino acid sequence shown in SEQ ID NO: 14, namely the PB2 protein. The NP gene fragment of this embodiment can encode a protein having selected amino acids (i.e., leucine at position 116 and lysine at position 294), namely an NP protein. The NP gene fragment can have the nucleotide sequence shown in SEQ ID NO: 1. The NP gene fragment can encode a protein having the amino acid sequence shown in SEQ ID NO: 6, namely an NP protein. The PA and NS gene fragments of this embodiment can also encode proteins, namely PA protein and NS1 and / or NS2 protein, which contain selected amino acids at positions 401 (PA protein), 30 (NS1 protein), and 118 (NS1 protein), namely lysine at position 401 (PA protein), proline at position 30 (NS1 protein), and lysine at position 118 (NS1 protein). The PA gene fragment can have the nucleotide sequence shown in SEQ ID NO: 15. The PA gene fragment can encode a protein having the amino acid sequence shown in SEQ ID NO: 16, namely a PA protein. The NS gene fragment can have the nucleotide sequence shown in SEQ ID NO: 17. The NS gene fragment can encode a protein having the amino acid sequence of SEQ ID NO: 18, namely the NS1 protein. The NS gene fragment can encode a protein having the amino acid sequence of SEQ ID NO: 19, namely the NS2 protein. The PB1, PB2 and PA gene fragments of this embodiment may also contain a cytosine-to-uracil promoter mutation at the 4th nucleotide position.

[0034] In another embodiment, the influenza virus backbone includes a PB1 gene fragment encoding a protein having selected amino acids at positions 40, 180, and 607 (i.e., leucine at position 40, tryptophan at position 180, and serine at position 607), namely the PB1 protein. The PB1 gene fragment may have the nucleotide sequence shown in SEQ ID NO: 4. The PB1 gene fragment may encode a protein having the amino acid sequence of SEQ ID NO: 9, namely the PB1 protein. In another aspect of this embodiment, the influenza virus backbone may include a PB2 gene fragment encoding a protein having selected amino acids at positions 504, 467, and 529 (i.e., valine at position 504, isoleucine at position 467, and valine at position 529), namely the PB2 protein. The PB2 gene fragment may have the nucleotide sequence shown in SEQ ID NO: 5. The PB2 gene fragment may encode a protein having the amino acid sequence of SEQ ID NO: 10, namely the PB2 protein. The NP gene fragment of this embodiment can encode a protein having selected amino acids (i.e., leucine at position 116 and arginine at position 311), namely an NP protein. The NP gene fragment can have the nucleotide sequence shown in SEQ ID NO: 3. The NP gene fragment can encode a protein having the amino acid sequence shown in SEQ ID NO: 8, namely an NP protein. The PA and NS gene fragments can also encode proteins, namely PA protein and NS1 and / or NS2 protein, which contain selected amino acids at positions 401 (PA protein), 30 (NS1 protein), and 118 (NS1 protein), namely lysine at position 401 (PA protein), proline at position 30 (NS1 protein), and lysine at position 118 (NS1 protein). The PA gene fragment can have the nucleotide sequence shown in SEQ ID NO: 15. The PA gene fragment can encode a protein having the amino acid sequence shown in SEQ ID NO: 16, namely a PA protein. The NS gene fragment can have the nucleotide sequence shown in SEQ ID NO: 17. The NS gene fragment can encode a protein having the amino acid sequence of SEQ ID NO: 18, namely the NS1 protein. The NS gene fragment can encode a protein having the amino acid sequence of SEQ ID NO: 19, namely the NS2 protein. The PB1, PB2 and PA gene fragments of this embodiment may also contain a cytosine-to-uracil promoter mutation at the 4th nucleotide position.

[0035] The influenza virus backbone may include an M (matrix protein) gene segment. In one embodiment, the M gene segment may be a mutant gene segment from influenza A, resulting in the virus lacking expression of the functional M2 protein. Such a virus is referred to herein as an "M2SR" virus. As used herein, "M2SR" and "AM2SR" are interchangeable. The M2SR virus is a single-replicating influenza virus. The M gene segment of the M2SR virus may be as shown in SEQ ID NO: 11. The M gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 12, i.e., a truncated M2 protein. The M2SR virus can proliferate in Vero cells (i.e., M2VeroA cells) that stably express the wild-type M2 protein to allow for multi-cycle replication. High yields in Vero cells are independent of mutations in the M gene segment. Therefore, the influenza virus backbone may include an M gene segment encoding the functional M2 protein (SEQ ID NO: 33).

[0036] Influenza B backbone protein

[0037] In one embodiment, the recombinant virus comprises an influenza virus backbone containing PA, NP, and NS gene fragments, wherein (a) the PA gene fragment contains thymine at nucleotide 2272; (b) the NP gene fragment encodes an NP protein having an amino acid sequence comprising selected amino acids, wherein the selected amino acids comprise serine at position 40, asparagine or glycine at position 161, threonine at position 204, and optionally valine at position 93; and (c) the NS gene fragment contains guanine at nucleotide 39, and the NS gene fragment encodes an NS protein having an amino acid sequence comprising selected amino acids, wherein the selected amino acids comprise glutamine at position 176.

[0038] The PB1 (polymerase basic protein 1) gene fragment can encode a protein containing at least one selected amino acid, i.e., the PB1 protein. The selected amino acid can be obtained by genetically mutating a parental PB1 sequence, which is, for example, a sequence identical to the PB1 amino acid sequence except for the position corresponding to the selected amino acid. The PB2 (polymerase basic protein 2) gene fragment can also encode a protein containing at least one selected amino acid, i.e., the PB2 protein.

[0039] The PA (polymerase acidic protein) gene fragment can encode a protein containing at least one selected amino acid, i.e., a PA protein. In a preferred embodiment, the gene fragment contains thymine at nucleotide position 2272. The NP (nucleoprotein) gene fragment can encode a protein containing at least one selected amino acid, i.e., an NP protein. In a preferred embodiment, the NP fragment contains thymine at position 177, adenine at position 540, and thymine at position 670, and the NP gene fragment encodes a protein having selected amino acids, said selected amino acids including serine at position 40, asparagine or glycine at position 161, threonine at position 204, and optionally valine at position 93.

[0040] The NS (non-structural) gene fragment can encode a protein containing at least one selected amino acid, namely the NS1 and / or NS2 protein. In a preferred embodiment, the NS fragment contains guanine at position 39 and cytosine at position 570, and the NS gene fragment encodes an NS protein (NS1 protein) having selected amino acids, said selected amino acids including glutamine at position 176.

[0041] In one embodiment, the influenza virus includes a PB1 gene fragment encoding a protein having selected amino acids, namely the PB1 protein. The PB1 gene fragment may have the nucleotide sequence shown in SEQ ID NO: 20. The PB1 gene fragment may encode a protein having the amino acid sequence shown in SEQ ID NO: 25, namely the PB1 protein. In another embodiment, the influenza virus may include a PB2 gene fragment encoding a protein having selected amino acids, namely the PB2 protein. The PB2 gene fragment may have the nucleotide sequence shown in SEQ ID NO: 21. The PB2 gene fragment may encode a protein having the amino acid sequence shown in SEQ ID NO: 26, namely the PB2 protein. In another embodiment, the influenza virus may include an NP gene fragment encoding a protein having selected amino acids (serine at position 40, asparagine or glycine at position 161, threonine at position 204, and optionally valine at position 93), namely the NP protein. The NP gene fragment may have the nucleotide sequence shown in SEQ ID NO: 23. The NP gene fragment can encode a protein having the amino acid sequence of SEQ ID NO: 28, i.e., the NP protein. In another embodiment, the influenza virus can include an NS gene fragment encoding a protein having a selected amino acid (i.e., glutamine at position 176), i.e., the NS1 and / or NS2 proteins. The NS gene fragment can include guanine at position 39 and cytosine at position 570. The NS gene fragment can have the nucleotide sequence shown in SEQ ID NO: 24. The NS gene fragment can encode a protein having the amino acid sequence of SEQ ID NO: 29, i.e., the NS1 and / or NS2 proteins. In another embodiment, the influenza virus can include a PA gene fragment encoding a protein (i.e., the PA protein). The PA gene fragment can have the nucleotide sequence shown in SEQ ID NO: 22. The PA gene fragment can encode a protein having the amino acid sequence of SEQ ID NO: 27, i.e., the PA protein.

[0042] Influenza viruses may contain an M (matrix protein) gene segment. In one embodiment, the M gene segment may be a mutant gene segment from influenza B, resulting in the virus lacking expression of the functional BM2 protein. Such a virus is referred to herein as a "BM2SR" virus. BM2SR virus is a single-replicating influenza virus. The M gene segment of BM2SR virus may be as shown in SEQ ID NO: 30. The M gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 32, i.e., a truncated BM2 protein. BM2SR virus can proliferate in Vero cells (i.e., BM2VeroA cells) that stably express the BM2 protein to allow for multi-cycle replication. High yields in Vero cells do not depend on mutations in the M gene segment. Therefore, influenza viruses may contain an M gene segment encoding the functional BM2 protein (SEQ ID NO: 34).

