Vaccine using M2 / BM2 deletion influenza vector

Abstract

The present invention provides a recombinant virus comprising an influenza virus backbone, the influenza backbone comprising the gene segments PB1, PB2, PA, NP, M, NS, HA, and NA, wherein at least one of the gene segments PB1, PB2, PA, NP, M, NS, HA, and NA comprises at least one nucleotide sequence encoding one or more antigens. The present invention also provides a recombinant virus wherein the antigen is an immunogenic fragment of the SARS-CoV-2 spike glycoprotein. The present invention also provides pharmaceutical formulations and methods for eliciting an immune response.

Description

[Technical Field]

[0001] Incorporation by reference of electronically submitted materials A computer-readable nucleotide / amino acid sequence listing, submitted concurrently with this specification and identified as a single 271,121-byte ASCII (text) file named "755022SequenceListing.txt" created on July 20, 2021, is incorporated herein by reference in its entirety. [Background technology]

[0002] Background of the Invention Vaccines are a vital tool for preventing infectious diseases. Infectious diseases can infect millions of people worldwide. Therefore, it is crucial to develop vaccines for many different types of diseases, and to do so quickly and efficiently. For example, the novel coronavirus infection 2019 (COVID-19) is a global pandemic caused by a newly emerged viral severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). More than 10 million people worldwide have been diagnosed with the disease, and hundreds of thousands have died from it. In its severe form, the disease is characterized by acute respiratory distress syndrome (ARDS), for which there are currently no targeted intervention strategies to treat or prevent it. The immune response to the virus is thought to contribute to the development of the disease and provide protection during its resolution. Therefore, there is an unprecedented need to develop a vaccine that is safe and effective in sensitizing a very large number of individuals. [Overview of the project] [Problems that the invention aims to solve]

[0003] The present invention provides a recombinant virus comprising an influenza virus backbone, wherein the influenza backbone comprises the gene segments PB1, PB2, PA, NP, M, NS, HA, and NA, and at least one of the gene segments PB1, PB2, PA, NP, M, NS, HA, and NA comprises at least one nucleotide sequence encoding at least one antigen. In a preferred embodiment, the antigen is an immunogenic fragment of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) spike glycoprotein. [Brief explanation of the drawing]

[0004] [Figure 1] This is a schematic diagram of an influenza A NS segment engineered to express NS1 against a SARS-CoV-2 spike receptor-binding domain fusion protein. This construct includes the full-length influenza A PR / 8 / 1934 NS1 protein, a first linker (GSG1), amino acids 331-530 of the SARS-CoV-2 Wuhan-Hu-1 spike S1 protein encoding the RBD (receptor-binding domain), a second linker (GSG2), a cleavage site (P2A), and cDNA of the essential PR8 nuclear export protein (NEP or NS2) having both exons 1 and 2. [Figure 2] This is a schematic diagram of an influenza A NS segment engineered to express the SARS-CoV-2 spike receptor binding domain as a separate polypeptide. This construct includes the full-length influenza A PR / 8 / 1934 NS1 protein, a first linker (GSG1), a first cleavage site (T2A), amino acids 331-530 of the SARS-CoV-2 Wuhan-Hu-1 spike S1 protein encoding RBD, a second linker (GSG2), a second cleavage site (P2A), and the cDNA of the essential PR8 nuclear export protein (NEP or NS2) having both exons 1 and 2. [Figure 3]The images show immunoblots of cell lysates of Vero cells infected with CoV2 NS M2SR, M2SR control, and mock medium alone. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblot analysis. The primary antibody was anti-SARS-CoV-2 RBD (Sino Biological Inc., Beijing, China), and the secondary antibody was anti-rabbit IgG horseshoe peroxidase (HRP) using 3,3',5,5'-tetramethylbenzidine (TMB) detection. [Figure 4] This is a set of images showing that cells infected with both CoV2 NS1 M2SR and standard M2SR express detectable levels of influenza A NP protein. On the other hand, FITC labeling of RBD could only be detected in CoV2 NS1 M2SR-infected cells, which exhibited significant detectable fluorescence. [Figure 5] This is a schematic diagram of influenza B M-segment 7 engineered to express a BM2 SARS-CoV-2 spike RBD fusion to the amino and carboxyl terminals of the BM2 protein (SEQ ID NOs: 84, SEQ ID NOs: 96). This construct includes the full-length influenza B / Florida / 4 / 2006M1 protein, a 5-mer translation termination / start site, a BM2 open reading frame (ORF) of amino acids 1-8, amino acids 330-524 of the SARS-CoV-2 Wuhan-Hu-1 spike S1 protein encoding the RBD, and the BM2 RBD fusion protein. [Figure 6] This is a schematic diagram of influenza B M-segment 7 engineered to express a BM2 SARS-CoV-2 spike RBD fusion at the amino terminus of the BM2 protein (SEQ ID NO: 83, SEQ ID NO: 95). This construct includes the full-length influenza B / Florida / 4 / 2006M1 protein, a BM2 RBD fusion protein containing a 5-mer translation termination / start site and a BM2 ORF of amino acids 1-3, and 330-524 of the SARS-CoV-2 Wuhan-Hu-1 spike S1 protein encoding the RBD. [Figure 7] The images show immunoblots of Vero cell lysates. These include whole cell lysates of cells infected with two SARS-CoV-2 BM2SR strains (SEQ ID NOs. 83, 84, 95, and 96), and whole cell lysates of cells infected with a negative control in mock medium only. Proteins were isolated by SDS-PAGE and then subjected to immunoblot analysis. The primary antibody was anti-SARS-CoV-2 RBD (Sino Biological Inc.), and the secondary antibody was anti-rabbit IgG-HRP using TMB detection. The location of the RBD fusion protein is indicated in the image by a number. [Figure 8A] This graph shows the percentage change in body weight of mice after sensitization with the M2SR recombinant virus. [Figure 8B] This graph shows the percentage change in body weight of mice after sensitization with the BM2SR recombinant virus. [Figure 9] This bar graph shows the multiplier increase in enzyme-linked immunosorbent assay (ELISA) titer from baseline before immunization. [Figure 10A] This is a set of graphs showing the test results, in which mice (N=8) were intranasally sensitized with monovalent H1N1 FGHY1-M2SR, monovalent H3N2 FGHY1-M2SR, bivalent H1N1 FGHY1-M2MR and H3N2 FGHY1-M2MR, monovalent BM2SR-Vic, monovalent BM2SR-Yam, bivalent BM2SR, trivalent H1N1 FGHY1-M2MR and H3N2 FGHY1-M2SR and BM2SR Victoria or Yamagata, or tetravalent H1N1 and H3N2 FGHY1-M2SR and BM2SR Victoria or Yamagata vaccine, or a control (SPG). Figure 10A shows anti-H1 HA serum IgG ELISA titer data, Figure 10B shows anti-H3 HA data, Figure 10C shows anti-influenza B-Vic HA data, and Figure 10D shows anti-influenza B-Yam HA data. [Figure 10B]This is a set of graphs showing the test results, in which mice (N=8) were intranasally sensitized with monovalent H1N1 FGHY1-M2SR, monovalent H3N2 FGHY1-M2SR, bivalent H1N1 FGHY1-M2MR and H3N2 FGHY1-M2MR, monovalent BM2SR-Vic, monovalent BM2SR-Yam, bivalent BM2SR, trivalent H1N1 FGHY1-M2MR and H3N2 FGHY1-M2SR and BM2SR Victoria or Yamagata, or tetravalent H1N1 and H3N2 FGHY1-M2SR and BM2SR Victoria or Yamagata vaccine, or a control (SPG). Figure 10A shows anti-H1 HA serum IgG ELISA titer data, Figure 10B shows anti-H3 HA data, Figure 10C shows anti-influenza B-Vic HA data, and Figure 10D shows anti-influenza B-Yam HA data. [Figure 10C] This is a set of graphs showing the test results, in which mice (N=8) were intranasally sensitized with monovalent H1N1 FGHY1-M2SR, monovalent H3N2 FGHY1-M2SR, bivalent H1N1 FGHY1-M2MR and H3N2 FGHY1-M2MR, monovalent BM2SR-Vic, monovalent BM2SR-Yam, bivalent BM2SR, trivalent H1N1 FGHY1-M2MR and H3N2 FGHY1-M2SR and BM2SR Victoria or Yamagata, or tetravalent H1N1 and H3N2 FGHY1-M2SR and BM2SR Victoria or Yamagata vaccine, or a control (SPG). Figure 10A shows anti-H1 HA serum IgG ELISA titer data, Figure 10B shows anti-H3 HA data, Figure 10C shows anti-influenza B-Vic HA data, and Figure 10D shows anti-influenza B-Yam HA data. [Figure 10D]A set of graphs showing the test results, where mice (N = 8) were intranasally sensitized with monovalent H1N1 FGHY1-M2SR, monovalent H3N2 FGHY1-M2SR, bivalent H1N1 FGHY1-M2MR and H3N2 FGHY1-M2MR, monovalent BM2SR-Vic, monovalent BM2SR-Yam, bivalent BM2SR, trivalent H1N1 FGHY1-M2MR and H3N2 FGHY1-M2SR and BM2SR Victoria or Yamagata, or quadrivalent H1N1 and H3N2 FGHY1-M2SR and BM2SR Victoria and Yamagata vaccines, or a control (SPG). Figure 10A shows the anti-H1 HA serum IgG ELISA titer data, Figure 10B shows the anti-H3 HA data, Figure 10C shows the anti-influenza B type-Vic HA data, and Figure 10D shows the data of anti-influenza B type-Yam HA. [Figure 11] A histogram showing the total cluster count versus the number of hits in one cluster. As indicated by the gray shading between 10.0 hits and 20.0 hits, very few clusters had more than 10 hits. [Figure 12] A graph showing the virus titer TCID50 curves for two strains, indicating that virus growth is not conferred by a synthetic segment expressing NS1 and NEP as a single self-cleaving peptide. [Figure 13] A graph showing a growth curve indicating that segment 8 with NS1 fusion to the unmodified SARS-CoV-2 helix antigen impairs virus growth compared to the wild type. [Figure 14] A schematic diagram of influenza A virus M segment 7 engineered to express a SARS-CoV-2 spike receptor-binding domain fusion to the amino terminus of the M2 protein. This construct contains the full-length influenza A virus / PR / 8 / 34 M1 protein, a splicing site, the M2 ORF of amino acids 1-25, the SARS-CoV-2 MHC I-compatible RBD antigen, an M2 RBD FLAG fusion protein containing a FLAG tag, and a stop codon. [Figure 15] Schematic diagram of influenza HA gene segment design for the production of M2SR influenza virus capable of driving the expression of an antigen scaffolded on the extracellular membrane of infected cells. For FIGS. 15 to 20, "UTR" refers to "untranslated region", "2A" refers to "2A self-cleaving peptide", "MD" refers to "multimerization domain", "TM" refers to "transmembrane domain", and "ncr" refers to "non-coding region". [Figure 16] Schematic diagram of influenza HA gene segment design for the production of M2SR influenza virus capable of driving the expression of an antigen scaffolded on the extracellular membrane of infected cells. [Figure 17] Schematic diagram of influenza NS gene segment design for the production of M2SR influenza virus capable of driving the expression of an antigen scaffolded on the extracellular membrane of infected cells. [Figure 18] Schematic diagram of influenza gene NS segment design for the production of M2SR influenza virus capable of driving the expression of an antigen scaffolded on the extracellular membrane of infected cells. [Figure 19] Schematic diagram of influenza gene NA segment design for the production of M2SR influenza virus capable of driving the expression of an antigen scaffolded on the extracellular membrane of infected cells. [Figure 20] Schematic diagram of influenza gene NA segment design for the production of M2SR influenza virus capable of driving the expression of an antigen scaffolded on the extracellular membrane of infected cells. [Figure 21]The sequences of the duplicated regions encoding the NS1 ORF and NEP exon 1 are shown. Lowercase letters indicate A / PR / 8 / 34 mutations. Bases 1-6 are deleted in the second copy of NEP exon 1 in the NEP delta 2N mutant (SEQ ID NO: 110). The second copy of the duplicated region of the NS segment encoding NEP exon 1 is 63% identical to the first copy, which is a wild-type A / PR / 8 / 34 NS segment cDNA sequence with a single nucleotide mutation that eliminates the splicing donor site (SEQ ID NO: 109). [Figure 22] The sequences of the NS1 ORF and NEP exon 2 are shown. Lowercase letters indicate A / PR / 8 / 34 mutations. The first copy of the overlapping region of the NS segment of NEP exon 2 (SEQ ID NO: 111) is 88% identical to the second copy (SEQ ID NO: 112), which is the cDNA sequence of the wild-type A / PR / 8 / 34 NS segment. [Figure 23] The images show fluorescence microscopy images taken over three consecutive days after inoculation of M2VeroA cells with MOI=10 using M2SR, with engineered NS segments (SEQ ID NOs. 111, 112, and 114) expressing a three-part polyprotein (SEQ ID NOs. 113) consisting of NS1, EGFP, and NEP peptides separated by the T2A and P2A sites, respectively. [Figure 24] Flow cytometry analysis of immunostained live M2VeroA cells infected with M2SR vector virus alone, or with M2SR virus having an NS1 segment designed to direct the expression of the SARS-CoV-2 S1 RBD minispike protein trimer on the cell surface using only 12 amino acids, T4 Foldon, RSV-derived TM, and the SARS-CoV-2 spike signal sequence (SEQ ID NO: 115). [Figure 25]Flow cytometry analysis of immunostained live M2VeroA cells infected with M2SR vector virus alone or with M2SR virus having an HA segment with a direct fusion of SARS-CoV-2 S1 RBD to the amino terminus of hemagglutinin derived from A / Singapore / 2016 H3N2 influenza virus (SEQ ID NO: 116). [Figure 26] Flow cytometry analysis of human 293T cells that have been transferred with a replicon DNA plasmid system containing an HA segment encoding the direct fusion of respiratory syncytial virus surface glycoprotein G (RSV G) antigen to the amino terminus of a hemagglutinin derived from the H3N2 influenza virus (SEQ ID NO: 117). [Figure 27] Flow cytometry analysis of live M2VeroA cells infected with either the M2SR vector virus alone or with an M2SR virus having an NS1 segment designed to direct the expression of the SARS-CoV-2 minispike protein on the cell surface using the SARS-CoV-2 S protein signal sequence, and an S2 helix conjugate domain containing the SARS-CoV-2 S protein™ is shown (SEQ ID NO: 119). [Figure 28] This graph shows the mean serum anti-SARS-CoV-2 RBD IgG titer for four drug regimens based on pre-vaccination and post-initial and booster antigen stimulation, as described in Example 6. [Modes for carrying out the invention]

[0005] Detailed description of the invention The recombinant virus of the present invention may be any type of virus. As used herein, a recombinant virus (e.g., a reassortant virus or a different virus) is a virus that comprises genetic material (e.g., a gene segment) derived from a genetically different virus (e.g., a heterogeneous gene segment).

[0006] As used herein, the term “gene segment” refers to a nucleotide sequence encoding a viral protein. This gene segment may be represented by viral RNA (vRNA) encoding a viral protein, i.e., cDNA (complementary DNA) sequences encoding SEQ ID NOs. 43–47, 53, 56, 58, 60, 63–67, and 73.

[0007] As used herein, the term “backbone” refers to the influenza gene segment encoding the proteins PB1, PB2, PA, NP, NS1, and / or NS2, as well as M. The gene segment of the present invention encodes a protein having selected amino acids. This viral backbone is an influenza virus backbone. There are four types of influenza viruses (i.e., A, B, C, and D) classified based on their core proteins, but seasonal epidemics most often result from the circulation of influenza A and B viruses. In one embodiment, the influenza virus backbone is an influenza A backbone. In another embodiment, the influenza virus backbone is an influenza B backbone.

[0008] 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.

[0009] Recombinant viruses (A) Influenza A backbone protein The PB1 (polymerase basic protein 1) gene segment of the present invention may encode a protein comprising at least one selected amino acid, i.e., the PB1 protein. In a preferred embodiment, the selected amino acid comprises leucine at position 40 and tryptophan at position 180. The selected amino acid of the PB1 protein further comprises at least one of asparagine at position 464 or serine at position 607. The PB1 gene segment may optionally include a promoter mutation from cytosine to uracil at nucleotide position 4.

[0010] The selected amino acids can be acquired by gene mutation to a sequence identical to the PB1 amino acid sequence of the present invention, except for the position corresponding to the selected amino acid, for example. Amino acid 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 very well 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, affect its interaction with host cell factors, and therefore affect influenza polymerase activity in Vero cells. Furthermore, influenza RNA polymerase is a heterotrimer composed of the subunits PA, PB1, and PB2. Histidine at position 465 of the PB1 protein interacts with glutamic acid at position 243 of the PA protein, and 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, but it is suggested that this amino acid is located between the RNA-dependent RNA polymerase region and the PB2 binding region, and that it may alter the interaction between PB1 and PB2, thereby influencing polymerase activity in Vero cells.