[0043] Influenza A surface protein

[0044] In another embodiment, the influenza virus backbone comprises NA (neuraminidase) and HA (hemagglutinin) gene fragments. In one embodiment, the HA gene fragment encodes an HA protein having an amino acid sequence that includes at least one selected amino acid (e.g., an amino acid mutation) in the HA1 subunit of the protein and / or at least one selected amino acid (e.g., an amino acid mutation) in the HA2 subunit. For example, at least one amino acid mutation in the HA2 subunit could be asparagine at position 107. Such mutations can also contribute to enhanced viral growth during production.

[0045] In one implementation, the PB1, PB2, PA, NP, and NS gene fragments are derived from a single influenza strain. The HA gene fragment may be derived from an influenza strain that is different from the single influenza strain from which the PB1, PB2, PA, NP, and NS gene fragments originate. Similarly, the NA gene fragment may be derived from an influenza strain that is different from the single influenza strain from which the PB1, PB2, PA, NP, and NS gene fragments originate. Therefore, the recombinant virus can be a pandemic virus (e.g., H5N1 and H7N9) or a seasonal virus (e.g., H1N1, H3N2, and influenza B).

[0046] Influenza B surface protein

[0047] In another embodiment, the recombinant virus comprises an influenza virus backbone, which further comprises NA (neuraminidase) and HA (hemagglutinin) gene fragments. In one embodiment, the HA gene fragment encodes an HA protein having an amino acid sequence in which at least one selected amino acid (e.g., an amino acid mutation) is contained in the HA1 subunit of the protein and / or in the HA2 subunit of the protein (e.g., an amino acid mutation). For example, at least one amino acid mutation in the HA2 subunit could be glutamic acid at position 61. In another embodiment, at least one amino acid mutation in the HA2 subunit could be glutamic acid at position 112. Amino acid mutations can be present in any subtype or lineage of influenza B virus (i.e., Victoria or Yamagata). In a preferred embodiment, the amino acid mutation in the HA2 subunit could be glutamic acid at position 61 in the Victoria lineage of influenza B virus. In another preferred embodiment, the amino acid mutation in the HA2 subunit could be glutamic acid at position 112 in the Yamagata lineage of influenza B virus. Such mutations can also contribute to enhanced viral growth during production.

[0048] In one implementation, the PB1, PB2, PA, NP, and NS gene fragments are derived from a single influenza strain. The HA gene fragment may be derived from an influenza strain that is different from the single influenza strain from which the PB1, PB2, PA, NP, and NS gene fragments originate. Similarly, the NA gene fragment may be derived from an influenza strain that is different from the single influenza strain from which the PB1, PB2, PA, NP, and NS gene fragments originate. Therefore, the influenza virus may be a seasonal influenza virus (e.g., influenza B).

[0049] Any suitable amount of influenza virus backbone can be present in the pharmaceutical formulation. In some implementations, the amount of influenza virus backbone per dose is approximately 5 × 10⁻⁶. 7 TCID 50 From approximately 1 × 10 10 TCID 50 For example, 5 × 10 7 TCID 50 6 × 10 7 TCID 50 7 × 10 7 TCID 50 8 × 10 7 TCID 50 9 × 10 7 TCID 50 1 × 10 8 TCID 50 2 × 108 TCID 50 3 × 10 8 TCID 50 4 × 10 8 TCID 50 5 × 10 8 TCID 50 6 × 10 8 TCID 50 7 × 10 8 TCID 50 8 × 10 8 TCID 50 9 × 10 8 TCID 50 1 × 10 9 TCID 50 2 × 10 9 TCID 50 3 × 10 9 TCID 50 4 × 10 9 TCID 50 5 × 10 9 TCID 50 6 × 10 9 TCID 50 7 × 10 9 TCID 50 8 × 10 9 TCID 50 9 × 10 9 TCID 50 Or 1 × 10 10 TCID 50 Or a range defined by any two of the aforementioned values. In some embodiments, the amount of the influenza virus backbone is at least 10. 9 TCID 50 / dose. In some implementations, the amount of the influenza virus backbone is approximately 5 × 10⁻⁶. 9 / mL to approximately 5 × 10 10 / mL, for example 5 × 10 9 / mL, 6 × 10 9 / mL, 7 × 10 9 / mL, 8 ×10 9 / mL, 9 × 10 9 / mL, 1 × 10 10 / mL, 2 × 10 10 / mL, 3 × 10 10 / mL, 4 × 10 10 / mL or 5 × 10 10 / mL, or a range defined by any two of the foregoing values. In some embodiments, the pharmaceutical formulation comprises one or more doses, two or more doses, three or more doses, four or more doses, five or more doses, six or more doses, seven or more doses, or eight or more doses of the influenza virus backbone. In some embodiments, the number of doses of the influenza virus backbone is the same as the number of influenza virus backbones present in the pharmaceutical formulation.

[0050] In some embodiments, the pharmaceutical formulation also comprises a polyol. Any suitable polyol may be present in the pharmaceutical formulation. In some embodiments, the polyol is mannitol or sorbitol.

[0051] Polyols can be present in pharmaceutical formulations at any suitable concentration. In some embodiments, the concentration of the polyol is from about 1.0% w / v to about 5.0% w / v, such as about 1.0% w / v, about 1.2% w / v, about 1.4% w / v, about 1.6% w / v, about 1.8% w / v, about 2.0% w / v, about 2.2% w / v, about 2.4% w / v, about 2.6% w / v, about 2.8% w / v, about 3.0% w / v, about 3.2% w / v, about 3.4% w / v, about 3.6% w / v, about 3.8% w / v, about 4.0% w / v, about 4.2% w / v, about 4.4% w / v, about 4.6% w / v, about 4.8% w / v, or about 5.0% w / v, or a concentration within a range defined by any two of the foregoing values.

[0052] In some embodiments, the pharmaceutical preparation further comprises one or more salts. In some embodiments, the one or more salts are selected from magnesium sulfate, magnesium chloride, sodium chloride, and potassium chloride.

[0053] One or more salts may be present in the pharmaceutical formulation at any suitable concentration. In some embodiments, the concentration of one or more salts is from about 0.01% w / v to about 0.15% w / v, such as about 0.01% w / v, about 0.02% w / v, about 0.03% w / v, about 0.04% w / v, about 0.05% w / v, about 0.06% w / v, about 0.07% w / v, about 0.08% w / v, about 0.08% w / v, about 0.09% w / v, about 0.10% w / v, about 0.11% w / v, about 0.12% w / v, about 0.13% w / v, about 0.14% w / v, or about 0.15% w / v, or a concentration within the range defined by any two of the foregoing values. In some embodiments, the concentration of one or more salts is from about 1 mM to about 150 mM, for example about 1 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM or about 150 mM, or a concentration within a range defined by any two of the foregoing values.

[0054] In some embodiments, the pharmaceutical formulation also includes a mucosal adhesive. Any suitable mucosal adhesive may be present in the pharmaceutical formulation. In some embodiments, the mucosal adhesive is carboxymethyl cellulose (CMC).

[0055] Mucosal adhesives can be present in pharmaceutical formulations at any suitable concentration. In some embodiments, the concentration of the mucosal adhesive is from about 0.1% w / v to about 0.5% w / v, such as about 0.1% w / v, about 0.15% w / v, about 0.20% w / v, about 0.25% w / v, about 0.30% w / v, about 0.35% w / v, about 0.40% w / v, about 0.45% w / v, or about 0.50% w / v, or a concentration within the range defined by any two of the foregoing values.

[0056] In some embodiments, the pharmaceutical formulation also includes a surfactant. Any suitable surfactant may be present in the pharmaceutical formulation. In some embodiments, the surfactant includes polysorbate 20, polysorbate 80, deoxycholic acid (DOC), sodium deoxycholate, poloxamer 188 (P188), and / or polyacrylic acid.

[0057] Surfactants can be present in the pharmaceutical formulation at any suitable concentration. In some embodiments, the concentration of the surfactant is from about 0.01% w / v to about 0.15% w / v, such as about 0.01% w / v, about 0.02% w / v, about 0.03% w / v, about 0.04% w / v, about 0.05% w / v, about 0.06% w / v, about 0.07% w / v, about 0.08% w / v, about 0.09% w / v, about 0.10% w / v, about 0.11% w / v, about 0.12% w / v, about 0.13% w / v, about 0.14% w / v, or about 0.15% w / v, or a concentration within the range defined by any two of the foregoing values.

[0058] In some embodiments, the pharmaceutical formulation also includes a chelating agent. Any suitable chelating agent may be present in the pharmaceutical formulation. In some embodiments, the chelating agent is EDTA.

[0059] Chelating agents can be present in the pharmaceutical formulation at any suitable concentration. In some embodiments, the concentration of the chelating agent is from about 1 μM to about 1000 μM, such as about 1 μM, about 50 μM, about 100 μM, about 150 μM, about 200 μM, about 250 μM, about 300 μM, about 350 μM, about 400 μM, about 450 μM, about 500 μM, about 550 μM, about 600 μM, about 650 μM, about 700 μM, about 750 μM, about 800 μM, about 850 μM, about 900 μM, about 950 μM, or about 1000 μM, or a concentration within the range defined by any two of the foregoing values.

[0060] In some embodiments, the pharmaceutical preparation has a pH of about 7.0 to about 7.6, such as about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, or about 7.6, or a pH within a range defined by any two of the foregoing values. In some embodiments, the pharmaceutical preparation has a pH of about 7.2 to about 7.4, and in a preferred embodiment, the pharmaceutical preparation has a pH of about 7.2 or about 7.4.

[0061] In some embodiments, the pharmaceutical formulation comprises (a) about 20 mM sodium phosphate; (b) about 134 mM sodium chloride and 4.5 mM potassium chloride; (c) about 10% w / v sucrose; (d) about 0.5% w / v rHSA; (e) about 5 mM glutamic acid and about 1% w / v arginine; and (f) about 10 9 TCID 50 / The influenza virus skeleton of the dose.

[0062] In some embodiments, the pharmaceutical formulation comprises (a) about 50 mM histidine; (b) about 3.0% w / v mannitol; (c) about 3.0% w / v trehalose; (d) about 0.1% w / v PEG4600; (e) about 0.4% w / v CMC; (f) about 0.5% w / v glycine; and (g) about 10 9 TCID 50 / The influenza virus skeleton of the dose.

[0063] In some embodiments, the pharmaceutical formulation comprises (a) about 50 mM HEPES; (b) about 3.0% w / v mannitol; (c) about 3.0% w / v trehalose; (d) about 0.1% w / v PVP40K; (e) about 0.5% w / v glycine; (f) about 0.2% w / v polyacrylic acid; and (g) about 10 9 TCID 50 / The influenza virus skeleton of the dose.

[0064] In some embodiments, the pharmaceutical formulation comprises (a) about 50 mM potassium phosphate; (b) about 3.0% w / v mannitol; (c) about 1.0% w / v trehalose; (d) about 0.01% w / v dextran 70K; (e) about 1.0% w / v glycine; (f) about 0.05% w / v P188; (g) about 50 μM EDTA; and (f) about 10 μM potassium phosphate. 9 TCID 50 / The influenza virus skeleton of the dose.

[0065] This invention provides a pharmaceutical formulation that is stable for a period of time under specific conditions. As used herein, "stable" means a percentage of the original viral titer after a set period of time under specific conditions, such as at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or a percentage within a range defined by any two of the foregoing values.

[0066] In some embodiments, the pharmaceutical formulation is stable at -20°C for at least 16 to 56 months, such as at least 16 months, at least 18 months, at least 20 months, at least 22 months, at least 24 months, at least 26 months, at least 28 months, at least 30 months, at least 32 months, at least 34 months, at least 36 months, at least 38 months, at least 40 months, at least 42 months, at least 44 months, at least 46 months, at least 48 months, at least 50 months, at least 52 months, at least 54 months, or at least 56 months, or a period of time within the range defined by any two of the foregoing values. In some embodiments, the pharmaceutical formulation is stable at 4°C for at least 30 days to 12 months, such as at least 30 days, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months, or a period of time within the range defined by any two of the foregoing values. In some implementations, the pharmaceutical preparation is stable at ambient temperature for at least 12 to 40 days, such as 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or within a time period defined by any two of the foregoing values. In some implementations, the pharmaceutical preparation is stable at 33°C for at least 4 to 22 days, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 days, or within a time period defined by any two of the foregoing values.

[0067] In some formulations, the pharmaceutical preparation is an influenza virus vaccine. Influenza virus vaccines can be live, attenuated, or inactivated virus vaccines (e.g., whole-virus, split-virus, or subunit vaccines). Viral vaccines can be formulated using multiple influenza virus backbone subtypes (i.e., different hemagglutinin and neuraminidase subtypes of influenza A, and the Yamagata or Victoria lineage of influenza B) to create monovalent, bivalent, trivalent, or quadrivalent vaccines.

[0068] The present invention also provides a method for treating mammals in need of an influenza virus vaccine, the method comprising administering an effective amount of the pharmaceutical preparation of the present invention described herein. The present invention further provides a method for inducing an immune response in mammals, the method comprising administering an effective amount of the pharmaceutical preparation of the present invention described herein.

[0069] The pharmaceutical formulation can be administered by any suitable method. In some embodiments, the pharmaceutical formulation is administered as an oral formulation (e.g., capsules, tablets, or oral films), a spray (e.g., a nasal spray), or any composition suitable for intranasal or parenteral administration (e.g., intravenous, intramuscular, intradermal, or subcutaneous administration), such as an aqueous or non-aqueous emulsion, solution, or suspension. In some embodiments, the pharmaceutical formulation is administered nasally. In some embodiments, the pharmaceutical formulation has a droplet size of 30 micrometers to 75 micrometers, such as 30 micrometers, 35 micrometers, 40 micrometers, 45 micrometers, 50 micrometers, 55 micrometers, 60 micrometers, 65 micrometers, 70 micrometers, or 75 micrometers, or a droplet size within the range defined by any two of the foregoing values.

[0070] The pharmaceutical preparation can be administered to any suitable subject. In some embodiments, the pharmaceutical preparation is administered to mammals. In some embodiments, the pharmaceutical preparation is administered to humans.

[0071] Implementation Plan

[0072] 1. A stable pharmaceutical preparation comprising (a) a buffer, (b) a sugar, (c) a stabilizer, (d) one or more amino acids, and (e) at least one influenza virus backbone.

[0073] 2. The pharmaceutical formulation according to embodiment 1, wherein the buffer comprises potassium phosphate, sodium phosphate, imidazole, histidine, citric acid or HEPES.

[0074] 3. The pharmaceutical formulation according to embodiment 1 or embodiment 2, wherein the pharmaceutical formulation comprises about 20 mM to about 150 mM of the buffer.

[0075] 4. The pharmaceutical preparation according to any one of embodiments 1 to 3, wherein the sugar comprises sucrose, lactose or trehalose.

[0076] 5. A pharmaceutical formulation according to any one of embodiments 1 to 4, wherein the pharmaceutical formulation comprises about 1% w / v to about 15% w / v of the sugar.

[0077] 6. The pharmaceutical formulation according to any one of embodiments 1 to 5, wherein the stabilizer comprises dextran 40K, dextran 70K, polyvinylpyrrolidone (PVP) 40K, PVP 70K, hydroxyethyl starch (HES), gelatin, polyethylene glycol (PEG) 4600 or recombinant human serum albumin (rHSA).

[0078] 7. A pharmaceutical formulation according to any one of embodiments 1 to 6, wherein the pharmaceutical formulation comprises the stabilizer at about 0.01% w / v to about 3.0% w / v.

[0079] 8. A pharmaceutical preparation according to any one of embodiments 1 to 7, wherein one or more amino acids are selected from glutamic acid, histidine, glycine and arginine.

[0080] 9. A pharmaceutical formulation according to any one of embodiments 1 to 8, wherein the pharmaceutical formulation comprises about 1% w / v of the one or more amino acids.

[0081] 10. A pharmaceutical preparation according to any one of embodiments 1 to 9, wherein the pharmaceutical preparation comprises at least two, at least three, or at least four influenza virus skeletons.

[0082] 11. A pharmaceutical preparation according to any one of embodiments 1 to 10, wherein at least one, at least two, at least three, or at least four influenza virus skeletons are independently selected from influenza A virus skeletons and influenza B virus skeletons.

[0083] 12. A pharmaceutical preparation according to any one of embodiments 1 to 11, wherein the pharmaceutical preparation comprises two influenza A virus backbones and two influenza B virus backbones.

[0084] 13. A pharmaceutical preparation according to any one of embodiments 1 to 12, wherein the pharmaceutical preparation comprises at least 10 9 TCID 50 / The influenza virus skeleton of the dose.

[0085] 14. A pharmaceutical preparation according to any one of embodiments 1 to 13, wherein the pharmaceutical preparation further comprises a polyol.

[0086] 15. The pharmaceutical formulation according to embodiment 14, wherein the polyol is mannitol or sorbitol.

[0087] 16. The pharmaceutical formulation according to embodiment 14 or embodiment 15, wherein the pharmaceutical formulation comprises about 1% w / v to about 5% w / v of the polyol.

[0088] 17. A pharmaceutical preparation according to any one of embodiments 1 to 16, wherein the pharmaceutical preparation further comprises one or more salts.