[0011] The PB2 (polymerase basic protein 2) gene segment of the present invention may also encode a protein comprising at least one selected amino acid, i.e., the PB2 protein. In a preferred embodiment, the selected amino acid comprises valine at position 504, and optionally isoleucine at position 467 and valine at position 529. The PB2 gene segment may optionally include a promoter mutation from cytosine to uracil at nucleotide position 4. The amino acids at positions 467 and 529 of the PB2 protein are located in the PB2-C protein. Specifically, the amino acid at position 467 is located in the cap-binding region of the PB2 protein, and the amino acid at position 529 is located in the cap-627 linker domain. In some influenza viruses, the PB2 protein utilizes caps derived from host RNA to bind the cap structure of host-capped RNA and generate influenza mRNA. This process is known as "cap-snatching". Furthermore, the amino acid at position 627 of PB2 is known to be a key determinant in host range and viral pathogenicity. Therefore, amino acid changes closest to the cap-binding region may affect the efficiency of viral mRNA synthesis.

[0012] The PA (polymerase acid protein) gene segment of the present invention may also encode a protein comprising at least one selected amino acid, i.e., a PA protein. In a preferred embodiment, the selected amino acid comprises lysine at position 401. The PA gene segment may optionally include a promoter mutation from cytosine to uracil at nucleotide position 4.

[0013] The NP (nucleoprotein) gene segment of the present invention may also encode a protein containing at least one selected amino acid, i.e., an NP protein. In a preferred embodiment, the selected amino acid includes leucine at position 116 and at least one of 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 body of the NP protein and therefore do not function as either a nuclear localization signal or a nuclear export signal.

[0014] The NS (non-structural) gene segment of the present invention may also encode a protein comprising at least one selected amino acid, i.e., an NS1 and / or NS2 protein. In a preferred embodiment, the selected amino acid comprises proline at position 30 (NS1 protein) and lysine at position 118 (NS1 protein).

[0015] In one embodiment of the present invention, the influenza virus backbone includes a PB1 gene segment encoding a protein having selected amino acids at positions 40, 180, and 464, i.e., leucine at position 40, tryptophan at position 180, and asparagine at position 464, i.e., the PB1 protein. The PB1 gene segment may have a nucleotide sequence represented by SEQ ID NO: 44. The PB1 gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 49, i.e., the PB1 protein. In another embodiment of this embodiment, the influenza virus backbone may include a PB2 gene segment encoding a protein having selected amino acid at position 504, i.e., valine at position 504, i.e., the PB2 protein. The PB2 gene segment may have a nucleotide sequence represented by SEQ ID NO: 56. The PB2 gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 57, i.e., the PB2 protein. 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 a nucleotide sequence represented by SEQ ID NO: 43. The NP gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 48, i.e., the NP protein. The PA gene segment and NS gene segment of this embodiment may also encode a protein 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 a nucleotide sequence represented by SEQ ID NO: 58. The PA gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 59, i.e., the PA protein. The NS gene segment may have a nucleotide sequence represented by SEQ ID NO: 60.The NS gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 61, i.e., the NS1 protein. The NS gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 62, i.e., the NS2 protein. The gene segments PB1, PB2, and PA in this embodiment may also contain a promoter mutation from cytosine to uracil at nucleotide position 4.

[0016] In another embodiment of the present invention, the influenza virus backbone includes a PB1 gene segment encoding a protein having selected amino acids at positions 40, 180, and 607, i.e., leucine at position 40, tryptophan at position 180, and serine at position 607, i.e., the PB1 protein. The PB1 gene segment may have a nucleotide sequence represented by SEQ ID NO: 46. The PB1 gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 51, i.e., the PB1 protein. In another embodiment of this embodiment, the influenza virus backbone may include a PB2 gene segment 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, i.e., the PB2 protein. The PB2 gene segment may have a nucleotide sequence represented by SEQ ID NO: 47. The PB2 gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 52, i.e., the PB2 protein. The NP gene segment of this embodiment may encode a protein having selected amino acids at positions 116 and 311, i.e., leucine at position 116 and arginine at position 311, i.e., the NP protein. The NP gene segment may have a nucleotide sequence represented by SEQ ID NO: 45. The NP gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 50, i.e., the NP protein. The gene segments PA and NS 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 a nucleotide sequence represented by SEQ ID NO: 58. The PA gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 59, i.e., the PA protein.The NS gene segment may have the nucleotide sequence represented by SEQ ID NO: 60. The NS gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 61, i.e., the NS1 protein. The NS gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 62, i.e., the NS2 protein. The gene segments PB1, PB2, and PA in this embodiment may also include a promoter mutation from cytosine to uracil at nucleotide position 4.

[0017] The selected amino acids in this embodiment confer enhanced growth characteristics to the influenza virus backbone, particularly in most of the proteins of the backbone, compared to an influenza virus backbone that is identical under the same conditions except for the absence of the selected amino acids. For example, the influenza virus backbone of the present invention exhibits enhanced growth in Vero cells.

[0018] The influenza virus backbone of the present invention may also include an M (matrix protein) gene segment. In one embodiment of the present invention, the M gene segment may be a gene segment of a mutant derived from influenza A, thereby the virus lacks the expression of a functional M2 protein. Such a virus is referred to herein as the "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 represented by Sequence ID No. 53. The M gene segment may encode a protein having the amino acid sequence of Sequence ID No. 54, for example, a truncated M2 protein. The M2SR virus can proliferate in Vero cells that stably express wild-type M2 protein (i.e., M2VeroA cells) and allow for multiple-cycle replication. The high yield in Vero cells does not depend on mutations in the M gene segment. Therefore, the influenza virus backbone of the present invention may include an M gene segment encoding a functional M2 protein (Sequence ID No. 1).

[0019] (B) Influenza B backbone protein In one embodiment of the present invention, the recombinant virus comprises an influenza virus backbone including gene segments PA, NP, and NS, wherein (a) the PA gene segment comprises thymine at nucleotide position 2272, (b) the NP gene segment encodes an NP protein having an amino acid sequence containing selected amino acids, the selected amino acids comprising serine at position 40, asparagine or glycine at position 161, threonine at position 204, and optionally valine at position 93, and (c) the NS gene segment comprises guanine at nucleotide position 39, and the NS gene segment encodes an NS protein having an amino acid sequence containing selected amino acids, the selected amino acid comprising glutamine at position 176.

[0020] The PB1 (polymerase basic protein 1) gene segment of the present invention may encode a protein containing at least one selected amino acid, i.e., a PB1 protein. The selected amino acid can be obtained by gene mutation into a sequence identical to the parental PB1 sequence, for example, the PB1 amino acid sequence of the present invention, except for the position corresponding to the selected amino acid. The PB2 (polymerase basic protein 2) gene segment of the present invention may also encode a protein containing at least one selected amino acid, i.e., a PB2 protein.

[0021] The PA (polymerase acid protein) gene segment of the present invention may also encode a protein containing at least one selected amino acid, i.e., a PA protein. In a preferred embodiment, the gene segment contains thymine at nucleotide position 2272.

[0022] The NP (nucleoprotein) gene segment of the present invention may also encode a protein comprising at least one selected amino acid, i.e., an NP protein. In a preferred embodiment, the NP segment comprises thymine at position 177, adenine at position 540, and thymine at position 670, and the NP gene segment encodes a protein having selected amino acids comprising serine at position 40, asparagine or glycine at position 161, threonine at position 204, and optionally valine at position 93.

[0023] The NS (non-structural) gene segment of the present invention may also encode a protein containing at least one selected amino acid, i.e., an NS1 and / or NS2 protein. In a preferred embodiment, the NS segment contains guanine at nucleotide position 39 and cytosine at position 570, and the NS gene segment encodes an NS protein having a selected amino acid including glutamine at position 176 (NS1 protein).

[0024] In one embodiment of this invention, the influenza virus comprises a PB1 gene segment encoding a protein having selected amino acids, i.e., the PB1 protein. The PB1 gene segment may have a nucleotide sequence represented by SEQ ID NO: 63. The PB1 gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 68, i.e., the PB1 protein. In another embodiment of this invention, the influenza virus comprises a PB2 gene segment encoding a protein having selected amino acids, i.e., the PB2 protein. The PB2 gene segment may have a nucleotide sequence represented by SEQ ID NO: 64. The PB2 gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 69, i.e., the PB2 protein. In another embodiment of this invention, the influenza virus comprises an NP gene segment encoding a protein having selected amino acids at positions 40, 161, and 204, i.e., serine at position 40, asparagine or glycine at position 161, threonine at position 204, and optionally valine at position 93, i.e., the NP protein. The NP gene segment may have a nucleotide sequence represented by SEQ ID NO: 66. The NP gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 71, i.e., the NP protein. In another embodiment of this embodiment, the influenza virus may include an NS gene segment encoding a protein having a selected amino acid at position 176, i.e., glutamine at position 176, i.e., the NS1 and / or NS2 proteins. The NS gene segment may include guanine at nucleotide position 39 and cytosine at position 570. The NS gene segment may have the nucleotide sequence represented by SEQ ID NO: 67. The NS gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 72, i.e., the NS1 and / or NS2 proteins. In another embodiment of this embodiment, the influenza virus may include a PA gene segment encoding a protein, i.e., the PA protein. The PA gene segment may have the nucleotide sequence represented by SEQ ID NO: 65.The PA gene segment may encode a protein having the amino acid sequence of SEQ ID NO: 70, i.e., the PA protein.

[0025] The selected amino acids in this embodiment confer enhanced growth characteristics to the influenza virus, particularly in most of the backbone proteins, compared to an influenza virus that is identical under the same conditions except for the absence of the selected amino acids. For example, the influenza virus of the present invention exhibits enhanced growth in Vero cells.

[0026] The influenza virus of the present invention may also include an M (matrix protein) gene segment. In one embodiment of the present invention, the M gene segment may be a gene segment of a mutant derived from influenza B, thereby the virus lacks the expression of a functional BM2 protein. Such a virus is referred to herein as the "BM2SR" virus. The BM2SR virus is a single-replicating influenza virus. The M gene segment of the BM2SR virus may be represented by Sequence ID No. 73. The M gene segment may encode a protein having the amino acid sequence of Sequence ID No. 78, for example, a truncated BM2 protein. The BM2SR virus can proliferate in Vero cells that stably express the BM2 protein (i.e., BM2VeroA cells) and allow for multiple-cycle replication. The high yield in Vero cells does not depend on mutations in the M gene segment. Therefore, the influenza virus of the present invention may include an M gene segment encoding a functional BM2 protein (Sequence ID No. 2).

[0027] (C) Influenza A surface protein In a further embodiment of the present invention, the influenza virus backbone includes NA (neuraminidase) and HA (hemagglutinin) gene segments. In one embodiment of the present invention, the HA gene segment may encode an HA protein having an amino acid sequence comprising at least one selected amino acid (e.g., an amino acid mutation) in the HA1 subunit of the HA protein, and / or at least one selected amino acid (e.g., an amino acid mutation) in the HA2 subunit of the HA protein. For example, the at least one amino acid mutation in the HA2 subunit could be asparagine at position 107. Such mutations may also contribute to enhanced growth of the virus during development.

[0028] In one embodiment of the present invention, the gene segments PB1, PB2, PA, NP, and NS 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 gene segments PB1, PB2, PA, NP, and NS are derived. Similarly, the NA gene segment may be derived from an influenza strain different from the single influenza strain from which the gene segments PB1, PB2, PA, NP, and NS are derived. Therefore, the recombinant virus of the present invention may be a pandemic virus (e.g., H5N1 and H7N9) or a seasonal virus (e.g., H1N1, H3N2, and influenza B).

[0029] (D) Influenza B surface protein In a further embodiment of the present invention, the recombinant virus comprises an influenza virus backbone further comprising NA (neuraminidase) and HA (hemagglutinin) gene segments. In one embodiment of the present invention, the HA gene segment may encode an HA protein having an amino acid sequence comprising at least one selected amino acid (e.g., an amino acid mutation) in the HA1 subunit of the HA protein, and / or at least one selected amino acid (e.g., an amino acid mutation) in the HA2 subunit of the HA protein. For example, at least one amino acid mutation in the HA2 subunit may be glutamic acid at position 61. In another embodiment, at least one amino acid mutation in the HA2 subunit may be glutamic acid at position 112. This amino acid mutation may be present in either a subtype or lineage of influenza B virus (i.e., Victoria or Yamagata). In a preferred embodiment, the amino acid mutation in the HA2 subunit may be glutamic acid at position 61 in the Victoria lineage of influenza B virus. In another preferred embodiment, the amino acid mutation in the HA2 subunit may be glutamic acid at position 112 in the Yamagata lineage of influenza B virus. Such a mutation may also contribute to enhanced growth of the virus during development.

[0030] In one embodiment of the present invention, the gene segments PB1, PB2, PA, NP, and NS 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 gene segments PB1, PB2, PA, NP, and NS are derived. Similarly, the NA gene segment may be derived from an influenza strain different from the single influenza strain from which the gene segments PB1, PB2, PA, NP, and NS are derived. Therefore, the influenza virus of the present invention may be a seasonal influenza virus (e.g., influenza B).

[0031] (E) Antigen In one embodiment, the recombinant virus comprises an influenza virus backbone including the gene segments PB1, PB2, PA, NP, M, NS, HA, and NA, wherein at least one of the gene segments PB2, PB2, PA, NP, M, NS, HA, and NA contains a nucleotide sequence encoding one or more antigens. As used herein, the term “antigen” refers to a heterologous antigen with respect to the HA gene segment. This antigen may be viral (including influenza), bacterial, fungal, or protozoan. For example, a viral antigen or epitope sequence inserted into a gene segment (e.g., the gene segments PB1, PB2, PA, NP, M, NS, HA, or NA) would be an antigen for that virus. In one embodiment, the antigen is an immunogenic fragment of the SARS-CoV-2 spike glycoprotein (e.g., the S1 protein). In another embodiment, the antigen is an influenza gene segment or fragment thereof that is heterologous to the HA gene segment in the influenza virus backbone (i.e., the gene segments or fragments PB1, PB2, PA, NP, M, NS, HA, or NA). In another embodiment, the antigen is respiratory syncytial virus (RSV) or a fragment thereof. In another embodiment, the antigen is parainfluenza virus (PIV) or a fragment thereof. In some embodiments, one or more antigens are expressed from within a viral gene segment.

[0032] In one embodiment of the present invention, at least one gene segment comprising a nucleotide sequence encoding one or more antigens further comprises a nucleotide sequence encoding at least one flexible linker protein, at least one cleavable sequence, and / or at least one FLAG protein. Such a gene segment may encode at least two flexible linker proteins, at least two cleavable sequences, and / or at least two FLAG proteins.

[0033] In one embodiment, the cleavable cleavable sequence includes a “self-cleaving” sequence. In one embodiment, the “self-cleaving” sequence is a “self-cleaving” 2A peptide. The “self-cleaving” 2A peptide is described, for example, in Liu et al., Sci.Rep., 7(1):2193 (2017) and Szymczak et al., Nature Biotechnol., 22(5):589-594 (2004). This 2A peptide is a viral oligopeptide that mediates polypeptide cleavage during translation in eukaryotic cells. The name “2A” refers to a specific region of the viral genome. Although not tied to a specific theory or mechanism, the mechanism of 2A-mediated “self-cleavage” is thought to be ribosome skipping of the C-terminal glycyl-prolyl peptide bond of the 2A peptide. Different 2A peptides may contain the consensus amino acid sequence GDVEXNPGP (SEQ ID NO: 19) at their C-terminus, where X in SEQ ID NO: 19 is any naturally occurring amino acid residue. In one embodiment of the present invention, the cleavable ribosome skip sequence is the porcine rhinovirus type 1 2A (P2A) amino acid sequence, the equine rhinitis virus type A (E2A) amino acid sequence, the tesea signalavirus 2A (T2A) amino acid sequence, or the foot-and-mouth disease virus (F2A) amino acid sequence. In one embodiment of the present invention, the ribosome skip sequence is a 2A peptide amino acid sequence that includes the P2A amino acid sequence, consists of such amino acid sequence, or is essentially composed of such amino acid sequence.

[0034] In one embodiment of the present invention, the flexible linker protein is independently 1 to 20 amino acid residues selected from the group consisting of glycine and serine. In some embodiments, the flexible linker protein is (Xaa1) rIt is defined as follows, where each Xaa1 is independently selected from glycine and serine, and r is an integer from 1 to 20. Examples of such linkers include, but are not limited to, GSG (SEQ ID NO: 75), GGGGSGGGGSGGGGS (SEQ ID NO: 76), and (G4S)3. In one embodiment of the present invention, at least one gene segment containing an amino acid sequence encoding an antigen further comprises at least one flexible linker protein. In another embodiment, such gene segment further comprises at least two flexible linker proteins.

[0035] In a preferred embodiment, the antigen is an immunogenic fragment of the SARS-CoV-2 spike glycoprotein (e.g., the S1 protein). In one embodiment, the M gene segment encodes a nucleotide sequence encoding at least one immunogenic fragment of the SARS-CoV-2 spike glycoprotein. The M gene segment may encode a mutated M2 protein or a BM2 protein. The M gene segment may further encode at least one flexible linker protein and at least one FLAG protein. In one embodiment, the M gene segment encodes a fusion protein comprising a mutated M2 protein, a flexible linker protein, and a FLAG epitope tag protein. For example, the M gene segment may have a nucleotide sequence represented by any one of SEQ ID NOs. 79 and 81-84. The M gene segment may encode a protein comprising any one of SEQ ID NOs. 1-14 and 92-96.