[0089] 18. The pharmaceutical preparation according to embodiment 17, wherein the one or more salts are selected from magnesium sulfate, magnesium chloride, sodium chloride and potassium chloride.

[0090] 19. The pharmaceutical formulation according to embodiment 17 or embodiment 18, wherein the pharmaceutical formulation comprises about 0.01% w / v to about 0.15% w / v of the one or more salts.

[0091] 20. The pharmaceutical formulation according to embodiment 17 or embodiment 18, wherein the pharmaceutical formulation comprises about 1 mM to about 150 mM of the one or more salts.

[0092] 21. The pharmaceutical formulation according to any one of embodiments 1 to 20, wherein the pharmaceutical formulation further comprises an mucosal adhesive.

[0093] 22. The pharmaceutical formulation according to embodiment 21, wherein the mucosal adhesive is carboxymethyl cellulose (CMC).

[0094] 23. The pharmaceutical formulation according to embodiment 21 or embodiment 22, wherein the pharmaceutical formulation comprises about 0.1% w / v to about 0.5% w / v of a mucosal adhesive.

[0095] 24. A pharmaceutical formulation according to any one of embodiments 1 to 23, wherein the pharmaceutical formulation further comprises a surfactant.

[0096] 25. A pharmaceutical formulation according to any one of embodiments 1 to 24, wherein the surfactant comprises polysorbate 20, polysorbate 80, deoxycholic acid (DOC), or poloxamer 188 (P188) or polyacrylic acid.

[0097] 26. A pharmaceutical formulation according to any one of embodiments 1 to 25, wherein the pharmaceutical formulation comprises about 0.01% w / v to about 0.15% w / v of the surfactant.

[0098] 27. A pharmaceutical preparation according to any one of embodiments 1 to 26, wherein the pharmaceutical preparation further comprises a chelating agent.

[0099] 28. The pharmaceutical formulation according to embodiment 27, wherein the chelating agent is EDTA.

[0100] 29. The pharmaceutical formulation according to embodiment 27 or 28, wherein the pharmaceutical formulation comprises about 1 μM to about 1000 μM of the chelating agent.

[0101] 30. The pharmaceutical preparation according to any one of embodiments 1 to 29, wherein the pH is about 7.0 to about 7.6.

[0102] 31. The pharmaceutical preparation according to any one of embodiments 1 to 30, wherein the pH is about 7.2 to about 7.4.

[0103] 32. The pharmaceutical preparation according to any one of embodiments 1 to 31, wherein the pH is about 7.2.

[0104] 33. The pharmaceutical preparation according to any one of embodiments 1 to 31, wherein the pH is about 7.4.

[0105] 34. A stable pharmaceutical preparation comprising: (a) about 20 mM sodium phosphate; (b) about 134 mM sodium chloride and 4.5 mM potassium chloride; (c) about 10% w / v sucrose; (d) about 0.5% w / v rHSA; (e) about 5 mM glutamate and about 1% w / v arginine; and (f) about 10 9 TCID 50 / dose of influenza virus backbone; or (a) about 50 mM histidine; (b) about 3.0% w / v mannitol; (c) about 3.0% w / v trehalose; (d) about 0.1% w / v PEG4600; (e) about 0.4% w / v CMC; (f) about 0.5% w / v glycine; and (g) about 10 9 TCID 50 / dose of influenza virus backbone; or (a) about 50 mM HEPES; (b) about 3.0% w / v mannitol; (c) about 3.0% w / v trehalose; (d) about 0.1% w / v PVP40K; (e) about 0.5% w / v glycine; (f) about 0.2% w / v polyacrylic acid; and (g) about 10 9 TCID 50 / dose of influenza virus backbone; or (a) about 50 mM potassium phosphate; (b) about 3.0% w / v mannitol; (c) about 1.0% w / v trehalose; (d) about 0.01% w / v dextran 70K; (e) about 1.0% w / v glycine; (f) about 0.05% w / v P188; (g) about 50 μM EDTA; and (f) about 10 9 TCID 50 / The influenza virus skeleton of the dose.

[0106] 35. A pharmaceutical preparation according to any one of embodiments 1 to 34, wherein the pharmaceutical preparation is stable at -20°C for at least 16 to 56 months.

[0107] 36. A pharmaceutical preparation according to any one of embodiments 1 to 34, wherein the pharmaceutical preparation is stable at 4°C for at least 30 days to 12 months.

[0108] 37. A pharmaceutical preparation according to any one of embodiments 1 to 34, wherein the pharmaceutical preparation is stable at ambient temperature for at least 12 to 40 days.

[0109] 38. A pharmaceutical preparation according to any one of embodiments 1 to 34, wherein the pharmaceutical preparation is stable at 33°C for at least 4 to 22 days.

[0110] 39. A pharmaceutical preparation according to any one of embodiments 1 to 38, wherein the pharmaceutical preparation is an influenza virus vaccine.

[0111] 40. A method for treating mammals that require an influenza virus vaccine, the method comprising administering an effective amount of any one of embodiments 1 to 39 of the pharmaceutical preparation.

[0112] 41. A method for inducing an immune response in mammals, the method comprising administering an effective amount of the pharmaceutical preparation described in any one of embodiments 1 to 39.

[0113] 42. The method according to embodiment 40 or embodiment 41, wherein the pharmaceutical preparation is administered nasally.

[0114] 43. The method according to any one of embodiments 40 to 42, wherein the pharmaceutical formulation has a droplet size of 30 micrometers to 75 micrometers.

[0115] 44. The method according to any one of embodiments 40 to 43, wherein the mammal is a human.

[0116] The following embodiments further illustrate the invention, but should not be construed as limiting its scope in any way.

[0117] Examples of the influenza A backbone use one of two backbones. In Examples 1 to 3, 5 to 8, 10 and 11, the backbone comprises a PB1 gene fragment encoding a PB1 protein containing leucine at position 40, tryptophan at position 180, asparagine at position 464, and proline at position 607; a PB2 gene fragment encoding a PB2 protein containing valine at position 504, methionine at position 467, and isoleucine at position 529; a PA gene fragment encoding a PA protein containing lysine at position 401; an NP gene fragment encoding an NP protein containing leucine at position 116, lysine at position 294, and arginine at position 311; and an NS1 gene fragment encoding an NS1 protein containing proline at position 30 and lysine at position 118. In Example 4, as described in U.S. Patent Application Publication No. 2014 / 0127249A1, an influenza backbone lacking the M2 gene fragment was used.

[0118] The following examples of influenza B backbones (see Examples 2, 5, 6, 7, and 8) use a backbone comprising a PA gene segment containing thymine at nucleotide 2272; an NP gene segment encoding an NP protein containing serine at position 40, asparagine at position 161, and threonine at position 204; an NS gene segment containing guanine at position 39; an NS gene segment encoding an NS protein containing glutamine at position 176; and the HA2 subunit of an HA gene segment encoding an HA protein containing glutamic acid at positions 61 and 112.

[0119] Example 1

[0120] This example demonstrates the stability of various pharmaceutical preparations containing the M2SR virus at elevated temperatures.

[0121] The properties (identity) of all five buffers were evaluated: potassium phosphate, sodium phosphate, imidazole, histidine, and citric acid, as well as their pH and concentration; five sugars and polyols: sucrose, lactose, trehalose, mannitol, and sorbitol; six polymers and proteins: dextran 40K, dextran 70K, PVP, hydroxyethyl starch (HES), gelatin, and recombinant human serum albumin (rHSA); four amino acids: glutamic acid, histidine, glycine, and arginine; four surfactants: polysorbate-20 (PS-20), polysorbate-80 (PS-80), deoxycholic acid (DOC), and poloxamer 188 (P188); and two salts: magnesium sulfate and magnesium chloride. Vaccine samples were prepared and incubated at 33°C for 10 days. Samples were retrieved on the initial day 0, and on days 5 and 10, and administered using a 50% tissue culture infectious dose (TCID). 50 The viability test is performed by measuring the viral titer, which is expressed as log10. 50 TCID / mL. Table 1 shows the drug formulations tested. The eight drug formulations with the highest titers on day 10 are shown in Table 2.

[0122] Table 1

[0123] Table 2

[0124] Several conclusions can be drawn from this data analysis. The surfactant poloxamer 188 (P188) is more beneficial than other surfactants, and it was found in 75% (6 / 8) of the best-performing formulations listed in Table 2. Magnesium sulfate was also observed in 75% (6 / 8) of the formulations listed in Table 2. Interestingly, the two best-performing formulations are almost identical in composition, except for their buffering properties. Lactose was not identified in any of the top eight formulations, and no other clearly preferred sugars were found, as 50% (4 / 8) of the formulations listed in Table 2 contain sucrose or trehalose. The benefits of polyols are uncertain: 38% (3 / 8) of the formulations listed in Table 2 contain mannitol, 25% (2 / 8) contain sorbitol, and 38% (3 / 8) do not contain polyols. Magnesium chloride is not included in the formulations listed in Table 2. 38% (3 / 8) of the formulations listed in Table 2 contain MgSO4, while 62% (5 / 8) of the formulations listed in Table 2 contain no magnesium salts at all; therefore, any benefit from Mg is likely negligible. Most of the tested buffer types appear to be compatible, with the exception of imidazole, which is absent from the eight best-performing drug formulations. Finally, no obvious preference was found for amino acid properties.