[0036] In another embodiment, the NS gene segment encodes a nucleotide sequence encoding at least one immunogenic fragment of the SARS-CoV-2 spike glycoprotein. The NS gene segment may encode the NS1 protein and the NS2 (i.e., NEP) protein, or fragments thereof. The NS gene segment may also encode at least one flexible linker protein or a fragment thereof. The NS gene segment may also encode at least one cleavable sequence. The NS gene segment may have a nucleotide sequence represented by any one of sequence numbers 80 and 85-91. The NS gene segment may encode proteins including sequence numbers 97-104.

[0037] In one embodiment of the present invention, the cleavable sequence is a P2A peptide sequence. In this embodiment, the P2A peptide sequence binds to the C-terminus of an NS1 protein at one end and to an antigen at the other end. In this embodiment, the antigen can bind to an NEP open reading frame (ORF). In another embodiment of the present invention, the P2A peptide sequence binds to the C-terminus of an NS1 protein at one end and to a first flexible linker protein at the other end. In this embodiment, the first flexible linker protein can bind to an antigen, which then attaches to an NEP ORF. In another embodiment, a second cleavable sequence exists. In one embodiment, the second cleavable sequence is a peptide sequence called P2A or T2A. Any second cleavable sequence can bind to an antigen at one end and to an NEP ORF at the other end. In another embodiment, a second cleavable cleavable sequence may bind to a flexible linker protein, which then binds to either an antigen or an NEP ORF.

[0038] In one embodiment, at least one (i.e., PB2, PB2, PA, NP, M, NS, HA, or NA) gene segment will encode a nucleotide sequence encoding one or more antigens. In some embodiments, at least two of the eight influenza virus backbone segments (i.e., the gene segments PB1 and PB2, PB1 and PA, PB1 and NP, PB1 and M, PB1 and NS, PB1 and HA, and PB1 and NA, PB2 and PA, PB2 and NP, PB2 and M, PB2 and NS, PB2 and HA, PB2 and NA, PA and NP, PA and M, PA and NS, PA and HA, PA and NA, NP and M, NP and NS, NP and HA, NP and NA, M and NS, M and HA, M and NA, NS and HA, NS and NA, or HA and NA) will encode a nucleotide sequence encoding one or more antigens, such as an immunogenic fragment of the SARS-CoV-2 spike glycoprotein (e.g., the S1 protein).

[0039] In some embodiments, a gene segment containing at least one nucleotide sequence encoding one or more antigens further includes a downstream duplication, where the downstream duplication contains at least one silent nucleotide mutation. In one embodiment, a gene segment containing at least one nucleotide sequence encoding one or more antigens further includes a downstream direct tandem duplication, where the downstream duplication contains at least one silent nucleotide mutation. A downstream duplication refers to a nucleotide sequence in which a portion of the nucleotide sequence is repeated one or more times in the same orientation. The repeating nucleotide sequences can be directly linked one after another, or can contain any nucleotide sequences between each of the repeating nucleotide sequences. In addition, the number of duplicated bases is not limited.

[0040] In some embodiments, downstream duplication of the nucleotide sequence of a gene segment occurs during the insertion of an antigen-coding nucleotide sequence. In some embodiments, downstream duplication can reduce the stability of the nucleotide sequence, as well as the encoded amino acid sequence and protein. To improve stability, at least one silent mutation (i.e., a mutation that does not affect the amino acid sequence encoded by the nucleotide sequence) is introduced to reduce the homology between the first nucleotide sequence and the second downstream duplicated nucleotide sequence. For example, in a preferred embodiment, the NS gene segment includes a nucleotide sequence that codes for an antigen. During the insertion of an antigen-coding nucleotide sequence, a portion of the nucleotide sequence is duplicated, creating a downstream duplication. The first copy of the nucleotide sequence is part of the packaging sequence, and the second copy may interfere with the packaging. To prevent interference, a silent mutation is added to the downstream duplication to reduce homology with the first copy. In one embodiment, the downstream duplication has at least one (i.e., 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, or at least ten) silent mutations.

[0041] A recombinant virus may have one or more (i.e., at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight) gene segments, each comprising at least one nucleotide sequence encoding one or more antigens, and further comprising downstream duplications which include at least one silent nucleotide mutation. For example, in one embodiment, such one or more gene segments may be the gene segments PB1, PB2, PA, NP, NS, M, HA, or NA. In another embodiment, such one or more gene segments may be PB1 and PB2, PB2 and PA, PB1 and NP, PB1 and NS, PB1 and M, PB1 and HA, PB1 and NA, PB2 and PA, PB2 and NP, PB2 and NS, PB2 and M, PB2 and HA, PB2 and NA, PA and NP, PA and NS, PA and M, PA and HA, PA and NA, NP and NS, NP and M, NP and HA, NP and NA, NS and M, NS and HA, NS and NA, M and HA, M and NA, or HA and NA.

[0042] (F) Characteristics of the influenza virus backbone The recombinant virus backbone of the present invention confers high growth characteristics to influenza viruses, particularly in Vero cells, regardless of the type of influenza virus (e.g., influenza A or B, seasonal or pandemic influenza virus). The influenza viruses of the present invention exhibit high yields even in manufacturing processes using low MOI (e.g., 0.001). MOI refers to the average number of active agents (e.g., viruses) per infectious target (e.g., cell). Lower MOIs are used when multiple cycle infections are required (e.g., viral vaccine production). Current ministerial ordinances on manufacturing control and quality control standards for pharmaceuticals, enforced by the U.S. Food and Drug Administration, generally require the use of the lowest MOI to still produce high-yield viruses. This is because master seed stocks are expensive, and the resulting toxicity from non-infectious particles and excess cellular proteins can reduce virus production.

[0043] In a further embodiment of the present invention, influenza viruses are generally stable, and thereafter, selected amino acids of the backbone proteins, particularly the proteins PB1, PB2, PA, NP, and NS1, are highly conserved even when growing 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 proteins PB1, PB2, and NP 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 a 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 (SEQ ID NO: 74) of influenza B virus (i.e., BM2Vero cells). BM2 is known to be a functional counterpart to influenza A virus M2. The influenza B virus M2 protein can functionally replace its influenza A virus counterpart in promoting viral replication (Wanitchang et al., Virology 498:99-108 (2016)). In such embodiments, the selected amino acids can be conserved even when the influenza virus is influenza A virus.

[0044] Genetically modified Vero cells (i.e., Vero cells expressing influenza M2 or BM2 proteins) behave similarly to normal Vero cells and support the growth of influenza A or B viruses comparable to that of normal Vero cells. The viral titer for M2SR virus in M2VeroA cells is comparable to that of influenza virus expressing functional M2 in unmodified Vero cell lines. Furthermore, the viral titer for BM2SR virus (i.e., influenza virus containing a mutant M gene segment derived from influenza B and consequently not expressing the functional BM2 protein) in BM2Vero cells is comparable to that of influenza virus expressing functional BM2 in unmodified Vero cell lines. Therefore, M2SR virus and BM2SR virus behave similarly to influenza virus replication in M2VeroA cell lines and BM2Vero cell lines, respectively.

[0045] In one embodiment of the present invention, the influenza virus can replicate in human cells.

[0046] Pharmaceutical preparations The present invention provides pharmaceutical formulations (e.g., vaccines or other immunogenic compositions) comprising the recombinant virus of the present invention as described herein.

[0047] The pharmaceutical formulation may further include at least one pharmaceutically acceptable carrier or pharmaceutical additive. As used herein, the term “pharmaceutically acceptable carrier or pharmaceutical additive” refers to any component of the pharmaceutical formulation other than the influenza virus of the present invention. The pharmaceutically acceptable carrier or pharmaceutical additive can enhance the efficacy of the recombinant virus of the present invention or maintain the stability of the pharmaceutical formulation, preferably without significantly inactivating the recombinant virus of the present invention.

[0048] At least one pharmaceutically acceptable carrier or pharmaceutical additive may be any suitable pharmaceutically acceptable carrier or pharmaceutical additive, many of which are known in the art. Exemplary pharmaceutically acceptable carriers or pharmaceutical additives include components that maintain the pH of a pharmaceutical formulation (e.g., buffers), components that adjust tonicity (e.g., tonic modifiers such as inorganic salts), components that improve the stability and / or immunogenicity of proteins (e.g., viruses), components that improve mucosal adhesion, components that prevent protein aggregation, and / or components that hold the pharmaceutical formulation (e.g., preservatives). For example, a pharmaceutically acceptable carrier or pharmaceutical additive 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 specific immune response), many of which are known in the art. A particular carrier or pharmaceutical additive may function for more than one purpose in a pharmaceutical formulation; therefore, embodiments are not limited to the descriptions enumerated herein.

[0049] Any suitable buffer may be present in a pharmaceutical formulation. In one embodiment, the buffer comprises at least one of imidazole buffer, potassium phosphate buffer, phosphate-buffered saline (PBS), Dulbecco's phosphate-buffered saline (DPBS) (e.g., 1×DPBS), histidine buffer, sodium citrate buffer, and sucrose phosphate glutamate buffer (SPG). The PBS preparation and / or DPBS preparation may comprise, for example, sodium chloride, potassium chloride, monobasic potassium phosphate, and dibasic sodium phosphate, and may further comprise calcium chloride and / or magnesium chloride. In some embodiments, the PBS preparation and / or DPBS preparation comprises about 136.9 mM sodium chloride, about 2.67 mM potassium chloride, about 1.47 mM monobasic potassium phosphate, and about 8.1 mM dibasic sodium phosphate, but any suitable PBS preparation and / or DPBS preparation, many of which are known in the art, may be used as a buffer in a pharmaceutical formulation.

[0050] The buffering agent may be present in the pharmaceutical preparation at any appropriate concentration. The buffering agent may be present in the pharmaceutical preparation at concentrations of approximately 0.1 mM or higher, approximately 1 mM or higher, approximately 10 mM or higher, approximately 20 mM or higher, approximately 30 mM or higher, approximately 40 mM or higher, approximately 50 mM or higher, approximately 60 mM or higher, approximately 70 mM or higher, approximately 80 mM or higher, approximately 90 mM or higher, approximately 100 mM or higher, approximately 120 mM or higher, approximately 140 mM or higher, approximately 160 mM or higher, approximately 180 mM or higher, approximately 200 mM or higher, approximately 250 mM or higher, approximately 300 mM or higher, approximately 350 mM or higher, approximately 400 mM or higher, approximately 450 mM or higher, or approximately 500 mM or higher. Alternatively, or in addition, the buffering agent may be present in the pharmaceutical preparation at concentrations of approximately 1,000 mM or less, approximately 500 mM or less, approximately 450 mM or less, approximately 400 mM or less, approximately 350 mM or less, approximately 300 mM or less, approximately 250 mM or less, approximately 200 mM or less, approximately 180 mM or less, approximately 160 mM or less, approximately 140 mM or less, approximately 120 mM or less, approximately 100 mM or less, approximately 90 mM or less, approximately 80 mM or less, approximately 70 mM or less, approximately 60 mM or less, approximately 50 mM or less, approximately 40 mM or less, approximately 30 mM or less, approximately 20 mM or less, approximately 10 mM or less, or approximately 1 mM or less. The buffering agent may be present in the pharmaceutical preparation at any concentration within the range defined by any of the endpoints described above. For example, the buffering agent may be present in pharmaceutical preparations at concentrations such as approximately 0.1 mM to approximately 1000 mM, approximately 0.1 mM to approximately 500 mM, approximately 0.1 mM to approximately 100 mM, approximately 1 mM to approximately 1000 mM, approximately 1 mM to approximately 500 mM, approximately 1 mM to approximately 100 mM, approximately 100 mM to approximately 1000 mM, and approximately 100 mM to approximately 500 mM.

[0051] In further embodiments, the buffer is present in the pharmaceutical formulation at a percentage concentration (e.g., volume / volume percentage (%v / v), weight / volume percentage (%w / v), or weight / weight percentage (%w / w)). The buffer may be present in the pharmaceutical formulation at a percentage concentration of about 0.1% or more, about 1% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, or about 50% or more. Alternatively, or in addition, the buffer may be present in the pharmaceutical formulation at a percentage concentration of about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, or about 1% or less. The buffer may be present in the pharmaceutical formulation at any percentage concentration within the range defined by any of the endpoints described above. For example, buffering agents may be present in pharmaceutical preparations at percentage concentrations such as approximately 0.1% to 60%, approximately 1% to 60%, approximately 10% to 60%, approximately 0.1% to 50%, approximately 1% to 50%, approximately 10% to 50%, approximately 20% to 60%, approximately 20% to 50%, approximately 20% to 40%, approximately 20% to 30%, approximately 30% to 40%, and approximately 40% to 50%.

[0052] A buffer can maintain the pH of a pharmaceutical formulation at any appropriate pH. For example, a buffer can maintain the pH of a pharmaceutical formulation at a pH of approximately 4 or higher, approximately 4.5 or higher, approximately 5 or higher, approximately 5.5 or higher, approximately 6 or higher, approximately 6.5 or higher, approximately 7 or higher, or approximately 7.5 or higher. Alternatively, or in addition, a buffer can maintain the pH of a pharmaceutical formulation at a pH of approximately 8 or lower, approximately 7.5 or lower, approximately 7 or lower, approximately 6.5 or lower, approximately 6 or lower, approximately 5.5 or lower, approximately 5 or lower, or approximately 4.5 or lower. A buffer can maintain the pH of a pharmaceutical formulation within a range defined by any of the endpoints described above. For example, buffering agents can maintain the pH of pharmaceutical formulations at pH levels such as approximately 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, and 6-7.

[0053] 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 may be at least one of sodium chloride (NaCl), magnesium sulfate (MgSO4), and magnesium chloride (MgCl2). The tonic modifier, e.g., the inorganic salt, may be present in the pharmaceutical formulation in any suitable amount. The tonic modifier, e.g., the inorganic salt, may be present in amounts of about 0.1 mM or more, about 0.2 mM or more, about 0.4 mM or more, about 0.6 mM or more, about 0.8 mM or more, about 1 mM or more, about 1.2 mM or more, about 1.4 mM or more, about 1.6 mM or more, about 1.8 mM or more, about 2 mM or more, about 3 mM or more, about 4 mM or more, about 5 mM or more, about 6 mM or more, about 7 mM or more, about 8 mM or more, about 9 mM or more, It may be present in pharmaceutical preparations at concentrations of approximately 10 mM or higher, approximately 20 mM or higher, approximately 30 mM or higher, approximately 40 mM or higher, approximately 50 mM or higher, approximately 100 mM or higher, approximately 200 mM or higher, approximately 300 mM or higher, approximately 400 mM or higher, approximately 500 mM or higher, approximately 600 mM or higher, approximately 700 mM or higher, approximately 800 mM or higher, approximately 900 mM or higher, approximately 1000 mM or higher, or approximately 1500 mM or higher. Alternatively, or in addition, a tonicity regulator, such as an inorganic salt, can be used in the following concentrations: approximately 2000 mM or less, approximately 1500 mM or less, approximately 1000 mM or less, approximately 900 mM or less, approximately 800 mM or less, approximately 700 mM or less, approximately 600 mM or less, approximately 500 mM or less, approximately 450 mM or less, approximately 400 mM or less, approximately 350 mM or less, approximately 300 mM or less, approximately 250 mM or less, approximately 200 mM or less, approximately 150 mM or less, approximately 100 mM or less, approximately 50 mM or less, approximately 45 mM or less, approximately 40 mM The following concentrations may be present in the pharmaceutical preparation: approximately 35 mM or less, approximately 30 mM or less, approximately 25 mM or less, approximately 20 mM or less, approximately 10 mM or less, approximately 9 mM or less, approximately 8 mM or less, approximately 7 mM or less, approximately 6 mM or less, approximately 5 mM or less, approximately 4 mM or less, approximately 3 mM or less, approximately 2 mM or less, approximately 1.8 mM or less, approximately 1.6 mM or less, approximately 1.4 mM or less, approximately 1.2 mM or less, approximately 1 mM or less, approximately 0.8 mM or less, approximately 0.6 mM or less, approximately 0.4 mM or less, or approximately 0.2 mM or less. Tonicity modifiers, such as inorganic salts, may be present in the pharmaceutical preparation at any concentration within the range defined by any of the endpoints described above.For example, tonicity regulators, such as inorganic salts, are available in concentrations of approximately 0.1 mM to 2000 mM, 0.1 mM to 1500 mM, 0.1 mM to 1000 mM, 0.1 mM to 500 mM, 0.1 mM to 250 mM, 0.1 mM to 100 mM, 0.1 mM to 50 mM, 0.1 mM to 10 mM, 1 mM to 2000 mM, 1 mM to 1500 mM, 1 mM to 1000 mM, 1 mM to 500 mM, 1 mM to 250 mM, 1 mM to 100 mM, 1 mM to 50 mM, and 1 mM to 10 mM. It can be present in pharmaceutical preparations at concentrations such as approximately 10 mM to approximately 2000 mM, approximately 10 mM to approximately 1500 mM, approximately 10 mM to approximately 1000 mM, approximately 10 mM to approximately 500 mM, approximately 10 mM to approximately 250 mM, approximately 10 mM to approximately 100 mM, approximately 10 mM to approximately 50 mM, approximately 100 mM to approximately 2000 mM, approximately 100 mM to approximately 1500 mM, approximately 100 mM to approximately 1000 mM, approximately 100 mM to approximately 500 mM, approximately 100 mM to approximately 250 mM, approximately 500 mM to approximately 2000 mM, approximately 500 mM to approximately 1500 mM, and approximately 500 mM to approximately 1000 mM.

[0054] 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)). Tonic modifiers, such as inorganic salts, may be present in the pharmaceutical formulation at a percentage concentration of about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, or about 10% or more. Alternatively, or in addition, tonic modifiers, such as inorganic salts, may be present in the pharmaceutical formulation at a percentage concentration of about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less. Tonicity modifiers, such as inorganic salts, may be present in pharmaceutical formulations at any percentage concentration within the range defined by any of the endpoints described above. For example, tonicity modifiers, such as inorganic salts, may be present in pharmaceutical formulations at percentage concentrations such as approximately 0.1% to approximately 1%, approximately 0.1% to approximately 2%, approximately 0.1% to approximately 5%, approximately 0.1% to approximately 10%, approximately 1% to approximately 2%, approximately 1% to approximately 5%, approximately 1% to approximately 10%, approximately 2% to approximately 10%, approximately 3% to approximately 10%, approximately 4% to approximately 10%, and approximately 5% to approximately 10%.

[0055] Any suitable surfactant may be present in the pharmaceutical formulation. In certain embodiments, the surfactant may include at least one of polysorbate 20, polysorbate 80, sodium deoxycholate, and poloxamer 188. The surfactant may be present in the pharmaceutical formulation in any suitable amount. In some embodiments, the surfactant 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)). Surfactants may be present in pharmaceutical formulations at a percentage concentration of approximately 0.01% or more, approximately 0.02% or more, approximately 0.03% or more, approximately 0.04% or more, approximately 0.05% or more, approximately 0.06% or more, approximately 0.07% or more, approximately 0.08% or more, approximately 0.09% or more, approximately 0.1% or more, approximately 0.2% or more, approximately 0.3% or more, approximately 0.4% or more, approximately 0.5% or more, approximately 0.6% or more, approximately 0.7% or more, approximately 0.8% or more, approximately 0.9% or more, or approximately 1% or more. Alternatively, or in addition, surfactants may be present in pharmaceutical formulations at a percentage concentration of approximately 1% or less, approximately 0.9% or less, approximately 0.8% or less, approximately 0.7% or less, approximately 0.6% or less, approximately 0.5% or less, approximately 0.4% or less, approximately 0.3% or less, approximately 0.2% or less, or approximately 0.1% or less. Surfactants may be present in pharmaceutical formulations at any percentage concentration within the range defined by any of the endpoints described above. For example, surfactants may be present in pharmaceutical formulations at percentage concentrations such as approximately 0.01% to 1%, approximately 0.01% to 0.1%, approximately 0.05% to 1%, approximately 0.05% to 0.1%, approximately 0.1% to 1%, approximately 0.1% to 0.5%, approximately 0.2% to 1%, and approximately 0.5% to 1%.

[0056] 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 may be present in the pharmaceutical formulation in any suitable amount. The amino acid may be present in the pharmaceutical formulation at concentrations of about 1 mM or more, about 2 mM or more, about 3 mM or more, about 5 mM or more, about 6 mM or more, about 7 mM or more, about 8 mM or more, about 9 mM or more, or about 10 mM or more. Alternatively, or in addition, the amino acid may be present in the pharmaceutical formulation at concentrations of about about 100 mM or less, about 90 mM or less, about 80 mM or less, about 70 mM or less, about 60 mM or less, about 50 mM or less, about 40 mM or less, about 30 mM or less, about 20 mM or less, or about 10 mM or less. Amino acids can be present in pharmaceutical formulations at any concentration within the range defined by one of the endpoints mentioned above. For example, amino acids can be present in pharmaceutical formulations at concentrations such as approximately 1 mM to 10 mM, approximately 1 mM to 50 mM, approximately 1 mM to 100 mM, approximately 5 mM to 50 mM, approximately 10 mM to 50 mM, and approximately 20 mM to 50 mM.

[0057] In some embodiments, amino acids are present in the pharmaceutical formulation at percentage concentrations (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 percentage concentrations of about 0.1% or more, about 0.2% or more, about 0.3% or more, about 0.4% or more, about 0.5% or more, about 0.6% or more, about 0.7% or more, about 0.8% or more, about 0.9% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, or about 5% or more. Alternatively, or in addition, amino acids may be present in the pharmaceutical formulation at percentage concentrations of about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less. Amino acids may be present in pharmaceutical formulations at any percentage concentration within the range defined by one of the endpoints mentioned above. For example, amino acids may be present in pharmaceutical formulations at percentage concentrations such as approximately 0.1% to 10%, approximately 0.2% to 10%, approximately 0.5% to 10%, approximately 0.1% to 5%, approximately 0.1% to 2%, approximately 0.2% to 2%, and approximately 0.5% to 1%.

[0058] 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. The polymer may preferably be polyanionic, such as carboxymethylcellulose or poly(acrylic acid). 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).

[0059] Polymers or polymer compounds may be present in pharmaceutical formulations in any appropriate amount. Polymers or polymer compounds may be present in pharmaceutical formulations at percentage concentrations (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 pharmaceutical formulations at percentage concentrations of approximately 0.1% or more, approximately 0.2% or more, approximately 0.3% or more, approximately 0.4% or more, approximately 0.5% or more, approximately 0.6% or more, approximately 0.7% or more, approximately 0.8% or more, approximately 0.9% or more, approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, or approximately 5% or more. Alternatively, or in addition, polymers or polymer compounds may be present in the pharmaceutical formulation at percentage concentrations of approximately 10% or less, approximately 9% or less, approximately 8% or less, approximately 7% or less, approximately 6% or less, approximately 5% or less, approximately 4% or less, approximately 3% or less, approximately 2% or less, or approximately 1% or less. Polymers or polymer compounds may be present in the pharmaceutical formulation at any percentage concentration within the range defined by any of the endpoints described above. For example, polymers or polymer compounds may be present in the pharmaceutical formulation at percentage concentrations such as approximately 0.1% to approximately 10%, approximately 0.2% to approximately 0%, approximately 0.5% to approximately 10%, approximately 0.1% to approximately 5%, approximately 0.1% to approximately 2%, approximately 0.2% to approximately 2%, approximately 0.5% to approximately 2%, approximately 0.1% to approximately 1%, approximately 0.2% to approximately 1%, and approximately 0.5% to approximately 1%.

[0060] Any suitable chelating agent may be present in the pharmaceutical formulation. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), amidooxime 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, approximately 20 μM or higher, approximately 30 μM or higher, approximately 40 μM or higher, approximately 50 μM or higher, approximately 60 μM or higher, approximately 70 μM or higher, approximately 80 μM or higher, approximately 90 μM or higher, approximately 100 μM or higher, approximately 120 μM or higher, or approximately 150 μM or higher. Alternatively, or in addition, chelating agents may be present in the pharmaceutical formulation at concentrations of approximately 500 μM or less, approximately 400 μM or less, approximately 300 μM or less, approximately 200 μM or less, approximately 150 μM or less, approximately 140 μM or less, approximately 130 μM or less, approximately 120 μM or less, approximately 110 μM or less, approximately 100 μM or less, approximately 80 μM or less, approximately 70 μM or less, approximately 60 μM or less, or approximately 50 μM or less. Chelating agents may be present in the pharmaceutical formulation at any concentration within the range defined by any of the endpoints described above. For example, chelating agents can be present in pharmaceutical preparations at concentrations such as approximately 10 μM to 500 μM, approximately 10 μM to 200 μM, approximately 10 μM to 150 μM, approximately 10 μM to 100 μM, approximately 50 μM to 500 μM, approximately 50 μM to 200 μM, approximately 50 μM to 150 μM, and approximately 50 μM to 100 μM.

[0061] 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 may be present in the pharmaceutical formulation at any suitable concentration. The sugar may be present at concentrations of approximately 0.1 mM or higher, approximately 0.2 mM or higher, approximately 0.4 mM or higher, approximately 0.6 mM or higher, approximately 0.8 mM or higher, approximately 1 mM or higher, approximately 1.2 mM or higher, approximately 1.4 mM or higher, approximately 1.6 mM or higher, approximately 1.8 mM or higher, approximately 2 mM or higher, approximately 3 mM or higher, approximately 4 mM or higher, approximately 5 mM or higher, approximately 6 mM or higher, approximately 7 mM or higher, approximately 8 mM or higher, approximately 9 mM or higher, approximately 10 mM or higher, approximately 20 mM or higher, and approximately 30 mM or higher. It may be present in pharmaceutical preparations at concentrations of approximately 40 mM or higher, approximately 50 mM or higher, approximately 60 mM or higher, approximately 70 mM or higher, approximately 80 mM or higher, approximately 90 mM or higher, or approximately 100 mM or higher, approximately 200 mM or higher, approximately 300 mM or higher, approximately 400 mM or higher, approximately 500 mM or higher, approximately 600 mM or higher, approximately 700 mM or higher, approximately 800 mM or higher, approximately 900 mM or higher, approximately 1000 mM or higher, or approximately 1500 mM or higher. Alternatively, or in addition, sugars are approximately 2000 mM or less, approximately 1500 mM or less, approximately 1000 mM or less, approximately 900 mM or less, approximately 800 mM or less, approximately 700 mM or less, approximately 600 mM or less, approximately 500 mM or less, approximately 450 mM or less, approximately 400 mM or less, approximately 350 mM or less, approximately 300 mM or less, approximately 250 mM or less, approximately approximately 200 mM or less, approximately 150 mM or less, approximately 100 mM or less, approximately 50 mM or less, approximately 45 mM or less, approximately 40 mM or less, approximately 35 Sugars may be present in pharmaceutical formulations at concentrations of mM or less, approximately 30 mM or less, approximately 25 mM or less, approximately 20 mM or less, approximately 10 mM or less, approximately 9 mM or less, approximately 8 mM or less, approximately 7 mM or less, approximately 6 mM or less, approximately 5 mM or less, approximately 4 mM or less, approximately 3 mM or less, approximately 2 mM or less, approximately 1.8 mM or less, approximately 1.6 mM or less, approximately 1.4 mM or less, approximately 1.2 mM or less, approximately 1 mM or less, approximately 0.8 mM or less, approximately 0.6 mM or less, approximately 0.4 mM or less, or approximately 0.2 mM or less. Sugars may be present in pharmaceutical formulations at any concentration within the range defined by any of the endpoints described above.For example, sugars are found in concentrations of approximately 0.1mM to 2000mM, 0.1mM to 1500mM, 0.1mM to 1000mM, 0.1mM to 500mM, 0.1mM to 250mM, 0.1mM to 100mM, 0.1mM to 50mM, 0.1mM to 10mM, 1mM to 2000mM, 1mM to 1500mM, 1mM to 1000mM, 1mM to 500mM, 1mM to 250mM, 1mM to 100mM, 1mM to 50mM, 1mM to 10mM, and 10mM. It can be present in pharmaceutical preparations at concentrations such as approximately 2000 mM, approximately 10 mM to approximately 1500 mM, approximately 10 mM to approximately 1000 mM, approximately 10 mM to approximately 500 mM, approximately 10 mM to approximately 250 mM, approximately 10 mM to approximately 100 mM, approximately 10 mM to approximately 50 mM, approximately 100 mM to approximately 2000 mM, approximately 100 mM to approximately 1500 mM, approximately 100 mM to approximately 1000 mM, approximately 100 mM to approximately 500 mM, approximately 100 mM to approximately 250 mM, approximately 500 mM to approximately 2000 mM, approximately 500 mM to approximately 1500 mM, and approximately 500 mM to approximately 1000 mM.

[0062] In other embodiments, sugars are present in the pharmaceutical formulation at percentage concentrations (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 percentage concentrations of about 0.1% or more, about 1% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, or about 40% or more. Alternatively, or in addition, sugars may be present in the pharmaceutical formulation at percentage concentrations of about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, or about 1% or less. Sugars may be present in the pharmaceutical formulation at any percentage concentration within the range defined by any of the endpoints described above. For example, sugars can be present in pharmaceutical preparations at percentage concentrations such as approximately 0.1% to 50%, approximately 1% to 50%, approximately 10% to 50%, approximately 0.1% to 20%, approximately 1% to 20%, approximately 10% to 20%, approximately 0.1% to 10%, and approximately 1% to 10%.

[0063] 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 approximately 0.1 mM or higher, approximately 1 mM or higher, approximately 10 mM or higher, approximately 20 mM or higher, approximately 30 mM or higher, approximately 40 mM or higher, approximately 50 mM or higher, approximately 60 mM or higher, approximately 70 mM or higher, approximately 80 mM or higher, approximately 90 mM or higher, approximately 100 mM or higher, approximately 120 mM or higher, approximately 140 mM or higher, approximately 160 mM or higher, approximately 180 mM or higher, approximately 200 mM or higher, approximately 250 mM or higher, approximately 300 mM or higher, approximately 350 mM or higher, approximately 400 mM or higher, approximately 450 mM or higher, or approximately 500 mM or higher. Alternatively, or in addition, polyols may be present in pharmaceutical formulations at concentrations of approximately 1000 mM or less, approximately 500 mM or less, approximately 450 mM or less, approximately 400 mM or less, approximately 350 mM or less, approximately 300 mM or less, approximately 250 mM or less, approximately 200 mM or less, approximately 180 mM or less, approximately 160 mM or less, approximately 140 mM or less, approximately 120 mM or less, approximately 100 mM or less, approximately 90 mM or less, approximately 80 mM or less, approximately 70 mM or less, approximately 60 mM or less, approximately 50 mM or less, approximately 40 mM or less, approximately 30 mM or less, approximately 20 mM or less, approximately 10 mM or less, or approximately 1 mM or less. Polyols may be present in pharmaceutical formulations at any concentration within the range defined by any of the endpoints described above. For example, polyols can be present in pharmaceutical formulations at concentrations such as approximately 0.1 mM to 1000 mM, approximately 0.1 mM to 500 mM, approximately 0.1 mM to 100 mM, approximately 1 mM to 1000 mM, approximately 1 mM to 500 mM, approximately 1 mM to 100 mM, approximately 100 mM to 1000 mM, and approximately 100 mM to 500 mM.

[0064] In other embodiments, polyols 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)). Polyols may be present in the pharmaceutical formulation at a percentage concentration of about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, or about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, or about 45% or more. Alternatively, or in addition, polyols may be present in the pharmaceutical preparation at a percentage concentration of approximately 50% or less, approximately 45% or less, approximately 40% or less, approximately 35% or less, approximately 30% or less, approximately 25% or less, approximately 20% or less, approximately 15% or less, approximately 10% or less, approximately 5% or less, approximately 4% or less, approximately 3% or less, approximately 2% or less, or approximately 1% or less. Polyols may be present in the pharmaceutical preparation at any percentage concentration within the range defined by any of the endpoints described above. For example, polyols can be present in pharmaceutical preparations at percentage concentrations such as approximately 0.1% to 50%, approximately 1% to 50%, approximately 5% to 50%, approximately 10% to 50%, approximately 15% to 50%, approximately 0.1% to 25%, approximately 1% to 25%, approximately 5% to 25%, approximately 10% to 25%, approximately 15% to 25%, approximately 0.1% to 15%, approximately 1% to 15%, approximately 5% to 15%, approximately 10% to 15%, approximately 0.1% to 10%, approximately 1% to 10%, approximately 5% to 10%, approximately 0.1% to 5%, and approximately 1% to 5%.

[0065] In one embodiment, the pharmaceutical formulation comprises the influenza virus of the present invention, about 0.5 M sucrose, about 0.1 M or about 0.5 M mannose, about 0.3 M or about 0.5 M trehalose, about 50% SPG, and about 0.05% polysorbate 20. In another embodiment, the pharmaceutical formulation comprises the influenza virus of the present invention, about 0.5 M sucrose, about 0.3 M trehalose, and about 0.05% polysorbate 20.

[0066] At least one pharmaceutically acceptable carrier or pharmaceutical additive may be a component that functions to bind the components of a pharmaceutical formulation (e.g., a binder). Binders may be, 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 pharmaceutical additive may be a component that increases the bulk of a pharmaceutical formulation (e.g., a bulking agent, diluent, and / or filler). Such bulking agents may be, but are not limited to, polysaccharides or their derivatives, sugars, and / or inorganic compounds. A pharmaceutically acceptable carrier or pharmaceutical additive may be a component that enhances the taste and / or appearance of a pharmaceutical formulation (e.g., a flavoring agent, a sweetener, and / or a coloring agent). A pharmaceutically acceptable carrier or pharmaceutical additive may be a component that makes a pharmaceutical formulation moisture-resistant by absorbing or adsorbing liquids or gases (e.g., an adsorbent). Adsorbents include, but are not limited to, starch, calcium phosphate, and / or colloidal silicon dioxide. Pharmaceutically acceptable carriers or pharmaceutical additives may be components that promote the dissolution of pharmaceutical formulations (e.g., disintegrants), such as starch, cellulose, and / or any other polymers known in the art, or derivatives thereof (e.g., cross-linked polyvinylpyrrolidone or sodium carboxymethylcellulose).