[0125] Example 2

[0126] This embodiment demonstrates the beneficial effect of sucrose on the stability of influenza A M2SR and influenza B BM2SR at 4°C.

[0127] In the stability study of this embodiment, the stability of influenza A Sing2016 H3N2M2SR and influenza B CO / 06 (VL) BM2SR viruses in two formulations, which differed due to the addition of sucrose, was tested at a storage temperature of 4°C. Sucrose was added at 10% (w / v) to a phosphate pharmaceutical formulation containing sodium phosphate (pH = 7.2), 134 mM NaCl, and 2% arginine.

[0128] Vaccine samples containing live Sing2016 H3N2 M2SR and CO / O6(VL) BM2SR vaccine viruses were prepared in pharmaceutical formulations with and without 10% sucrose, and then stored at 4°C to assess their stability. Samples were removed at the initial t = 0, and after storage periods of 1 day, 3 days, 9 days, 20 days, and 73 days (t = 1 day, 3 days, 9 days, 20 days, 73 days), and frozen at -80°C until the study was completed. The samples were then tested using a 50% tissue culture infectious dose (TCID). 50 The viral titer was determined by testing samples at all time points in duplicate to assess viability. The viral titer was expressed as log10 TCID. 50 / mL. Figure 1 The viral titer plots for each virus at each time point after storage in phosphate formulations and phosphate-10% sucrose formulations are presented. Adding sucrose to the formulations stabilized both M2SR and BM2SR. Without sucrose, the CO / O6 BM2SR virus was unstable under these conditions (cross-shaded bars). After 1 day of storage, the virus was completely inactivated, and the detection limit was log10 = 1.5 TCID. 50 Virus titers above / mL (as shown by the dashed line) are undetectable. Meanwhile, Sing2016 H3N2 M2SR (gray column) is more stable in the absence of sucrose, but still loses viability, and after 9 days of storage at 4°C, the titer drops to a level of LOD or close to LOD that is almost undetectable.

[0129] Both viral strains were more stable in the presence of sucrose. The Sing2016 H3N2 M2SR strain in sucrose (black column) maintained a titer of log10 TCID at day 20. 50 / mL = 3.67. CO / 06 BM2SR with sucrose (white column) had a detectable titer throughout the 73-day testing period, with a final titer of log 10 TCID. 50 / mL = 4.00. Therefore, adding sucrose to the stored drug formulation stabilized both influenza A M2SR and influenza B BM2SR viruses at 4°C, but the benefits of sucrose were more pronounced for influenza B virus strains.

[0130] Example 3

[0131] This example demonstrates the stability of pharmaceutical formulations using various brands and amounts of rHSA.

[0132] The stability of two influenza A M2SR strains, MT50 H1N1 and Sing2016 H3N2, and influenza B BM2SR virus CO / 06 / 2017, was tested at a storage temperature of 4°C. Twelve pharmaceutical formulations were prepared, all containing 20 mM sodium phosphate, 154 mM sodium chloride, and 4.5 mM potassium chloride. Sucrose was added at three levels: 10%, 12%, and 14% (w / v), according to the amounts given in Table 3, and recombinant human serum albumin (rHSA) was added at 0.5% (w / v), 0.95% (w / v), and 1.4% (w / v). rHSA was commercially available from a supplier, and the brand name RECOMBUMIN PRIME was evaluated. TM RECOMBUMIN ELITE TM and OPTIBUMIN TM The three forms of materials.

[0133] Table 3

[0134] Vaccine samples containing live MT50 H1N1 M2SR, Sing2016 H3N2 M2SR, and CO / O6 (VL) BM2SR vaccine viruses were prepared by diluting the concentrated stock virus 100-fold using the 12 pharmaceutical formulations described in Table 3, and then stored at 4°C to assess their stability. Samples were retrieved at the initial t = 0, and after storage periods of 1 day, 21 days, 91 days, 161 days, and 231 days (t = 1 day, 21 days, 91 days, 161 days, and 231 days), and frozen at -80°C until the study was completed. The samples were then tested using a 50% tissue culture infectious dose (TCID50). 50 The viral titer was measured by performing viability testing on samples from all time points in triplicate. The viral titer was expressed as log [missing value]. 10 TCID 50 / mL. All three viruses exhibited a degree of stability throughout the study. Even after approximately 7.5 months (t = 231 days), levels above the detection limit log = 1.5 TCID remained detectable. 50 Viral titer per mL. Overall MT50 H1N1 M2SR titer ranged from 5.81 + / - 0.31 log 10 TCID 50 The initial average titer / mL + / - standard deviation decreased to 3.40 + / - 0.33 log 10 TCID 50 The average value per mL. H3N2 strain Sing2016 M2SR from 7.10 + / - 0.32 log 10 TCID 50 The initial average titer / mL + / - standard deviation decreased to 5.83 + / - 0.35 log 10 TCID 50 The average value per mL. CO / O6 (VL) BM2SR titer ranged from 6.18 + / - 0.38 log 10 TCID 50 Starting at / mL, it decreased to 3.59 + / - 0.23 log during the experiment. 10 TCID 50 Average titer per mL.

[0135] No significant differences in stability were observed among the different drug formulations. All drug formulations were statistically identical within the assessed concentrations of sucrose and rHSA. Figure 2 shows the mean titers (in logarithmic form) from all 12 drug formulations.10 TCID 50 The viral titers (in mL) are summarized on a viral titer plot at various time points after storage at 4°C for each virus. The slope of the best-fit line (plotted with a dashed line) is used to estimate the loss rate for each of the three strains. For Figure 2A MT50 H1N1 M2SR and Figure 2B The CO / O6 BM2SR in the samples decreased at similar rates, approximately 0.010 log₂S. 10 TCID 50 / mL / day and 0.011 log 10 TCID 50 / mL / day, or approximately 1 log loss of titer every 90 to 100 days. 10 . Figure 2C The Sing2016 H3N2 M2SR is more stable, with an apparent slope of approximately -0.005 log. 10 TCID 50 / mL / day, which is equivalent to a titer loss of about 10 times every 200 days.

[0136] Example 4

[0137] This example demonstrates the effectiveness of recombinant human serum albumin (rHSA) in pharmaceutical formulations.

[0138] Two formulations were directly compared using Bris10 H3N2 M2SR influenza A virus stored at 4°C. The only difference between the two formulations was the addition of recombinant human serum albumin (rHSA). rHSA was added at 1.40% (w / v) to a sucrose phosphate glutamate (SPG) formulation prepared with sodium phosphate buffer (pH = 7.2), 134 mM NaCl, 4.5 mM KCl, 10% sucrose, and 5 mM sodium glutamate (MSG). The formulations are shown in Table 4.

[0139] Table 4

[0140] Vaccine samples containing live Bris10 M2SR were prepared in two pharmaceutical formulations, SPG and SPG+rHSA, and then stored at 4°C to assess long-term storage stability. Samples were removed at the initial t = 0, and after storage periods of 1, 2, 3, 4, and 6 months (t = 1 month, 2 months, 3 months, 4 months, and 6 months), and frozen at -80°C until the study was completed. The samples were then subjected to a 50% tissue culture infectious dose (TCID50). 50The viral titer was measured by performing viability testing on samples at all time points in duplicate. The viral titer was expressed as log... 10 TCID 50 / mL. Viral titer profiles at each time point after storage in SPG and SPG+rHSA formulations. Figure 3 The results are given in [reference needed]. For both drug formulations, the titer values ​​decreased over time; however, the +rHSA formulation appeared to be superior to the formulation without rHSA. For the SPG formulation alone, the slope was -0.32 log [value missing]. 10 TCID 50 / mL / month, while the titer of SPG + rHSA decreased by only -0.18 log. 10 TCID 50 / mL / month. Therefore, the addition of recombinant human serum albumin stabilized the Bris10 H3N2 M2SR influenza virus vaccine for a duration of 6 months during storage at 4°C.

[0141] Example 5

[0142] This embodiment demonstrates the stability of the pharmaceutical formulation of the present invention, which contains influenza A M2SR and influenza B BM2SR, at an ambient temperature of approximately 21°C.