[0067] In some embodiments, pharmaceutically acceptable carriers or pharmaceutical additives are components (e.g., fluidizers) that reduce interparticle adhesion and / or optimize the flow of the product in and during the manufacture of a pharmaceutical formulation. Examples of fluidizers include, but are not limited to, talc, colloidal silicon dioxide, and corn starch. Pharmaceutically acceptable carriers or pharmaceutical additives may be components that provide non-stick properties, such as reducing adhesion between components, for example, punch faces or lubricants in and during the manufacture of a pharmaceutical formulation, particularly when the pharmaceutical formulation is formulated as an oral preparation. For example, an anti-adhesion agent may include magnesium stearate. In other embodiments, pharmaceutically acceptable carriers or pharmaceutical additives may be components that reduce the aggregation of components and / or reduce friction between the surface of a pharmaceutical formulation, i.e., a pharmaceutical formulation being formulated as an oral preparation, and the die wall during manufacturing (e.g., lubricants). Water-soluble or water-insoluble lubricants such as magnesium stearate, stearic acid, vegetable oil, mineral oil, polyethylene glycol, and / or sodium lauryl sulfate may be used in certain embodiments. Pharmaceutically acceptable carriers or pharmaceutical additives may be components that act as coating agents. Coating agents include, but are not limited to, gelatin and / or cellulosic coating agents (e.g., hydroxypropyl methylcellulose).

[0068] Other suitable binders, flavorings, sweeteners, colorants, disintegrants, fluidizers, anti-adhesion agents, lubricants, and coating agents are well known in the art and readily identifiable.

[0069] The pharmaceutical formulation may further include therapeutic agents (e.g., chemotherapeutic agents or anti-inflammatory agents). The pharmaceutical formulation may also include activators that induce an immune response independently of the influenza virus. Such additional components other than the influenza virus of the present invention may be present in any appropriate amount.

[0070] Additional components can be mixed with other components to form a pharmaceutical formulation prior to presentation to the immune system. Additional components can also be presented to the immune system separately from the pharmaceutical formulation. For example, additional components and the pharmaceutical formulation can be presented to the immune system separately (e.g., administered to an organism). When additional components and the pharmaceutical formulation are administered separately, they can be administered to the same site in the organism being immunized.

[0071] In one embodiment of the pharmaceutical formulation, the pharmaceutical formulation is a viral vaccine. The viral vaccine may be a live attenuated viral vaccine or an inactivated viral vaccine (e.g., a whole-virus vaccine, a split-virus vaccine, or a subunit vaccine). The viral vaccine may be formulated as a monovalent vaccine, a bivalent vaccine (e.g., H1H3, H1By, H1Bv, H3Bv, or BvBy), a trivalent vaccine (e.g., H1H3By, H1H3Bv, BvByH1, or BvByH3), or a tetravalent vaccine (e.g., H1H3ByBv) using multiple influenza virus backbone subtypes (i.e., different hemagglutinin subtypes and neuraminidase subtypes for influenza A, and either the Yamagata or Victoria lineage for influenza B). For example, the vaccine may comprise multiple embodiments of the recombinant virus of the present invention. In some embodiments, the vaccine may further comprise at least one recombinant virus different from the recombinant virus of the present invention.

[0072] Viral vaccines can be formulated into compositions for any suitable means of administration. For example, viral vaccines can be formulated as any composition suitable for intranasal or parenteral administration, such as intravenous, intramuscular, intradermal, or subcutaneous administration, including oral preparations (e.g., capsules, tablets, or oral films), sprays (e.g., intranasal sprays), or aqueous or non-aqueous emulsions, solutions, or suspensions.

[0073] Embodiments: (1) A recombinant virus comprising an influenza virus backbone, wherein the influenza virus backbone comprises gene segments PB1, PB2, PA, NP, M, NS, HA, and NA, and at least one of the gene segments PB1, PB2, PA, NP, M, NS, HA, and NA comprises at least one nucleotide sequence encoding one or more antigens, and (a) the PB1 gene segment encodes a PB1 protein having an amino acid sequence comprising a selected amino acid, wherein the selected amino acid comprises leucine at position 40 and tryptophan at position 180, and at least one of asparagine at position 464, isoleucine at position 563, or serine at position 607, and the PB1 gene segment optionally comprises a promoter mutation from cytosine to uracil at nucleotide position 4, and (b) the PB2 gene segment encodes a PB2 protein having an amino acid sequence comprising a selected amino acid, wherein the selected amino acid is at position 504 (c) a recombinant virus comprising valine, and optionally isoleucine at position 467 and valine at position 529, and the PB2 gene segment optionally comprising a promoter mutation from cytosine to uracil at nucleotide position 4, (d) a PA gene segment encoding a PA protein having an amino acid sequence comprising a selected amino acid, wherein the selected amino acid comprises lysine at position 401, and the PA gene segment optionally comprising a promoter mutation from cytosine to uracil at nucleotide position 4, (d) an NP gene segment encoding an NP protein having an amino acid sequence comprising a selected amino acid, wherein the selected amino acid comprises leucine at position 116 and at least one of lysine at position 294 or arginine at position 311, and (e) an NS gene segment encoding an NS1 protein having an amino acid sequence comprising a selected amino acid, wherein the selected amino acid comprises proline at position 30, lysine at position 55, and lysine at position 118.

[0074] (2) The recombinant virus of Embodiment 1, wherein the antigen is an immunogenic fragment of the SARS-CoV-2 spike glycoprotein.

[0075] (3) The recombinant virus of Embodiment 1 or 2, wherein the M gene segment comprises at least one nucleotide sequence encoding an antigen, where the antigen is an immunogenic fragment of the SARS-CoV-2 spike glycoprotein.

[0076] (4) A recombinant virus of any of Embodiments 1 to 3, wherein the M gene segment encodes a mutated M2 protein.

[0077] (5) A recombinant virus of Embodiment 4, wherein the M gene segment encodes a protein comprising at least one linker protein and a FLAG epitope tag.

[0078] (6) A recombinant virus according to any one of embodiments 1 to 5, wherein the M segment encodes a protein comprising any one of sequence numbers 1 to 14 and 92 to 96.

[0079] (7) Recombinant virus comprising an influenza virus backbone, wherein the influenza virus backbone comprises gene segments PB1, PB2, PA, NP, M, NS, HA, and NA, each comprising at least one nucleotide sequence encoding one or more antigens, wherein (a) the PA gene segment comprises thymine at nucleotide position 2272, (b) the NP gene segment encodes an NP protein having an amino acid sequence comprising a selected amino acid, wherein the selected amino acid comprises serine at position 40, asparagine or glycine at position 161, threonine at position 204, and optionally valine at position 93, and (c) the NS gene segment comprises guanine at nucleotide position 39, wherein the NS gene segment encodes an NS protein having an amino acid sequence comprising a selected amino acid, wherein the selected amino acid comprises glutamine at position 176.

[0080] (8) The recombinant virus of Embodiment 7, wherein the antigen is an immunogenic fragment of the SARSARS-CoV-2 spike glycoprotein.

[0081] (9) The recombinant virus of Embodiment 7 or 8, wherein the M gene segment comprises at least one nucleotide sequence encoding an antigen, where the antigen is an immunogenic fragment of the SARS-CoV-2 spike glycoprotein and further encodes a mutated BM2 protein.

[0082] (10) A recombinant virus according to any one of embodiments 1 to 9, wherein the NS gene segment comprises at least one nucleotide sequence encoding one or more antigens.

[0083] (11) A recombinant virus according to any one of Embodiments 1 to 10, wherein the antigen is an immunogenic fragment of the SARS-CoV-2 spike glycoprotein.

[0084] (12) A recombinant virus of any one of Embodiments 1 to 11, wherein the NS gene segment encodes (1) an NS1 protein, (2) at least one flexible linker protein, (3) an immunogenic fragment of the SARS-CoV-2 spike glycoprotein, (4) at least one cleavable sequence, and (5) a NEP protein.

[0085] A recombinant virus of Embodiment 12, wherein at least one cleavable sequence is a T2A peptide sequence or a P2A peptide sequence.

[0086] (14) A recombinant virus of any one of embodiments 1 to 13, wherein the NS gene segment encodes a protein comprising any one of sequence numbers 97 to 104.

[0087] (15) A recombinant virus of Embodiment 1 or 7, wherein each of the gene segments M and NS comprises at least one nucleotide sequence encoding one or more antigens, and the antigen is an immunogenic fragment of the SARS-CoV-2 spike glycoprotein.

[0088] (16) A recombinant virus of Embodiment 1 or 7, wherein each of the gene segments NA and NS comprises at least one nucleotide sequence encoding one or more antigens, and the antigen is an immunogenic fragment of the SARS-CoV-2 glycoprotein.

[0089] (17) A recombinant virus of Embodiment 1 or 7, wherein each of the gene segments M and NA comprises at least one nucleotide sequence encoding one or more antigens, and the antigen is an immunogenic fragment of the SARS-CoV-2 glycoprotein.

[0090] (18) A recombinant virus of Embodiment 1 or 7, wherein each of the gene segments M and HA comprises at least one nucleotide sequence encoding one or more antigens, and the antigen is an immunogenic fragment of the SARS-CoV-2 glycoprotein.

[0091] (19) A recombinant virus of Embodiment 1 or 7, wherein each of the gene segments NS and NA comprises at least one nucleotide sequence encoding one or more antigens, and the antigen is an immunogenic fragment of the SARS-CoV-2 glycoprotein.

[0092] (20) A recombinant virus of Embodiment 1 or 7, wherein each of the gene segments NS and HA comprises at least one nucleotide sequence encoding one or more antigens, and the antigen is an immunogenic fragment of the SARS-CoV-2 spike glycoprotein.

[0093] (21) A recombinant virus of any one of Embodiments 1 to 20, wherein the virus can replicate in human cells.

[0094] (22) Any one of the recombinant viruses from Embodiments 1 to 21 having enhanced growth compared to a recombinant virus that is identical to the recombinant virus except that it does not have selected amino acids in Vero cells under the same conditions.

[0095] (23) Any one of embodiments 1 to 22, wherein a gene segment comprising at least one nucleotide sequence encoding one or more antigens further comprises a downstream duplication, and the downstream duplication comprises at least one silent nucleotide mutation.

[0096] (24) A pharmaceutical formulation comprising any one recombinant virus from Embodiments 1 to 23.

[0097] (25) The pharmaceutical formulation of Embodiment 24, wherein the vaccine is formulated as a monovalent vaccine.

[0098] (26) The pharmaceutical formulation of Embodiment 24, wherein the vaccine is formulated as a bivalent vaccine.

[0099] (27) A pharmaceutical formulation of Embodiment 24, wherein the vaccine is formulated as a trivalent vaccine.

[0100] (28) The pharmaceutical formulation of Embodiment 24, wherein the vaccine is formulated as a quadrivalent vaccine.

[0101] (29) A method for inducing an immune response in a mammal, comprising administering to a mammal any one recombinant virus from Embodiments 1 to 23 or any one pharmaceutical formulation from Embodiments 24 to 28, thereby inducing an immune response to an antigen in the mammal.

[0102] (30) The method of Embodiment 29, wherein the mammal is a human. [Examples]

[0103] The following examples further illustrate the present invention, but it should be understood that they should not be construed as limiting the scope of the invention in any way.

[0104] Example 1 This example demonstrates a method used to select MHC I peptides for use in influenza vectors. The peptides were suitable for insertion into the genes M2, BM2, and NS.

[0105] Peptide antigens for vaccines were selected based on their ability to stimulate immune responses from the broadest possible number of MHC genotypes, thus providing benefits for the largest possible number of potent vaccines. This approach was adopted because of its high specificity for interactions between MHC class I molecules on the cell surface and homologous antigen peptides that bind and are presented to immune effector T cells. Antigen peptides with higher specific MHC I affinity induce a stronger immune response upon vaccination. Several models were developed that can predict the affinity of any peptide sequence to a given MHC class I molecule. This interaction is also allele-specific among the many known MHC class I genotypes recognized worldwide, within individuals from different genetic backgrounds. Therefore, any single peptide will have different affinities to MHC class I receptors, depending on the individual MHC I genotype.

[0106] The S1 or spike surface glycoprotein of SARS-CoV-2 coronavirus was used as the target antigen protein for identifying the best peptides. The amino acid sequence of the S1 protein (SEQ ID NO: 77) was predicted from the complete genome sequence of the severe acute respiratory syndrome coronavirus type 2 isolate Wuhan-Hu-1 (Genbank NC_045512.2) using a standard codon usage table. Using the primary S1 protein sequence, the best MHC class I compatible 9 MEPTED peptides were identified by predicting peptide affinity across a 27-member human MHC class I allele panel. Peptide predictions were grouped together across all predictors and ranked by their predicted consensus percentile rank, with those with a percentile rank of 1 or less being selected. Cluster analysis was applied to these peptides using known methods (see, e.g., Dhanda et al., Front. Immunol., 9:1369 (2018)) to identify epitope clusters predicted to have high affinity for many MHC class I molecules due to high genetic diversity. The top-scoring peptides were ranked using a two-step process. First, peptides were ranked by their clustering degree to locate peptide affinity "smears," which are regions of high predicted affinity where multiple 9-mers are tiled (i.e., peptides are aligned and partially overlapping). This method allows for the identification of peptide smears longer than 9 residues, which can be targeted for inclusion in vaccines. The number of times a given peptide was scored in the top 1% was compiled into a table, and the cumulative hit count and median epitope rank were scored to select top peptide smears predicted to bind MHC I in human subjects with high genetic diversity. This method is described in more detail below.

[0107] Epitope prediction using MHC-I activity Using TepiTool from the Immune Epitope Database and Analysis Resource (IEDB), we extracted MHC I-related epitope predictions from the amino acid sequence of the SARS-CoV-2 spike protein. Epitopes for human MHC I alleles were predicted using a 27-allele panel, resulting in epitope sizes ranging from 8 to 11 mars. Duplicate peptides were removed, and the IEDB-recommended prediction method was used. Peptides with predicted consensus percentile ranks of 1 or less were selected, generating 647 epitopes and 136,566 overall element sets (tuples) (epitope, allele, predictor, rank) that met the rank criteria.

[0108] Clustering of epitopes into smears Using the IEDB's epitope cluster analysis tool, already selected epitopes were clustered into clusters of related epitopes, referred herein as “epitope smears.” A minimum sequence identity threshold of 70% was selected, and no size filters were placed on the epitope list. Predicted epitopes were clustered into smears, with consensus percentile ranks of 1 or less. A cluster-breaking clustering algorithm was used to output clusters with epitope alignment to a comma-separated values ​​(CSV) file format. In this case, clusters were effectively ordered by reducing connectivity. Using this method, clusters were ranked in the order in which they were selected in lexicographically ordered clusters-subclusters.

[0109] Smear analysis Using Python scripts, clusters were incorporated, normalized, and then aligned to an open reading frame of the spike S1 protein obtained from the GenBank RefSeq genome NC045512.2. Various qualitative statistical analyses were performed on hit distribution, sequence length, etc., to obtain smear selection. Hit count (number of epitopes per cluster) and rank median (median consensus percentile rank for each epitope in the cluster) were added as columns. Total hit count was a useful indicator for selecting top candidate smear sequences. Only small subsets of peptide smears were found to contain more than 10 hits per cluster (Figure 11). By setting an arbitrary cutoff of 9 hits per cluster, only 8 total smears were identified within the SARS-CoV-2 S1 protein with a length of 1273 amino acids.

[0110] The results from the statistical analysis were edited and re-exported to CSV format for visualization. The top candidate smears were compared with each other and manually selected. In three cases, the candidate smears overlapped or were nearly adjacent, allowing them to join to a superconsensus smear sequence spanning two candidate sequences. The eight selected candidate smears are shown in Table 1.

[0111] [Table 1]

[0112] Designing the optimal spacer Epitopes were aggregated into concatemers by extension from a published algorithm already implemented in Fred2 (Schubert et al., Bioinformatics, 32(13):1367-4803, 2044(2016)) (Schubert et al., Genome Medicine, 8(1):9(2016)). Both spacer and epitope ordering are optimized to minimize neoepitope formation and maximize the probability of MHC processing cleavage in the spacer region. Theoretically, manipulating carefully selected entries in the cost matrix to positive or negative infinity could provide optimized spacers, but the ILP solver (CBC) used fails to solve problems with infinite coefficients in the target. In this novel method, the k-mer spacer was optimized for kmax={3,6}. To appropriately optimize cleavage and neoepitope formation at both ends of epitope chain inserts linked at the C-terminus or N-terminus by existing amino acid strings, four partial modifications were designed to improve upon conventional methods. 1. The C-terminal / N-terminal binding string is attached to the peptide set in the Traveling Salesman model (formulated as ILP in Miller-Tucker-Zemlin form), 2. Modify the spacer optimization to generate spacers for the boundary-defining sequences at the precise ends of all epitope peptides. 3. Add constraints to this model to enhance the precise ordering of the sequences that define the epitopes and boundaries. 4. The target object was modified, and missing entries in the TSP cost matrix introduced by the boundary-defining sequence were ignored.

[0113] [Table 2-1]

[0114] [Table 2-2]

[0115] [Table 2-3]

[0116] [Table 2-4]

[0117] [Table 3-1]

[0118] [Table 3-2]

[0119] Example 2 This example demonstrates the successful expression of the SARS-CoV-2 receptor-binding domain (RBD) antigen derived from an influenza A M2SR vector.

[0120] To express the SARS-CoV-2 RBD antigen from an influenza A M2SR vector, an engineered NS segment 8 was synthetically constructed (Figure 1). The designed gene was then inserted into an RNA PolI vector for expression as a negative sense vRNA. Segment 8 was designed to express three major open reading frames (ORFs): firstly, a single fusion polypeptide of the complete influenza A PR / 8 / 1934 NS1 protein, a flexible GSG linker, amino acids 331-530 of the SARS-CoV-2 Wuhan-Hu-1 spike S1 protein, another GSG linker, and the PR8 nuclear export protein (NEP or NS2) ORF. The NS1-RBD fusion protein was isolated from NEP by a P2A peptide derived from porcine teesshouvirus type 1 2A. During translation, the P2A site enables the expression of the downstream NEP protein as a separate polypeptide, through an unknown mechanism thought to involve ribosomal slippage. For example, the NEP protein can be represented by Sequence ID No. 108. Therefore, the required function of NEP was maintained, but the functionality of NS1 is considered conserved, as the entire NS1 ORF was also maintained. The artificial segment 8 may be unstable for at least two reasons. Two changes were made to improve sequence stability. Removing splicing would have required replication of parts of the segment encoding NEP exon 1 and part of exon 2 (see Figures 21 and 22). Therefore, several silent mutations were introduced throughout both the NS1 and NEP ORFs to reduce the homology between these replications. In addition, both the GSG and P2A sequences were optimized to reflect the AT-rich codon bias of influenza. The SARS-CoV-2 sequence could not be altered because it already had over 60% AT.

[0121] The NS1-RBD fusion may be unable to perform NS1 function, or its function may be impaired. In that case, the recombinant virus may lack the ability to suppress the innate response mediated by both interferon and RIG-I by altering mRNA polyadenylation and splicing. To address this possibility, we constructed an alternative construct with a cleavage site derived from teseasignavirus 2A (T2A) between NS1 and RBD (Figure 2). This design was intended to allow the expression of three distinct polypeptides: NS1, RBD, and NEP.

[0122] A vector encoding a novel CoV2 NS segment was used in a standard plasmid-based influenza virus reverse genetics procedure to rescue M2-deficient single-replicating (M2SR) viruses possessing the SARS-CoV-2 RBD segment 8. Both viruses were successfully obtained using the HA and NA segments from the WHO-recommended vaccine strain A / Singapore / INFIMH-16-0019 / 2016IVR-186(H3N2). The viruses were recovered using M2VeroA cells engineered to constitutively express the M2 protein (SEQ ID NOs: 1, 15, 17) lost from M2SR grown in animal-free (AOF) medium. This virus rescue and culture system was suitable for preparing viral seeds for cGMP production of M2SR vaccine candidates intended for testing in human clinical trials.

[0123] Expression of the NS1 SARS-CoV-2 fusion construct was examined by infecting Vero cells with the CoV2 NS1 M2SR virus strain at a high MOI (greater than 1.0). Both virus-free mocks and Singapore 2016 M2SR without RBD insert infection were performed. Eleven hours after inoculation, cells were collected for immunoblot analysis of whole cell lysates. The results showed that antiserum against SARS RBD bound proteins of the expected size. The band was detected only in extracts of cells infected with the RBD virus, but not in the control (Figure 3).

[0124] To confirm the expression of the NS1 SARS-CoV-2 fusion construct, the same Vero cells were infected with a high MOI and fixed in formalin for immunofluorescence staining. The cells were incubated with antiserum against SARS RBD. After washing, the cells were stained with secondarily labeled anti-rabbit fluorescein isothiocyanate (FITC) and anti-influenza A NP antibody directly labeled with ALEXA FLOUR™ 647. The images shown in Figure 4 show that cells infected with both CoV-2 NS1 M2SR and standard M2SR express detectable levels of influenza A NP protein. On the other hand, FITC labeling of RBD could only be detected in cells infected with CoV-2 NS1 M2SR, which produced significant detectable fluorescence. This staining indicates that the NS1-RBD fusion protein is cytoplasmic.

[0125] Example 3 This example demonstrates the successful expression of SARS-CoV-2 RBD from an influenza B BM2SR vector.

[0126] To express the SARS-CoV-2 RBD antigen from an influenza B BM2SR vector, an engineered influenza BM2-deficient segment 7s was synthetically constructed (Figures 5-6, SEQ ID NOs. 83, 84). The designed gene segment was then inserted into an RNA PolI vector for expression as a negative sense vRNA. Segment 7s was designed to express two polypeptide ORFs from a single viral mRNA: firstly, the complete influenza B / Florida / 4 / 2006M1 protein, the 5-Mar ribosome termination-start slippage site, and secondly, a BM2 fusion protein to amino acids 330-524 of the SARS-CoV-2 Wuhan-Hu-1 spike S1 protein. The naturally occurring 5-base (5-mer) sequence motif TT(bold)A(italic bold)TG(italic) (SEQ ID NO: 20) between BM1 and BM2 contains both the BM1 ORF translation termination codon (bold) and the BM2 ORF start codon (italic). Translational ribosome slippage and restart allow for viral expression of BM2 in the second leading frame without the need for splicing, in contrast to influenza A segment 7. In synthetic SARS-CoV-2 RBDs containing influenza B segment 7, a small portion of the BM2 ORF was fused to the S1 RBD (SEQ ID NOs: 95, 96).

[0127] Artificial influenza segments, which lead to poor viral growth in unsuitable cultures for manufacturing, can be unstable. This is due to at least two reasons. Firstly, the expression of essential activity, in this case the M1 matrix protein, may be affected. To maintain sequence stability, a local RNA structure of approximately 5 memars was maintained so as not to affect M1 translation. Therefore, the amino acids at the amino terminus of the BM2 ORF were fused with the SARS-CoV-2 RBD. Segments can be lost due to low packaging efficiency. Since all ends of an influenza genome segment are self-complementary, they can form pairs via hybridization, and both untranslated and translated sequences within 100 bp from both ends of the segment initiate the formation of a complex tertiary structure. Preservation of the exact UTR of the segment was of paramount importance. In vaccine segment 7, the coronavirus sequence was fused to the 3' UTR of influenza B (mRNA sense). This UTR was longer than the 85 bp UTR of influenza A. Two versions were constructed. A more conserved version encodes the insertion of RBD into BM2, thereby conserving longer chains from both ends of the BM2 ORF (Figure 5, SEQ ID NO: 84). The longer versions each possess 10 and 13 terminal amino acids of BM2, respectively (Figure 6, SEQ ID NO: 96).

[0128] The more trimmed version contains only the N-terminal 9bp, 3 residues of BM2, which is directly fused to the RBD (SEQ ID NO: 95) and then to the UTR segment (Figure 6, SEQ ID NO: 83). The SARS-CoV-2 sequence was examined to see if it reflects the AT-rich codon bias of influenza. The SARS-CoV-2 sequence could not be altered as it was already approximately 60% AT.

[0129] Vectors encoding two SARS-CoV-2M segments (SEQ ID NOs: 83, 84) were used in a standard plasmid-based influenza virus reverse genetics procedure to rescue BM2-deficient single-replicating (BM2SR) viruses containing SARS-CoV-2 RBD BM2SR segment 7. Both viruses were successfully obtained using the HA and NA segments from the WHO recommended vaccine strain B / CA / 12 / 2015(YL). The viruses were recovered using BM2Vero cells engineered to constitutively express the BM2 protein lost from BM2SR viruses grown in animal-free (AOF) medium (SEQ ID NOs: 2, 16, 18). This virus rescue and culture system was suitable for preparing viral seeds for cGMP production of BM2SR vaccine candidates intended for testing in human clinical trials.

[0130] Expression of the SARS-CoV-2 BM2 fusion protein construct (SEQ ID NOs. 95, 96) was examined by infecting Vero cells with the CoV-2 BM2SR virus strain at a high MOI (greater than 1.0). Both virus-free mocks and vectors containing only CA12 BM2SR without RBD insert infection were performed. Eleven hours after inoculation, cells were collected for immunoblotting analysis of whole cell lysates. The results showed that antiserum against SARS RBD bound the protein to the expected size. This band was detected only in extracts of cells infected with the RBD virus and not in control extracts (Figure 7). These results suggest that the minimum 22 kDa RBD construct is expressed at higher levels than the longer 24 kDa version.

[0131] Example 4 This example demonstrates that the viruses M2SR and BM2SR, which encode sequences derived from SARS-CoV-2, were attenuated in vivo.

[0132] Seven-week-old BALB / c female mice were intranasally immunized with the following viral constructs shown in Table 4. The sequences of the backbone sequences M2SR and BM2SR, as well as the segment encoding the SARS-CoV-2 sequence, are described in SEQ ID NOs. 43-47, 56, 58, 60, 63-67, 73, 80, 83, 95, 97, and 107. These viruses were administered to 1 × 10⁶ mice per mouse. 6 TCID 50 The drug was administered at the specified dose. Control mice were given DPBS (pH 7.2) containing 10% sucrose and 5 mM sodium glutamate (SPGNa). For 14 days after immunization, the mice were observed for any changes in body weight and symptoms of infection.

[0133] [Table 4]

[0134] No clinical symptoms of infection or weight loss were observed over a 14-day period in mice immunized with the M2SR or BM2SR mutants or SPG controls. Figure 8A shows the percentage change in post-immunization weight of mice for the M2SR recombinant virus, and Figure 8B shows the percentage change for the BM2SR recombinant virus. Furthermore, the change in weight was similar between the groups over the 14-day period. These results indicate that the M2SR and BM2SR viruses, which contain the SARS-CoV-2 sequence, were attenuated and not pathogenic in mice.

[0135] Example 5 This example demonstrates that the viruses M2SR and BM2SR from Example 4 induce an antibody response against SARS-CoV-2.

[0136] Serum was collected from mice before the initial immunostimulation and approximately 3 weeks after the first dose. Anti-spike RBD serum IgG antibody titers from pooled serum samples for each group were determined by enzyme-linked immunosorbent assay (ELISA).

[0137] ELISA was performed using C-terminal HIS-tagged soluble SARS-CoV-2 recombinant RBD protein expressed in 293T cells and purified using COMPLETE® His-Tag Purification resin (F. Hoffmann-La Roche AG, Basel, Switzerland). ELISA plates were coated overnight at 4°C with 100 μL of RBD protein at a concentration of 2 μg / mL in phosphate-buffered saline (PBS). After blocking the plates with PBS containing 0.1% polysorbate 20 (PBS-T) and 1% gelatin derived from cold-water fish skin, the plates were replicated and incubated with mouse serum diluted in PBS-T containing 1% gelatin derived from cold-water fish skin. After incubation at room temperature for 2 hours, the plates were washed six times with PBS-T and then incubated with anti-mouse IgG secondary antibody (KPL, diluted 1:2,000 in PBS-T containing 1% gelatin derived from cold-water fish skin) conjugated with horseradish peroxidase. After incubation with the secondary antibody for 1 hour, the plates were washed six times with PBS-T and then developed with 1-STEP® Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific, Waltham, MA). After incubation for 10 minutes, the reaction was stopped by adding 4N sulfuric acid. Absorbance was measured at a wavelength of 450 nm (OD 450 The titer was measured using ). The endpoint titer was defined as the reciprocal of the dilution that exceeded the cutoff value determined by subtracting the mean multiplied by the standard deviation of the blank plus 6.

[0138] Figure 9 shows the multiplier increase in ELISA titer from baseline before immunity. Empty vectors that did not encode the spike RBD sequence did not show an increase in ELISA titer at day 21. Viruses M2SR and BM2SR, which encoded the SARS-CoV-2 sequence, induced an increase in RBD spike ELISA titer.

[0139] Example 6 This example demonstrates that systemic antibodies are generated after a second dose of the vaccine used as the initial stimulating protein or spike protein.

[0140] The four viral administration treatment plans in Example 4 were: (1) mice initially stimulated with one of the M2SR-COVID-19 vaccine candidate (i.e., AM2SR-CovidS-1) or M2SR vector virus (i.e., M2SR-Sing V5), and then additionally stimulated nasally with the same (i.e., AM2SR-CovidS-1 or M2SR-Sing V5) approximately 4 weeks after the initial stimulation; and (2) mice initially stimulated with the M2SR-COVID-19 vaccine candidate (i.e., AM2SR-CovidS-1) or M2SR vector virus (i.e., M2SR-Sing V5). We evaluated mice that were initially stimulated intranasally with one of the following (V5) and then additionally stimulated intramuscularly with purified SARS-CoV-2 protein approximately 4 weeks after the initial stimulation; (3) mice that were initially stimulated with one of the following (BM2SR-COVID-19 vaccine candidates (i.e., BM2SR-CovidS-1) or BM2SR vector virus (i.e., BM2SR-CA12) and then additionally stimulated intranasally with the same approximately 4 weeks after the initial stimulation; and (4) mice that were initially stimulated intranasally with one of the following (BM2SR-COVID-19 vaccine candidates (i.e., BM2SR-CovidS-1) or BM2SR vector virus (i.e., BM2SR-CA12) and then additionally stimulated intramuscularly with purified SARS-CoV-2 protein approximately 4 weeks after the initial stimulation. All mice were induced to bleed in the terminal stage approximately 3 weeks after secondary immunity (additional stimulation), then euthanized, and serum samples were collected for analysis.

[0141] The serum sample was analyzed by ELISA as described above for Example 5.

[0142] Figure 28 shows the anti-SARS-CoV-2 RBD IgG titers. Two administrations of an empty vector that does not encode spike RBD sequences (i.e., M25R-Sing V5 and BM2SR-CA12) (initial stimulation - additional stimulation) did not induce an increase in RBD spike ELISA titers. Two administrations of M2SR and BM2SR viruses encoding SARS-CoV-2 sequences (i.e., AM2SR-CovidS-1 and BM2SR-CovidS-1) (initial stimulation - additional stimulation) increased RBD spike ELISA titers. Initial stimulation with M2SR and BM2SR viruses encoding SARS-CoV-2 sequences (i.e., AM2SR-CovidS-1 and BM2SR-CovidS-1) substantially increased the ELISA titer of the RBD spike when further stimulated with purified SARS-CoV-2 protein, whereas initial stimulation with empty M2SR vector viruses did not induce a substantial increase in the ELISA titer of the RBD spike when further stimulated with purified SARS-CoV-2.

[0143] Example 7 This example demonstrates that the vectors M2SR and BM2SR can be used in multivalent formulations and that each can possess immunogenicity.

[0144] Influenza A H1N1 or H3N2 FGHY1-M2SR, BM2SR-Vic, or BM2SR-Yam viruses induce an antibody response when formulated as monovalent, bivalent, trivalent, or tetravalent vaccines.

[0145] Seven-week-old BALB / c female mice (N=8) were intranasally immunized with the following vaccines: monovalent H1N1 FGHY1-M2SR, monovalent H3N2 FGHY1-M2SR, bivalent H1N1 and H3N2 FGHY1-M2MR, monovalent BM2SR-Victoria, monovalent BM2SR-Yamagata, bivalent BM2SR, trivalent H1N1 and H3N2 FGHY1-M2SR and BM2SRVictoria or Yamagata, or tetravalent H1N1 and H3N2 FGHY1-M2SR and BM2SRVictoria or Yamagata. A control group of mice was mock-immunized with SPG. On day 28 after vaccination, the mice were intranasally immunized with an additional stimulus vaccine consisting of the same vaccine administered for initial stimulus immunity. Serum samples were collected on days 7, 14, and 21 after the initial immune stimulation, and on days 35, 42, and 49 after additional immune stimulation (day 28). Serum IgG antibody titers for anti-H1 HA, anti-H3 HA, anti-influenza B-Vic HA, and anti-influenza B-Yam HA derived from the serum samples were determined by ELISA.

[0146] The resulting anti-H1 HA data is shown in Figure 10A. The resulting anti-H3 HA data is shown in Figure 10B. The resulting anti-influenza B-Vic HA data is shown in Figure 10C. The resulting anti-influenza B-Yam HA data is shown in Figure 10D. These results demonstrate that all vaccines were able to increase anti-influenza virus antibodies more than the SPG control, and that these increases were similar across multiple vaccine formulations. Furthermore, these results demonstrate that monovalent components retain their ability to induce an immune response to monovalent components when formulated into a multivalent vaccine.

[0147] Example 8 This example is expected to demonstrate that the M2SR-SARS-CoV-2 vaccine induces an elevated in vivo antibody response upon re-administration, which is non-toxic to the host.

[0148] To demonstrate that the M2SR-SARS-CoV-2 vaccine virus induces an immune response against the components without causing toxicity to the host, 15 male and 15 female ferrets will be intranasally immunized with the M2SR-SARS-CoV-2 vaccine at dose levels of 1×10 8 TCID 50 (low dose) or 1×10 9 TCID 50 (high dose). A third group of ferrets will be intranasally mock-immunized with SPG as a placebo control. A three-dose vaccination treatment schedule will be utilized for each treatment group. The ferrets will be administered a primary immunization (day 1 of the study) and two additional booster immunizations 13 and 27 days later (day 14 and day 28 of the study). After each immunization, the ferrets will be observed for 7 days for death, and body weight, body temperature, and clinical signs will be measured daily. Blood will be collected from all surviving ferrets before the study and on days 14, 16, 30, and 49 of the study to evaluate clinical pathological properties. Serum samples will be collected before the study and on days 14, 30, and 49 of the study and the antibody levels will be evaluated over time by ELISA, hemagglutination inhibition (HAI) assay, and virus neutralization (VN) assay. Necropsies will be performed on 5 male and 5 female per group on days 3, 30, and 49 of the study, including examination of the outer surface of the body, all openings, the cranial cavity, thoracic cavity and peritoneal cavity, and their contents.