[0143] A histidine-buffered saline (HIS)-based storage formulation was prepared, containing 50 mM histidine (pH = 7.2), 0.5% (w / v) glycine, 3% (w / v) trehalose, 3% (w / v) mannitol, and 0.1% (w / v) polyethylene glycol (PEG4600) with an average molecular weight of 4600. The HIS storage formulation was supplemented with either 0.4% (w / v) carboxymethyl cellulose (+CMC) or 0.5% (w / v) recombinant human serum albumin (+rHSA) as a possible stabilizer. Three vaccine samples containing live MT50 H1N1 M2SR, Sing2016H3N2 M2SR, and CA / 12 (YL) BM2SR vaccine viruses were prepared by 100-fold dilution of the concentrated stock virus using three formulations: HIS only, HIS + CMC, and HIS + rHSA. The samples were then stored at an ambient laboratory temperature of 21°C to assess room temperature storage stability. Samples were retrieved at the initial t = 0, and after storage periods of 3, 6, 12, 24, and 48 days (t = 1, 3, 6, 12, and 24 days), and frozen at -80°C until the study was completed. They were then subjected to a 50% tissue culture infection dose (TCID). 50The viral titer was determined by viability testing of samples at all time points in at least two copies and, for some samples, in up to five copies. The viral titer was expressed as log10 TCID. 50 / mL. The average titer of each virus in the three formulations at all time points is plotted in three graphs, one graph for each tested strain. These graphs are shown in... Figures 4A to 4C middle.

[0144] Throughout the study of the additive-free HIS drug formulation, viral titers decreased for all three viruses. After 48 days at ambient temperature, titers above the limit of detection (LOD) of 1.50 log [value missing] were still detectable for two of the three viral strains. 10 TCID 50 Viral titer per mL (as shown by the horizontal dashed line). MT50 H1N1 M2SR titers range from 6.33 + / - 0.31 log [missing value]. 10 TCID 50 The initial average titer / mL decreased by + / - standard deviation until it was just above the determination LOD of 1.57 + / - 0.00log. 10 TCID 50 The average titer / mL. The initial average titer + / - standard deviation of H3N2 strain Sing2016 M2SR was 6.95 + / - 0.06 log 10 TCID 50 / mL, decreased to 3.50 + / - 0.12 log 10 TCID 50 The average value was 6.44 / mL. After 48 days, CA / 12(YL)BM2SR lost all detectable titers, which decreased from 6.44 + / - 0.29 log₂ / mL during the experiment. 10 TCID 50 / mL decreased to below 1.50 log 10 TCID 50 / mL LOD.

[0145] Adding 0.4% (w / v) CMC to the HIS drug formulation resulted in better environmental temperature stability for all three evaluated vaccine candidates. After 48 days, the final viral titers of the MT50 H1N1 M2SR, Sing2016 H3N2 M2SR, and CA / 12 (YL) BM2SR samples were 3.43 + / - 0.14 log₂ / ₃. 10 TCID 50 / mL, 5.67 + / - 0.94 log 10 TCID 50 / mL and 2.72 + / - 0.25 log10 TCID 50 / mL. For both H1N1 and H3N2 M2SR, the titers increased by approximately 100-fold or 2log. 10 TCID 50 / mL. Although the magnitude of the titer increase for CA / 12 (YL) BM2SR is unknown because the virus was not detected in the absence of CMC, the increased viral titer measured in the HIS + CMC formulation was at least 1 log higher than the LOD.

[0146] Adding rHSA to stored drug formulations improved viral stability under certain drug formulations and storage conditions (e.g., phosphate formulations and storage at 4°C). In this case, after 24 days, MT50 H1N1 M2SR and CA / 12 (YL) BM2SR samples with rHSA were at or below LOD, corresponding to a decrease of 3 log at t = 24 time points. 10 Or 100-fold titer. The Sing2016 H3N2 M2SR vaccine appears to be more stable across all tested formulations. The H3N2 candidate vaccine did not lose all titers, and after 24 and 48 days of storage, the titer loss of this strain was minimal, decreasing by only about 1 log.

[0147] Example 6

[0148] This example demonstrates the stability of pharmaceutical formulations containing both alpha-M2SR and beta-BM2SR backbones after freeze-thaw cycles. Samples were thawed at 4°C for 4 hours, then homogenized, collected, and refrozen at -80°C.

[0149] Sucrose phosphate (SP) storage formulations supplemented with 0.5% (w / v) recombinant rHSA and histidine-buffered phosphate (HIS) storage formulations were formulated. The HIS formulation contains 50 mM histidine (pH = 7.2), 0.5% (w / v) glycine, 3% (w / v) trehalose, 3% (w / v) mannitol, and 0.1% (w / v) polyethylene glycol (PEG4600) with an average molecular weight of 4600. The SP formulation contains 20 mM sodium phosphate buffer (pH = 7.2), 134 mM NaCl, 4.5 mM KCl, 10% (w / v) sucrose, and 0.5% (w / v) rHSA.

[0150] Three variations of each drug formulation were prepared. For the HIS storage formulation, the following stabilizers were added: 0.4% (w / v) carboxymethyl cellulose (HIS + CMC), 0.5% (w / v) recombinant human serum albumin (HIS + rHSA), or both CMC and rHSA (HIS + both). For the SP storage formulation, the following stabilizers were added: 0.4% (w / v) carboxymethyl cellulose (SP + CMC), 0.5% (w / v) polyethylene glycol with an average molecular weight of 6000 (SP + PEG), or both CMC and PEG (SP + both).

[0151] Vaccine samples containing three live virus vaccines, MT50H1N1 M2SR, Sing2016 H3N2 M2SR, and CA / 12 (YL) BM2SR, were prepared by diluting concentrated stock viruses 100-fold in eight pharmaceutical formulations: HIS, HIS + CMC, HIS + rHSA, HIS + both; SP, SP + CMC, SP + PEG, and SP + both. At the start of the experiment, all aliquots of the samples were frozen to -80°C at once. These samples were considered to have undergone 0 freeze-thaw cycles. All samples were then thawed by incubating at 4°C for 4 hours. The samples were then mixed until homogeneous and then re-frozen. These samples were considered to have undergone one freeze-thaw cycle. Freeze-thaw cycles were repeated until a total of 16 cycles were performed. After the set number of freeze-thaw cycles, groups of 24 samples representing the three viruses from the eight pharmaceutical formulations were isolated and no further freeze-thaw cycles were performed until the study was completed. Sample groups were removed after the initial 0th freeze-thaw cycle, and after 1, 2, 4, 8, and 16 freeze-thaw cycles. All samples were kept frozen at -80°C until the study was completed.

[0152] Then, samples of all viruses from the eight drug formulations and the specified freeze-thaw cycles were thawed, homogenized, and subjected to a 50% tissue culture infection dose (TCID). 50 The viability test is performed by measuring the viral titer, expressed as log... 10 TCID 50 / mL is used as the unit of measurement. After 0, 1, 8, or 16 freeze-thaw cycles, the titer values ​​for each of the three viral strains in eight test formulations were plotted in six graphs, and two graphs were plotted for each drug formulation tested against the three vaccine candidates. These graphs are presented in... Figures 5A to 5F The information is provided in the text.

[0153] The HIS formulation appears to confer less freeze-thaw stability than the SP formulation. Without supplementation, all three strains tested showed approximately 1.5 log [value missing] freeze-thaw stability after 16 freeze-thaw cycles in the HIS formulation. 10The decrease in titer was observed. Adding 0.5% (w / v) rHSA to the HIS+ formulation and both rHSA and HIS+ formulations conferred stability on all tested strains: no statistically relevant changes in titer were observed under those storage conditions. No change in stability was observed in the HIS+CMC formulation with the addition of 0.4% (w / v) CMC.

[0154] In the SP formulation, the vaccine candidate titer remained stable after 16 freeze-thaw cycles without the addition of stabilizers. Similar to the HIS formulation, no change in stability was observed with the addition of 0.4% (w / v) CMC in the SP formulation. The addition of 0.5% (w / v) PEG to both SP + PEG and SP + both formulations resulted in a loss of stability, particularly for the MT50 H1N1 M2SR and CA / 12 (YL) BM2SR vaccine candidates.

[0155] Example 7

[0156] This example demonstrates the effect of recombinant human serum albumin (rHSA) on the stability of sucrose phosphate glutamate (SPG) pharmaceutical compositions at 4°C.

[0157] A sucrose phosphate glutamate (SPG) formulation was prepared using sodium phosphate buffer (pH = 7.2), 134 mM NaCl, 4.5 mM KCl, 10% (w / v) sucrose, and 5 mM sodium glutamate (MSG). A second sucrose phosphate formulation (SP3) was prepared using sodium phosphate buffer (pH = 7.2), 134 mM NaCl, 4.5 mM KCl, 10% (w / v) sucrose, and 0.5% (w / v) rHSA instead of MSG.