[0149] Vaccine virus immunization. Ferrets will be intranasally immunized with three administrations of either the M2SR-SARS-CoV-2 vaccine at a dose of 1×10 8 TCID 50 or 1×10 9 TCID 50 . Vials of frozen vaccine virus stock were thawed at room temperature for at least 10 minutes and then stored refrigerated or on wet ice until use. Ferrets were anesthetized with ketamine / xylazine and the virus dose was administered intranasally in an amount of 500 μL (250 μL per nostril).

[0150] The M2SR-SARS-CoV-2 vaccine virus is a recombinant influenza A virus that does not express the functional M2 protein and encodes the HA and NA genes of influenza A / Singapore / INFIMH-16-0019 / 2016(H3N2), as well as the SARS-CoV-2 spike protein RBD.

[0151] Experimental Design: This study will utilize 90 ferrets (Triple F Farms, Sayre, PA) consisting of 45 males and 45 females, aged 16–22 weeks at the start of the study. All animal procedures will be performed in an animal biosafety level 2 facility according to protocols approved by the IIT Research Institute Animal Experimentation Committee. Prior to immunization, the ferrets will be monitored for 4 days to establish baseline body temperature. Temperature readings will be recorded daily in each ferret via a subcutaneously implanted transponder (BioMedic data systems, Seaford, DE). After blood collection, the study will begin, and serum will be tested for influenza antibodies. Pre-immunization serum samples will be treated with receptor-destroying enzymes (RDE) (Denka Seiken, Tokyo, Japan) to remove nonspecific inhibitors, then serially diluted and tested for specified 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 titers will be defined by the minimum serum dilution that produces inhibition of hemagglutination. Only ferrets with HAI titers less than 40 will be considered serologically negative and used in this study. Test animals will be randomized and divided into three groups (15 male ferrets and 15 female ferrets per group).

[0152] To evaluate the efficacy and toxicity of the vaccine, ferrets were subjected to 1 × 10⁶ M2SR-SARS-CoV-2. 8 TCID 50 Three doses or 1 x 10 9 TCID 50 Three doses of SPG will result in intranasal immunization on days 1, 14, and 28 of the study. The control group will be intranasal mock-immunized with SPG on days 1, 14, and 28 of the study. The ferrets' body temperature, weight, and clinical symptoms will be monitored daily for 7 days after immunization. Blood will be collected from all surviving ferrets 5 days prior to the study, 14, 16, 30, and 49 to assess their clinicopathological characteristics. Serum samples will be collected on days 5, 14, 30, and 49 of the study and frozen at approximately -70°C until antibody titer measurement by ELISA, viral neutralization assay, and HAI assay. All test animals will be euthanized and necropped on the planned day (day 3, 30, or 49, 5 males and 5 females per group). The necrop will consist of examination of the external surface of the body, all openings, and the cranial, thoracic, and peritoneal cavities, as well as their contents. The tissue will be collected, fixed, and histopathologically evaluated by a committee-certified animal pathologist.

[0153] Endangered / Death and Clinical Findings: All ferrets are expected to survive until the planned slaughter date. All ferrets are expected to have an activity level score of "0" (agile and playful) at all time points measured between day 1 and day 49.

[0154] Weight and weight change: No differences are expected to be observed.

[0155] Body temperature: No difference is expected to be observed.

[0156] Enzyme-linked immunosorbent assay (ELISA): Anti-HA IgG antibody titers derived from serum samples will be determined by ELISA. ELISA plates will be coated with recombinant HA protein derived from type A / Singapore / INFIMH-16-0019 / 2016(H3N2) (Immune Technology Corp., New York, NY) or spike RBD, blocked with skim milk, and then the samples will be applied. Ferret IgG antibodies were detected using horseradish peroxidase-labeled anti-ferret IgG goat antibody (SeraCare Life Sciences, Milford, MA) and 1-STEP® Ultra TMB-ELISA (Thermo Fisher Scientific Inc.) substrates.

[0157] Ferrets in each of the immunized groups are expected to show a significant increase in serum anti-H3 HA antibodies, but antibody levels in animals that received SPG alone are not expected to change from baseline. Anti-H3 HA antibody titers will likely be higher in the immunized groups than in the SPG control group two weeks after the initial stimulation dose. The mean antibody titer per immunized group will likely increase further after the first and second doses of the vaccine.

[0158] Hemagglutination Inhibition (HAI) Assay: Serum samples will be analyzed by an HAI assay to demonstrate the functional activity of antibodies detected by ELISA. Serum samples will be treated with RDE to remove nonspecific hemagglutination inhibitors. The RDE will be reconstituted according to the manufacturer's instructions. Serum will be 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 will be incubated in a 56°C ± 2°C water bath for 30-5 minutes. A solution consisting of 0.85% NaCl will be added to each sample to achieve a final serum dilution of 1:10 after RDE treatment. The samples will then be further diluted 2-fold in PBS (1:10 to 1:1,280) and incubated with 4 hemagglutination units of influenza A / Singapore / INFIMH-16-0019 / 2016(H3N2) virus. After incubation, 0.5% bird erythrocytes will be added to each sample and incubated for 30 ± 5 minutes. Then, the presence or absence of erythrocyte agglutination will be scored.

[0159] High dose (1×10 9 TCID 50 The ) group received a low dose (1 × 10 8 TCID 50 The SPG (control) group is expected to show higher HAI titers than the control group, and is not expected to induce any HAI titer. M2SR-SARS-CoV-2 immunized ferrets are expected to demonstrate an HAI titer equivalent to or higher than 80 for the test virus. The CDC has stated that a serum HAI antibody titer of 40 was associated with at least a 50% reduction in the risk of influenza infection or influenza illness in the population. Therefore, these results are expected to indicate that the M2SR-SARS-CoV-2 virus can induce a protective immune response.

[0160] Viral Neutralization Assay: Serum samples will be tested for influenza A / Singapore / INFIMH-16-0019 / 2016(H3N2) virus in a viral neutralization assay before the test and at the processing stages (days 3, 14, 30, and 49). Serum samples will be inactivated at 56°C for 30 minutes. The serum will then be 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 and virus mixture will then be transferred into individual wells of a 96-well plate containing monolayer MDCK cells. The plate (containing the samples) will then be incubated at 37±2°C and 5.0±1% CO2 for 18-22 hours. After incubation, cells will be fixed with paraformaldehyde and stained with a pool of anti-influenza A nucleoprotein monoclonal antibodies (1 part MAB8257:1 part MAB8258 (Millipore; Billerica, MA)), followed by peroxidase-conjugated goat anti-mouse IgG. Spots will be developed using TrueBlue peroxidase substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Plaques will be visualized and counted using an enzyme-conjugated immunosuppressant spot (ELISPOT®) instrument (AID GmbH, Strassberg, Germany). 50% plaque reduction neutralizing titer (PRNT) will be measured. 50 This would represent a 50% reduction in the input control viral plaque count based on back titration of control plaques, calculated by counting plaques and reporting the titer as the reciprocal of the final serum dilution.

[0161] Ferrets in the SPG group are expected to remain negative (titer ≤ 100) throughout the duration of this study. 1 × 10 8 TCID 50 Ferrets immunized with M2SR-SARS-CoV-2 at a dose of 1 × 10⁻¹⁰ are expected to have a high geometric mean titer (GMT). 9 TCID 50Ferrets immunized with M2SR-SARS-CoV-2 at a dose of 1 × 10⁻¹⁰ are expected to have a higher GMT VN titer. 9 TCID 50 All ferrets immunized with three doses of H3N2 M2SR-SARS-CoV-2 showed the highest PRNT (Progressive Research and Development) 50 It is expected to exhibit potency.

[0162] Clinicopathological Examination: Blood samples for clinicochemical, hematological, and coagulation parameter analysis will be 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 will be fasted for 4-6 hours prior to blood collection. Ethylenediaminetetraacetic acid (EDTA) will be used as an anticoagulant for hematological samples, while sodium citrate will be used for coagulation samples. Samples for clinicochemical examination will be collected without anticoagulants. Urine samples will be collected directly from the bladder of each ferret at necropsy.

[0163] No treatment-related or toxicologically significant findings are expected to be noteworthy for any clinical-chemical or hematological parameters evaluated during this study.

[0164] Gross autopsy and histopathological findings: Gross autopsies and histopathological examinations were performed on 5 males and 5 females per group on days 3, 30, and 49 of the study. 1 × 10 8 TCID 50 Intranasal immunization of M2SR-SARS-CoV-2 in ferrets at a dose of 1 × 10 is expected to result in no macroscopic findings. 9 TCID 50 At this dose, macroscopic findings are expected to be particularly noticeable in the lungs (pigmentation, dark or patchy), and microscopic findings are expected to be particularly noticeable in the lungs on days 3 and 30 (mixed cellular infiltration). After 3 weeks of recovery, no macroscopic lesions related to the test item are expected to be observed on day 49 of the study.

[0165] This embodiment is expected to demonstrate that intranasal immunization with the M2SR-SARS-COV-2 vaccine virus is not transmitted in vaccinated hosts and is not associated with any vaccine-related adverse events (e.g., elevated body temperature, weight loss, or clinical signs). These results are expected to indicate that the M2SR-SARS-COV-2 virus is useful as an intranasal coronavirus vaccine, as it induces a single-dose protective immune response against homologous viruses that can be further elevated with re-administration.

[0166] Example 9 This example demonstrates the successful design and generation of several M2SR virus strains expressing various antigens derived from the SARS-CoV-2 spike protein.

[0167] Influenza A NS segment 8, which encodes two non-structural proteins NS1 and NEP (nuclear export protein), can be modified to use influenza A as a vaccine vector for antigen expression. While NEP is required, the NS1 ORF may be absent for viral replication. NS1 cleavage can be isolated by repeated passaging in Vero cell culture, and even complete NS1 deletion strains can be constructed. NS1 plays a crucial role in promoting influenza infection, including variations in splicing and blocking of the host cell's innate response. NS1 mutations reduce viral titers, making production difficult, and, more importantly, impair viral replication in primary cells and in vivo. A second major obstacle to vectorizing NS segments is the expression of the essential NEP protein from the spliced ​​form of segment 8 mRNA. Splicing involves annexing a short exon 1 sequence to exon 2 in an alternative translational reading frame that overlaps with the NS1 ORF encoded by the unspliced ​​mRNA.

[0168] To encode the antigen, two key changes are made to the NS segment. First, the splice donor (SEQ ID NO: 109) and acceptor sites are eliminated. Next, sequences encoding NEP exon 1 and exon 2 are joined to construct an intron-free NEP ORF. The result is an ORF encoding a single polypeptide, where the C-terminus of NS1 is fused to the NEP ORF separated by the NS1 P2A peptide derived from porcine teesshou virus type 1 2A and the GSG flexible linker (SEQ ID NOs: 80, 85, 87-91, 97-104). During translation, the P2A site enables the expression of the NEP protein as a separate polypeptide by an unknown mechanism thought to involve ribosome slippage. The genetic information encoding the desired vaccine antigen is inserted between the influenza ORFs, either by fusion to NS1 or by the addition of a second P2A or T2A peptide, cleaving both sides of the antigen (SEQ ID NOs: 86, 99). This arrangement results in an expanded segment 8 that carries long repeats of nucleotide sequences from within the NS1 ORF.

[0169] Unfortunately, such replication results in gene instability. Atypical hybridization between two segments during genome replication can induce influenza RNA polymerase errors, typically leading to deletions and insertions, as well as defective genomic RNA synthesis. This instability can be exacerbated in this case because the gene duplication contains only 26 bp of the NEP exon 1 sequence from the segment 8 end, and this NEP exon 1 sequence is likely crucial for genome packaging. The assembly of the influenza genomic segment into a virion is mediated via a double-stranded "full-handle" RNA structure formed by the hybridization of reverse repeat sequences in the 5' and 3' UTR. The coding sequence near the segment end is also involved in packaging, and silent mutations near the segment end have been shown to block viral replication. The internal duplication region relative to this segment can compete with essential hybridization with the terminal UTR packaging sequence, impairing viral assembly. As a result, viral growth is poor, viral titer is low, and transgene expression is lost.

[0170] To improve gene stability by reducing homology between tandem duplications in the manipulated segment, critical mutations were introduced into the sequences encoding both NS1 and NEP ORFs (SEQ ID NOs. 110, 111). Silent mutations were performed at the third position of the codon encoding NS1, removing the start codon and adding a stop codon to either of the strong alternative translational reading frames, thereby cutting off the ability for unintended expression. This reduces the chance of unintended neoantigen production from either or both the vector and / or insert sequences.

[0171] The duplication of the exon 1 sequences encoding the 10 N-terminal amino acids of NEP was significantly altered in two ways to reduce inter-copy sequence identity (Figure 21, SEQ ID NOs: 80, 97, 110). NEP is expressed via this construct through the P2A cleavage site. Although the mechanism is unknown, cleavage always leaves a single prolyl residue as a protein trace attached to the N-terminus of the cleaved downstream peptide. The third amino acid of NEP is already proline, suggesting that these first two residues are structurally unrelated. Therefore, the constructed NS segment was designed to express a 6bp deletion N-terminal mutant of NEP that begins with proline and has no trace (SEQ ID NO: 117). Harmful homology was further improved by altering the position of the third codon, which still maintains a high %AT. In addition, both the GSG and P2A site sequences were codon-optimized to reflect the AT-rich codon bias of influenza.

[0172] Splice-negative NS segments containing duplication (SEQ ID NOs. 85, 98) were inserted into an RNA PolI plasmid vector for expression as negative sense vRNA. Using standard reverse genetics procedures for plasmid-based influenza viruses, M2 deletion single-replica (M2SR) viruses containing manipulated and control A-type PR / 8 / 1934 segment 8 were rescued. The recovered virus strains were amplified in M2 VeroA cells, and viral titers were determined. Using these strains, triplicate cultures were inoculated at MOI = 0.001 for comparison of growth dynamics. Four days after inoculation, virus cultures were sampled, and a certain amount was cryopreserved for subsequent titer analysis. TCID 50 After determining the viral titer, the average daily titer was calculated. The plot of curves for the two strains (Figure 12) shows that viral growth is not impaired by the synthetic segment that expresses NS1 and NEP as a single self-cleaving peptide.

[0173] Several M2SR virus strains were generated that were designed to express various antigens derived from the SARS-CoV-2 spike protein grown in M2VeroA cells, as shown in Table 5.

[0174] [Table 5-1]

[0175] [Table 5-2]

[0176] Example 10 This embodiment demonstrates the functionality of an NS vector segment for expressing the antigen, which is the spike helix of the SARS-CoV-2 S1 protein.

[0177] It is known that the spike helix of the SARS-CoV-2 S1 protein undergoes a major conformational change that drives fusion between the viral membrane and the cell membrane. Studies of other viral spike protein helices, including those of RSV and PIV, have identified a set of two tandem proline mutations that improve performance by stabilizing the spike protein as a recombinant antigen. These two proline residues (2P) are located, in order, between two shorter helices, which are present in the pre-fusion conformation of the spike protein. This change locks the protein in the pre-fusion conformation, resulting in a dual benefit of much better recombinant protein expression and neutralization of the immunological response to vaccination. The growth curves in Figure 13 show that segment 8 with NS1 fusion to the unmodified SARS-CoV-2 helical antigen impairs viral growth (SEQ ID NOs. 88, 101) compared to the wild type. Replacing the turn residue with 2P to lock the spike helix into the pre-fusion form likely improves growth by enhancing the functionality of NS1 (SEQ ID NOs: 88, 102).

[0178] Example 11 This embodiment demonstrates the successful expression of the SARS-CoV-2 receptor-binding domain (RBD) from the influenza A M2SR segment.

[0179] Various SARS-CoV-2 RBD antigens were expressed from synthetically constructed, engineered influenza A M2SR influenza A M2-deficient vector segment 7 (SEQ ID NOs: 122-124) (Figure 14). The designed gene segments were then inserted into an RNA Pol I vector for expression as negative sense vRNA. Segment 7 is designed to express two polypeptide open reading frames (ORFs) from spliced ​​viral mRNA: firstly, the complete influenza A / PR / 8 / 34 M1 protein, and secondly, a fusion protein of M2 with the SARS-CoV-2 Wuhan-Hu-1 spike S1 protein antigen. The viral expression of M2 in the second reading frame of influenza A segment 7 is due to splicing. To maintain the essential M1 protein function from synthetic SARS-CoV-2 containing influenza A segment 7, a portion of the M2 ORF is fused with the S1 RBD (SEQ ID NOs: 6, 8, 10, 79).

[0180] We rescued M2-deficient single-replicating (M2SR) viruses containing SARS-CoV-2 RBD M2SR segment 7 using a vector encoding the SARS-CoV-2 M2SR segment in a standard plasmid-based influenza virus reverse genetics procedure. Both viruses were successfully obtained using the HA and NA segments derived from the WHO-recommended vaccine strain of type A / Singapore / 2016 (H3N2). Viruses were recovered using M2Vero cells engineered to constitutively express the missing M2 protein from M2SR viruses grown in AOF medium. This virus rescue and culture system is suitable for preparing viral seeds for cGMP production of M2SR vaccine candidates intended for testing in human clinical trials.

[0181] Example 12 This example demonstrates that the M2SR influenza virus can drive the expression of antigens immobilized on the extracellular membrane of infected cells (see Figures 15-20 and 24-27).

[0182] The packaged virus produced in supportive M2-expressing substrate cells was expected to substantially lack the protein encoded by the target gene, multimerizing domain, or transmembrane domain, unless the antigen directly fused with a combined influenza subunit, such as hemagglutinin HA. The target gene (GOI) could be an element relevant as an antigen of viral (including influenza) origin, bacterial origin, fungal origin, or protozoan origin.

[0183] Proper antigen expression on the cell surface can be confirmed by immunofluorescence staining analyzed by flow cytometry of cells infected with monoclonal antibodies specific to the intended vaccine antigen encoded by the virus. The same antigen can be expressed using different segments. An example antigen is the human ACE2 receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. The RBD antigen can be expressed on the cell surface using the TM domain of another membrane protein. The fusion of RBD with the TM of the respiratory syncytial virus (RSV) F protein was encoded by the NS1 segment of a single open reading frame (ORF) containing translational slippage sites P2A and T2A with three peptides, namely the NS1, NEP, and T4 trimer domains, resulting in the RBD-RSV TM fusion. An alternative approach is direct fusion to the HA protein itself. HA is a membrane protein in which the thus fused antigen would appear as a trimer on the surface of the infected cell and be incorporated into the virion.

[0184] M2VeroA cells were inoculated with M2SR virus expressing RBD on the membrane from either the NS1 or HA segment at infection multiplicity (MOI) between 1 and 10. Infected cells were immunostained for SARS-CoV-2 S1 RBD surface expression 18 hours after inoculation in FACS buffer (1×DPBS, 1%FBS) at 4°C. Untreated viable cells were stained using neutralizing monoclonal antibody CR3022 isolated from convalescent SARS-CoV-1 patients as primary (ter Meulen et al., PLoS Med. 3(7): e237 (2006)), followed by detection with Alexa Fluor 488-labeled anti-human IgG secondary antibody, as shown in Figures 24-25. Surface expression above background was detected in cells infected with a virus expressing the SARS-CoV-2 RBD antigen from either the NS or HA segment.

[0185] Another respiratory virus that can be targeted is respiratory syncytial virus (RSV). Both of RSV's two main surface proteins, fusion (F) and surface glycoprotein (G), are important binding sites for monoclonal antibodies capable of neutralizing RSV. Human 293T cells were chemically translocated with influenza A replicons of four DNA plasmids to constitutively overexpress the viral subunits PA, PB1, PB2, and NP, which are required to express proteins encoded by the influenza RNA segment and by a single plasmid expressing the influenza HA genome segment from the RNA polymerase I promoter. The RSV G protein antigen was directly fused with the HA glycoprotein, which leads to the expression of the RSV G antigen on the cell membrane. Untreated live cells were immunostained 48 hours after translocation for surface expression of the RSV G surface glycoprotein antigen. Staining was performed in FACS buffer using the primary mouse monoclonal antibody 131-2G (Chemicon), followed by detection with an Alexa Fluor 488-labeled anti-mouse IgG secondary antibody. Surface expression exceeding the background level was detected in cells infected with a virus expressing the RSV G protein antigen.

[0186] Multiple antigens from a single pathogen can be displayed on the membrane. Cells inoculated with M2SR virus encoding the SARS-CoV-2 S2 antigen at MOI=1 express a second alternative COVID vaccine target other than RBD on their surface, as shown in Figure 27. Untreated live M2VeroA cells infected with the virus at MOI=1 were immunostained for surface expression of the SARS-CoV-2 spike S2 ​​subunit antigen 18 hours after inoculation. Staining was performed in FACS buffer using the primary rabbit monoclonal antibody 3C4 (Genscript) generated against the SARS-CoV-2 S2 immunogen, followed by detection with an Alexa Fluor 488-labeled anti-rabbit IgG secondary antibody. Surface expression above background was detected in cells infected with viruses expressing SARS-CoV-2 S2 conjugate factor and TM antigen.

[0187] The packaging signal is maintained upstream of the GOI for the constructs for both the HA and NS segments, and in the case of HA, the packaging signal can be replicated to maintain proper HA processing. The duplicated packaging signal shall have silent mutations to help eliminate secondary interactions with the 5' packaging signal and prevent undesirable recombination events. Other embodiments may employ direct fusion of the antigen with HA itself (e.g., respiratory syncytial virus (RSV) fusion (F) or glycoprotein (G) sequence or SARS-CoV-2 spike sequence), in which case the packaging sequence is not duplicated (Figures 24-27, SEQ ID NOs. 116-118). In many cases, the use of a multimerizing domain (MD) would be preferred to enhance immunogenicity. Such MDs can be selected from a variety of motifs, such as the C-terminal domain of T4 fibrintin (SEQ ID NOs. 115, Foldon) or the GCN4-pl leucine zipper domain. The transmembrane domain (TM) may be the transmembrane helix amino acids 1214-1246 (Genebank acceptance number: YP_009724390.1, SEQ ID NOs. 36-41, 77, 119) presumed to be the SARS-CoV-2 spike, and the use of at least amino acids 1201-1246 would deductively include the MHC I compatible epitope (SEQ ID NO: 21) ranked highly in Table 1. Other TMs may include those of the RSV fusion protein (SEQ ID NO: 115).

[0188] Table 6 shows the fusion of minispike proteins, which are portions of the SARS-CoV-2 S1 protein, or portions of S1 designed to adhere to the membrane using Spike™. The use of S1 signal sequences (SEQ ID NOs. 39-41, 42, 115, 119) would direct peptides to the cellular secretory system for display on the cell surface membrane and for post-translational modifications such as N-linked glycosylation.

[0189] [Table 6-1]

[0190] [Table 6-2]

[0191] [Table 6-3]

[0192] Example 13 This embodiment demonstrates the functionality of an M2SR NS vector segment containing a silent mutation (Figures 21-22) that expresses the antigen (SEQ ID NO: 113), which is also the EGFP fluorescent protein and a marker gene (SEQ ID NO: 114). M2VeroA cells were infected with an MOI of 10 using an M2SR virus containing an NS segment designed to express EGFP. Fluorescence microscopy over a 3-day period after inoculation showed that strong EGFP expression could be detected within 24 hours and spread to the point where nearly 100% of cells expressed the antigen by 48 hours. By 72 hours, cells had detached from the substrate and exhibited a strong cytopathic effect (CPE), as expected from influenza infection (Figure 23). The strong CPE observed on day 3 indicates that viral replication was not substantially inhibited by the inserted EGFP gene.

[0193] Example 14 This example demonstrates that the viruses M2SR and BM2SR, which encode the SARS-CoV-2 sequence of Example 4, possess the ability to induce an antibody response against influenza HA (hemagglutinin) surface proteins.

[0194] Serum was collected from mice before initial stimulation and approximately 3 weeks after the first dose. Serum samples were pooled for each group, and anti-HA IgG antibody titers were determined by enzyme-linked immunosorbent assay (ELISA).

[0195] ELISA was performed using recombinant HA protein as the capture antigen. Recombinant H3 HA(ΔTM)(Type A / Singapore / INFIMH-16-0019 / 2006)(H3N2)[Immune-Tech, New York, NY] was used for serum ELISA analysis of M2SR vector virus or M2SR virus administered to mice encoding the SARS-CoV-2 sequence. Recombinant Inf Type B HA1(Type B / Phuket / 3073 / 2013)[Immune-Tech, New York, NY]) was used for serum ELISA analysis of BM2SR vector virus or BM2SR virus administered to mice encoding the SARS-CoV-2 sequence.

[0196] ELISA plates were coated overnight at 4°C with 100 μL of capture antigen at a concentration of 2 μg / mL in phosphate-buffered saline (PBS). After blocking the plates with PBS-T containing 0.1% polysorbate 20 and 1% gelatin derived from cold-water fish skin, the plates were incubated in pairs with mouse serum diluted in PBS-T containing 1% gelatin derived from cold-water fish skin. After incubation at room temperature for 2 hours, the plates were washed six times with PBS-T and then incubated with anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (KPL; diluted 1:2,000 in PBS-T containing 1% gelatin derived from cold-water fish skin). After incubation with the secondary antibody for 1 hour, the plates were washed six times with PBS-T and then developed with 1-STEP® Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, Waltham, MA). After a 10-minute incubation, the reaction was stopped by adding 4N sulfuric acid. The absorbance was measured at a wavelength of 450 nm (OD). 450 ) was measured. The endpoint titer was blank plus 0.3 OD. 450 The reciprocal of the dilution that exceeded the cutoff value, which was determined by subtracting the average value, was used.

[0197] No differences in serum anti-HA IgG levels were observed between M2SR and BM2SR, which encode SARS-CoV-2 sequences, and their corresponding vector viruses, as shown in Table 7.

[0198] [Table 7]

[0199] All references cited herein, including publications, patent applications, and patents, are incorporated by reference to the same extent as they were described herein, with each reference being shown to be incorporated by reference individually and specifically.

[0200] The terms “a,” “an,” “the,” and “at least one,” as well as the use of similar referents in the context describing the present invention (particularly in the context of the claims described below), should be interpreted to encompass both singular and plural forms unless otherwise indicated herein or unless the context clearly contradicts it. 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 to mean one item (A or B) selected from the enumerated items, or any combination of two or more of the enumerated items (A and B), unless otherwise indicated herein or unless the context clearly contradicts it. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms unless otherwise stated (i.e., “including, but not limited to.” The enumeration of value ranges herein is intended merely as a convenient way to refer individually to each individual value falling within that range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually enumerated herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or unless otherwise clearly contradicted by the context. The use of any and all examples provided herein, or exemplary language (e.g., “like”), is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise asserted. No language herein shall be construed as indicating any element not described in any claim that is essential for the practice of the invention.

[0201] Preferred embodiments of the present invention, including the best modes known to the inventors for carrying out the invention, are described herein. Variations of such preferred embodiments may become apparent to those skilled in the art as they read the above description. The inventors expect that those skilled in the art will use such variations appropriately, and they intend that the invention will be carried out in ways other than those specifically described herein. Accordingly, the invention includes all variations and equivalents of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the elements described above in all possible variations is encompassed by the invention unless otherwise indicated herein or unless otherwise clearly contradicted by context.

[0202] The sequencing rules are based on DNA, which consists of four nucleotides: adenine (A), guanine (G), cytosine (C), and thymine (T). When referring to RNA or influenza viruses, T represents uracil (U).

Claims

1. A recombinant virus comprising an influenza virus backbone, wherein the influenza virus backbone comprises the gene segments PB1, PB2, PA, NP, M, NS, HA, and NA, (a) The PB1 gene segment encodes a PB1 protein having the amino acid sequence of SEQ ID NO: 49, the PB2 gene segment encodes a PB2 protein having the amino acid sequence of SEQ ID NO: 57, the PA gene segment encodes a PA protein having the amino acid sequence of SEQ ID NO: 59, the NP gene segment encodes an NP protein having the amino acid sequence of SEQ ID NO: 48, and the NS gene segment encodes an NS1 protein having the amino acid sequence of SEQ ID NO: 61, or (b) The PB1 gene segment encodes a PB1 protein having the amino acid sequence of SEQ ID NO: 51, the PB2 gene segment encodes a PB2 protein having the amino acid sequence of SEQ ID NO: 52, the PA gene segment encodes a PA protein having the amino acid sequence of SEQ ID NO: 59, the NP gene segment encodes an NP protein having the amino acid sequence of SEQ ID NO: 50, and the NS gene segment encodes an NS1 protein having the amino acid sequence of SEQ ID NO:

61. Here, at least one of the gene segments PB1, PB2, PA, NP, M, NS, HA, and NA comprises at least one nucleotide sequence encoding one or more antigens; Herein, a recombinant virus wherein the M gene segment encodes a protein comprising any one of sequence numbers 1-14, 95, and 96, and / or the NS gene segment encodes a protein comprising the NS1 protein and the antigen, wherein the protein comprises any one of sequence numbers 97-99 and 101-104.

2. The recombinant virus according to claim 1, wherein the antigen is an immunogenic fragment of the SARS-CoV-2 spike glycoprotein.

3. The recombinant virus according to claim 1 or 2, wherein the M gene segment comprises at least one nucleotide sequence encoding an antigen, wherein the antigen is an immunogenic fragment of the SARS-CoV-2 spike glycoprotein.

4. The recombinant virus according to any one of claims 1 to 3, wherein the M gene segment encodes a mutated M2 protein.

5. The recombinant virus according to claim 4, wherein the M gene segment encodes a protein comprising at least one linker protein and a FLAG epitope tag.

6. The recombinant virus according to any one of claims 1 to 5, wherein the M gene segment encodes a protein comprising any one of sequence numbers 1 to 14, 95, and 96.

7. The recombinant virus according to any one of claims 1 to 6, wherein the NS gene segment comprises at least one nucleotide sequence encoding one or more antigens.

8. The recombinant virus according to any one of claims 1 to 7, wherein the antigen is an immunogenic fragment of the SARS-CoV-2 spike glycoprotein.

9. The recombinant virus according to any one of claims 1 to 8, wherein the NS gene segment encodes (1) an NS1 protein, (2) at least one flexible linker protein, (3) an immunogenic fragment of a SARS-CoV-2 spike glycoprotein, (4) at least one cleavable sequence, and (5) a NEP protein.

10. The recombinant virus according to claim 9, wherein the at least one cleavable sequence is a T2A peptide sequence or a P2A peptide sequence.

11. The recombinant virus according to any one of claims 1 to 10, wherein the NS gene segment encodes a protein comprising the NS1 protein and the antigen, and the protein comprises one amino acid sequence of any one of SEQ ID NOs: 97-99 and 101-104.

12. The recombinant virus according to claim 1, wherein each of the gene segments M and NS comprises at least one nucleotide sequence encoding one or more antigens, and the antigen is an immunogenic fragment of the SARS-CoV-2 spike glycoprotein.

13. The recombinant virus according to claim 1, wherein each of the gene segments NA and NS comprises at least one nucleotide sequence encoding one or more antigens, and the antigen is an immunogenic fragment of the SARS-CoV-2 glycoprotein.

14. The recombinant virus according to claim 1, wherein each of the gene segments M and NA comprises at least one nucleotide sequence encoding one or more antigens, and the antigen is an immunogenic fragment of the SARS-CoV-2 glycoprotein.

15. The recombinant virus according to claim 1, wherein each of the gene segments M and HA comprises at least one nucleotide sequence encoding one or more antigens, and the antigen is an immunogenic fragment of the SARS-CoV-2 glycoprotein.

16. The recombinant virus according to claim 1, wherein each of the gene segments NS and NA comprises at least one nucleotide sequence encoding one or more antigens, and the antigen is an immunogenic fragment of the SARS-CoV-2 glycoprotein.

17. The recombinant virus according to claim 1, wherein each of the gene segments NS and HA comprises at least one nucleotide sequence encoding one or more antigens, and the antigen is an immunogenic fragment of the SARS-CoV-2 spike glycoprotein.

18. The recombinant virus according to any one of claims 1 to 17, wherein the virus can replicate in human cells.

19. The virus exhibits enhanced growth in Vero cells compared to a recombinant virus identical under the same conditions, except that the PB1 protein lacks leucine at position 40, tryptophan at position 180, and asparagine at position 464, the PB2 protein lacks valine at position 504, the PA protein lacks lysine at position 401, the NP protein lacks leucine at position 116 and lysine at position 294, and the NS1 protein lacks proline at position 30 and lysine at position 118, or A recombinant virus according to any one of claims 1 to 18, which, under the same conditions, exhibits enhanced growth in Vero cells compared to a recombinant virus that is identical except that the PB1 protein lacks leucine at position 40, tryptophan at position 180, and serine at position 607, the PB2 protein lacks valine at position 504, isoleucine at position 467, and valine at position 529, the PA protein lacks lysine at position 401, the NP protein lacks leucine at position 116 and arginine at position 311, and the NS1 protein lacks proline at position 30 and lysine at position 118.

20. A recombinant virus according to any one of claims 1 to 19, wherein the gene segment comprising at least one nucleotide sequence encoding one or more antigens further comprises a downstream duplication, and the downstream duplication comprises at least one silent nucleotide mutation.

21. A pharmaceutical preparation comprising a recombinant virus as described in any one of claims 1 to 20.

22. The pharmaceutical preparation according to claim 21, wherein the pharmaceutical preparation is a vaccine.

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

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

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

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

27. A recombinant virus according to any one of claims 1 to 20 or a pharmaceutical preparation according to any one of claims 22 to 26, for use in inducing an immune response to the antigen in mammals.

28. The recombinant virus or pharmaceutical preparation according to claim 27, wherein the mammal is a human.

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