[0158] Vaccine samples containing three live vaccine viruses—MT50 H1N1 M2SR, Sing2016 H3N2 M2SR, and CA / 12 (YL) BM2SR—were prepared by diluting concentrated stock viruses 100-fold in two pharmaceutical formulations: SPG and SP3. The samples were then stored at 4°C to assess their freezer stability. Samples were removed at the initial t = 0, and after storage periods of 28, 98, 210, and 365 days (t = 28, 98, 210, and 365 days), and frozen at -80°C until the study was completed. The samples were then tested using a 50% tissue culture infectious dose (TCID50). 50 The viral titer was determined by measuring the viability of samples at all time points in at least duplicates and, for some samples, in up to five copies. The viral titer was expressed as log 10 TCID 50 / mL. The average titer of each virus at all time points in both formulations is plotted in three graphs, one graph per test strain. These graphs are shown in... Figures 6A to 6C middle.

[0159] For all three viruses tested, the titer values ​​in both formulations trended downward over time; however, the SP3 formulation containing rHSA performed better than the SPG formulation containing 5 mM glutamate. After 98 days, the measured titers for all three strains in SPG decreased to the detection limit of 1.50 TCID. 50 / mL or close to the detection limit (shown as a horizontal dashed line). The titer was also almost undetectable at 210 days. SPG samples were not analyzed at day 365. The SP3 formulation showed significantly better stability than the SPG formulation, but the performance of all three tested lines was not comparable. Sing2016 H3N2 M2SR was the most stable line, with an initial titer of 7.49 + / - 0.47 log 10 TCID 50 / mL, and a final titer of 5.61 + / - 0.10 log 10 TCID 50 / mL, decreased by 1.88 log after 1 year. 10 TCID 50 / mL. MT50H1N1 M2SR is substable, with an initial titer of 6.03 + / - 0.30 log. 10 TCID 50 / mL, and decreased to 3.70 + / - 0.06 log 10 TCID 50 The titer of / mL decreased by 2.33 log within 1 year. 10 TCID 50 / mL. The CA / 12 (YL) BM2SR strain was relatively unstable, with an initial titer of 6.62 + / - 0.34 log 10 TCID 50 / mL, and decreased to 2.67 + / - 0.29 log 10 TCID 50 The titer was 3.95 log / mL, with a titer loss of 3.95 log. 10 TCID 50 / mL.

[0160] rHSA is an excellent stabilizer for influenza vaccine candidates in sucrose phosphate buffer. After storage at 4°C for 210 days in the SP3 formulation containing rHSA, compared to the SPG formulation without rHSA, the titer increase was approximately 5 log [value missing] for H3N2.10 TCID 50 / mL, the titers of MT50 H1N1 and CA / 12 (YL) are approximately 4 log high. 10 TCID 50 / mL. This is equivalent to a titer that is 10,000 to 100,000 times higher after 7 months of storage at 4°C.

[0161] Example 8

[0162] This example demonstrates the stability of the three pharmaceutical formulations under storage conditions of -20°C.

[0163] The efficacy of three drug formulations, SP3, HIS, and SPG, in stabilizing M2SR H3N2, H1N1, and BM2SR viruses at -20°C was compared. Vaccine samples were prepared for each of the three drug formulations and a sucrose phosphate glutamate (SPG) control, and then frozen at -20°C. Samples were retrieved on the initial day 0 and at specified time intervals, and subsequently transferred to -80°C for long-term storage until the end of the study. The sampling schedule was days 28, 98, 154, 210, and 365 of storage at -20°C. After all samples were collected, a 50% tissue culture infectious dose (TCID50) was used. 50 The viability test is performed by measuring the viral titer, which is expressed as log. 10 TCID 50 / mL.

[0164] like Figures 7A to 7C As shown, the SP3 formulation best supports the stability of all three influenza subtypes at -20°C. H3N2M2SR, H1N1 M2SR, and CA12 BM2SR are stable for at least 6 months under -20°C storage conditions. The SPG formulation is less stable, and all three tested items lose approximately 2 log titers within 90 days at -20°C. 10 TCID 50 / mL. The HIS formulation also outperformed SPG and, at the 1-year time point, provided similar stability to SP3 compared to SPG against two of the three vaccine candidates tested for stability: H1N1 M2SR and CA12 BM2SR. H3N2 M2SR was less stable compared to the other two viruses in the HIS formulation.

[0165] Example 9

[0166] This embodiment demonstrates the droplet size distribution, plume geometry, and spray pattern analysis of the SPGNa pharmaceutical formulation of the present invention.

[0167] TELEFLEX TMVAXINATOR TM The nasal spray device was used to determine the droplet size distribution, plume geometry, and spray pattern analysis of a pharmaceutical formulation (pH 7.2) containing 303 mM sucrose, 5 mM glutamate, 136.9 mM sodium chloride, 2.67 mM potassium chloride, 1.47 mM potassium dihydrogen phosphate, and 8.1 mM disodium hydrogen phosphate. Ten units were tested in each study at a tip-to-laser distance of 3 cm and a velocity of 70 mm / s. Pump delivery was also recorded. The average pump delivery during droplet size distribution, plume geometry, and spray pattern analysis were 0.1269 g, 0.1266 g, and 0.1698 g, respectively.

[0168] Using TELEFEX TM VAXINATOR TM The nasal spray device was successfully characterized for the SPGNa placebo formulation in terms of overall plume geometry (Table 5), droplet size distribution (DSD, Table 6), and spray pattern (SP, Table 7). The results of this study were compared with a dataset representing typical nasal spray performance at Next Breath. The spray pattern and plume geometry results were within the performance range of typical nasal sprays. Based on expected limits from the FDA, the percentage of DSDs with a diameter < 10 μm should be between 5% and 7%. The mean result in this study was 6%. Overall, the variability based on %CV was within the normal expectations for nasal spray devices.

[0169] Table 5. Overall plume geometry

[0170] Table 6. Overall droplet size distribution

[0171] Table 7. Overall Spray Patterns

[0172] Example 10

[0173] This example demonstrates the stability of various pharmaceutical formulations.

[0174] Forty storage formulations were screened using studies evaluating the accelerated stability of Bris10 2007 H3N2 M2SR virus at 35°C and the real-time stability at 4°C. In this study, the following were evaluated in single experiments: pH, concentration, and properties of two buffer / stabilizer combinations: histidine / PEG and HEPES / PVP; concentration and properties of four mucosal adhesion compounds: sodium alginate, carbopol, pectin, and carboxymethyl cellulose (CMC); and concentrations of mannitol, trehalose, and glycine. The 40 storage formulations are shown in Table 8. Vaccine samples were prepared in each of the 40 formulations and the sucrose phosphate glutamate (SPG) control and then incubated at 4°C and 35°C. Samples were removed on the initial day 0 and at specified time intervals and then frozen at -80°C until the study was completed. For storage at 35°C, the sampling schedule was days 5 and 10, and for storage at 4°C, the sampling schedule was days 15 and 30. After collecting all samples, a 50% tissue culture infection dose (TCID) was used. 50 The viability test is performed by measuring the viral titer, which is expressed as log. 10 TCID 50 / mL, and is given in Table 9. The weight osmolarity (mOsm / kg) and viscosity (cP) of each drug formulation were also measured.

[0175] Table 8. Pharmaceutical Preparations

[0176] Table 9. Virus titer, weight osmolarity, and viscosity

[0177] #: A value of 1.5 indicates that no detection was detected below the detection limit = 1.50 log 10 TCID 50 / mL of virus.

[0178] Example 11

[0179] This example demonstrates the stability of various pharmaceutical formulations.

[0180] The tested drug formulations are shown in Table 10.

[0181] Table 10. Pharmaceutical Preparations

[0182] Vaccine samples for Bris10 2007 M2SR virus were prepared in each of the 10 pharmaceutical formulations shown in Table 10 and then incubated at 4°C. Samples were removed on the initial day 0 and at specified time intervals and then frozen at -80°C until the study was completed. Sampling was performed at 4, 6, 9, and 12 months after storage at 4°C. After all samples were collected, a 50% tissue culture infectious dose (TCID50) was used. 50 The viability test is performed by measuring the viral titer, which is expressed as log. 10 TCID 50 / mL. In Figure 8 The results for five HEPES drug formulations, #1 to #5, are presented, and... Figure 9 The results for three HIS drug formulations #6 to #8 are presented, with the LOD measured as log 10 TCID 50 / mL = 1.50, displayed as a gray dashed line.

[0183] All references cited in this article, including publications, patent applications and patents, are incorporated herein by reference to the extent that each reference is individually and specifically indicated by reference and is listed in its entirety in this article.

[0184] In the context of describing the invention (particularly in the context of the appended claims), the terms “a,” “an,” “the,” and “at least one,” and similar designations, should be interpreted as encompassing both the singular and plural, unless otherwise indicated herein or clearly contradicted by the context. The use of the term “at least one” followed by a list of one or more items (e.g., “at least one of A and B”) should be interpreted as referring to one item (A or B) selected from the list or any combination of two or more of the list (A and B), unless otherwise indicated herein or clearly contradicted by the context. Unless otherwise stated, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”). Unless otherwise indicated herein, the description of ranges of values ​​herein is intended only as a concise way of referring to each individual value falling within that range, and each individual value is incorporated into this specification as if it were described separately herein. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by the context. Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the invention and does not constitute a limitation on the scope of the invention. The language in this specification should not be construed as indicating that any unclaimed element is necessary for the practice of the invention.

[0185] This document describes preferred embodiments of the invention, including the best modes known to the inventors for carrying out the invention. Variations of those preferred embodiments will be apparent to those skilled in the art after reading the foregoing description. The inventors expect those skilled in the art to appropriately employ such variations, and the inventors intend to practice the invention in ways different from those specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter set forth in the appended claims as permitted by applicable law. Furthermore, the invention includes any combination of the foregoing elements in all their possible variations, unless otherwise indicated herein or clearly contradicted by the context.

Claims

1. A stable pharmaceutical preparation comprising (a) a buffer, (b) a sugar, (c) a stabilizer, (d) one or more amino acids, and (e) at least one influenza virus backbone.

2. The pharmaceutical formulation according to claim 1, wherein the buffer comprises potassium phosphate, sodium phosphate, imidazole, histidine, citric acid, or HEPES.

3. The pharmaceutical formulation according to claim 1 or 2, wherein the pharmaceutical formulation comprises about 20 mM to about 150 mM of the buffer.

4. The pharmaceutical preparation according to any one of claims 1 to 3, wherein the sugar comprises sucrose, lactose, or trehalose.

5. The pharmaceutical formulation according to any one of claims 1 to 4, wherein the pharmaceutical formulation comprises about 1% w / v to about 15% w / v of the sugar.

6. The pharmaceutical formulation according to any one of claims 1 to 5, wherein the stabilizer comprises dextran 40K, dextran 70K, polyvinylpyrrolidone (PVP) 40K, PVP 70K, hydroxyethyl starch (HES), gelatin, polyethylene glycol (PEG) 4600, or recombinant human serum albumin (rHSA).

7. The pharmaceutical formulation according to any one of claims 1 to 6, wherein the pharmaceutical formulation comprises the stabilizer at about 0.01% w / v to about 3.0% w / v.

8. The pharmaceutical preparation according to any one of claims 1 to 7, wherein one or more amino acids are selected from glutamic acid, histidine, glycine and arginine.

9. The pharmaceutical formulation according to any one of claims 1 to 8, wherein the pharmaceutical formulation comprises about 1% w / v of the one or more amino acids.

10. The pharmaceutical preparation according to any one of claims 1 to 9, wherein the pharmaceutical preparation comprises at least two, at least three, or at least four influenza virus skeletons.

11. The pharmaceutical preparation according to any one of claims 1 to 10, wherein at least one, at least two, at least three, or at least four influenza virus skeletons are independently selected from influenza A virus skeletons and influenza B virus skeletons.

12. The pharmaceutical formulation according to any one of claims 1 to 11, wherein the pharmaceutical formulation comprises two influenza A virus backbones and two influenza B virus backbones.

13. The pharmaceutical preparation according to any one of claims 1 to 12, wherein the pharmaceutical preparation comprises at least 10 9 TCID 50 / The influenza virus skeleton of the dose.

14. The pharmaceutical preparation according to any one of claims 1 to 13, wherein the pharmaceutical preparation further comprises a polyol.

15. The pharmaceutical preparation according to claim 14, wherein the polyol is mannitol or sorbitol.

16. The pharmaceutical formulation of claim 14 or claim 15, wherein the pharmaceutical formulation comprises about 1% w / v to about 5% w / v of the polyol.

17. The pharmaceutical preparation according to any one of claims 1 to 16, wherein the pharmaceutical preparation further comprises one or more salts.

18. The pharmaceutical preparation according to claim 17, wherein one or more salts are selected from magnesium sulfate, magnesium chloride, sodium chloride, and potassium chloride.

19. The pharmaceutical formulation of claim 17 or claim 18, wherein the pharmaceutical formulation comprises about 0.01% w / v to about 0.15% w / v of the one or more salts.

20. The pharmaceutical formulation of claim 17 or claim 18, wherein the pharmaceutical formulation comprises one or more salts of about 1 mM to about 150 mM.

21. The pharmaceutical formulation according to any one of claims 1 to 20, wherein the pharmaceutical formulation further comprises an mucosal adhesive.

22. The pharmaceutical formulation according to claim 21, wherein the mucosal adhesive is carboxymethyl cellulose (CMC).

23. The pharmaceutical formulation of claim 21 or claim 22, wherein the pharmaceutical formulation comprises about 0.1% w / v to about 0.5% w / v of a mucosal adhesive.

24. The pharmaceutical formulation according to any one of claims 1 to 23, wherein the pharmaceutical formulation further comprises a surfactant.

25. The pharmaceutical formulation according to any one of claims 1 to 24, wherein the surfactant comprises polysorbate 20, polysorbate 80, deoxycholic acid (DOC), or poloxamer 188 (P188) or polyacrylic acid.

26. The pharmaceutical formulation according to any one of claims 1 to 25, wherein the pharmaceutical formulation comprises about 0.01% w / v to about 0.15% w / v of the surfactant.

27. The pharmaceutical preparation according to any one of claims 1 to 26, wherein the pharmaceutical preparation further comprises a chelating agent.

28. The pharmaceutical formulation of claim 27, wherein the chelating agent is EDTA.

29. The pharmaceutical formulation according to claim 27 or 28, wherein the pharmaceutical formulation comprises about 1 μM to about 1000 μM of the chelating agent.

30. The pharmaceutical preparation according to any one of claims 1 to 29, wherein the pH is about 7.0 to about 7.

6.

31. The pharmaceutical preparation according to any one of claims 1 to 30, wherein the pH is about 7.2 to about 7.

4.

32. The pharmaceutical preparation according to any one of claims 1 to 31, wherein the pH is about 7.

2.

33. The pharmaceutical preparation according to any one of claims 1 to 31, wherein the pH is about 7.

4.

34. Stable pharmaceutical preparations, comprising: (a) Approximately 20 mM sodium phosphate; (b) Approximately 134 mM sodium chloride and 4.5 mM potassium chloride; (c) Approximately 10% w / v sucrose; (d) Approximately 0.5% w / v rHSA; (e) Approximately 5 mM glutamate and approximately 1% w / v arginine; and (f) Approximately 10 9 TCID 50 / dose of influenza virus skeleton; or (a) Approximately 50 mM histidine; (b) Approximately 3.0% w / v mannitol; (c) Approximately 3.0% w / v trehalose; (d) Approximately 0.1% w / v PEG4600; (e) Approximately 0.4% w / v CMC; (f) Approximately 0.5% w / v glycine; and (g) Approximately 10 9 TCID 50 / dose of influenza virus skeleton; or (a) Approximately 50 mM HEPES; (b) Approximately 3.0% w / v mannitol; (c) Approximately 3.0% w / v trehalose; (d) Approximately 0.1% w / v PVP40K; (e) Approximately 0.5% w / v glycine; (f) Approximately 0.2% w / v polyacrylic acid; and (g) Approximately 10 9 TCID 50 / dose of influenza virus skeleton; or (a) Approximately 50 mM potassium phosphate; (b) Approximately 3.0% w / v mannitol; (c) Approximately 1.0% w / v trehalose; (d) Approximately 0.01% w / v dextran 70K; (e) Approximately 1.0% w / v glycine; (f) Approximately 0.05% w / v P188; (g) Approximately 50 μM EDTA; and (f) Approximately 10 9 TCID 50 / The influenza virus skeleton of the dose.

35. The pharmaceutical preparation according to any one of claims 1 to 34, wherein the pharmaceutical preparation is stable at -20°C for at least 16 to 56 months.

36. The pharmaceutical preparation according to any one of claims 1 to 34, wherein the pharmaceutical preparation is stable at 4°C for at least 30 days to 12 months.

37. The pharmaceutical preparation according to any one of claims 1 to 34, wherein the pharmaceutical preparation is stable at ambient temperature for at least 12 to 40 days.

38. The pharmaceutical preparation according to any one of claims 1 to 34, wherein the pharmaceutical preparation is stable at 33°C for at least 4 to 22 days.

39. The pharmaceutical preparation according to any one of claims 1 to 38, wherein the pharmaceutical preparation is an influenza virus vaccine.

40. A method for treating mammals in need of an influenza virus vaccine, the method comprising administering an effective amount of the pharmaceutical preparation of any one of claims 1 to 39.

41. A method for inducing an immune response in mammals, the method comprising administering an effective amount of the pharmaceutical preparation of any one of claims 1 to 39.

42. The method of claim 40 or claim 41, wherein the pharmaceutical preparation is administered nasally.

43. The method according to any one of claims 40 to 42, wherein the pharmaceutical formulation has a droplet size of 30 micrometers to 75 micrometers.

44. The method according to any one of claims 40 to 43, wherein the mammal is a human.

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