ACE2 EXPRESS!NG INFLUENZA VIRUS

Gross-Reference to Related! App!ications

This application claims the benefit of the filing date of U.S. application No.

63/029,417, filed on May 23, 2020, the disclosure of which is Incorporated by reference herein.

Summary

The disclosure provides a recombinant influenza virus expressing secreted (soluble) angiotensin-converting enzyme 2 (ACE2), the receptor of several coronaviruses including SARS-CoV and SARS-CoV-2. The recombinant influenza virus In a composition may be live virus, e.g., an influenza virus strain that has low pathogenicity but may, In one embodiment, be subjected to conditions to kill the virus (inactivated virus), a replication-incompetent influenza virus, e.g., an influenza virus that Is produced In a helper cell line but does not replicate in a wild-type cell but after infection of a wild-type cell deposits viral proteins and viral RNA (e.g. PB2 or NP knock- out virus), a single cycle influenza virus, e.g., a M2, HA, NA, or PB1 -F2 knock-out virus, which Is produced In a helper cell and after infection of a wild-type cell, undergoes one cycle of replication, resulting in one round of replication/transcription and one round of protein synthesis, and/or live attenuated influenza viruses, e.g., viruses that undergo multiple rounds of replication/transcription, but at low levels, for example, as a result of one or more viral proteins encoding mutations that confer temperature-sensitivity (ts), cold-αdaption (ca), or attenuation (aff), or have an epitope tag. Expression, e.g., overexpression, of ACE2 by cells infected with the virus results in a decoy receptor, e.g., one which is soluble, for instance, as a result of a signal peptide that allows for secretion and which, in one embodiment, is cleaved off from the ACE2 extracellular domain sequences, thereby neutralizing SARS-CoV-2. As a result of Increased ACE2 levels, the lung may also be protected from SARS-CoV- 2-mediated lung injury. Thus, the use of a recombinant virus such as a recombinant influenza virus to express a solubie portion of ACE2, may be more efficacious as it would deliver ACE2 to the respiratory tract, thereby providing a decoy to neutralize incoming virus and to protect the lung from coronavirus mediated lung injury. In one embodiment, the signal peptide is encoded by the ACE2 gene. In one embodiment, the signal peptide is a heterologous signal peptide. Thus, the recombinant soluble ACE2 expressing virus is a biologic therapeutic to combat or limit Infection progression caused by coronaviruses that utilize ACE2 as a receptor, in one embodiment, the disclosure provides for an intranasally administered composition for treating or preventing infections caused by coronaviruses that utilize ACE2 as a receptor, including SARS-GoV-2. in one embodiment, the disclosure provides compositions comprising recombinant influenza virus and methods that result in expression of soluble ACE2 from an influenza backbone viral segment (e.g., from one of the viral segments encoding the ‘internal’ viral proteins), or from one of the viral segments for a glycoprotein, HA or NA, for example, following intranasai administration to the respiratory tract, for treating or preventing infections caused by coro navi ruses that utilize ACE2 as a receptor.

In one embodiment, the disclosure provides a recombinant influenza virus expressing a soluble receptor for coronavirus, comprising: 7, 8 or 9 different viral segments including a viral segment that encodes a cell binding protein, for instance, a HA viral segment which encodes an influenza A HA, a chimeric HA, or a heterologous cel! binding protein (e.g., non-influenza virus host cell binding protein including but not limited to a fiiovirus protein, an antibody, an a!phavirus protein, a !entivirus protein, a retrovirus protein, albumin, or a rhabdovirus protein), and at least 6 of: a P.A viral segment, a PB1 viral segment, a PB2 viral segment, a HA viral segment, a NA viral segment, a NP viral segment, a M (M1 and M2) viral segment, or a NS (NS1 and NS2) viral segment, wherein a viral segment other than the HA viral segment has a heterologous nucleotide sequence encoding a soluble receptor for coronavirus or wherein a ninth viral segment comprises a heterologous nucleotide sequence encoding a soluble receptor for coronavirus. In one embodiment, the disclosure provides a recombinant influenza virus expressing a soluble receptor for coronavirus, comprising: at least 7, 8 or 9 different viral segments including a HA viral segment, e.g., which encodes an Influenza B HA, a chimeric HA, or a heterologous cell binding protein, and at least 6 of: a PA viral segment, a PB1 viral segment, a PB2 viral segment, a HA viral segment, a NA (NA and NB) viral segment, a NP viral segment, a M (M1 and BM2) viral segment or a NS (NS1 and NS2) viral segment, wherein a viral segment other than the HA viral segment has a heterologous nucleotide sequence encoding a soluble receptor for coronavirus or wherein a ninth viral segment comprises a heterologous nucleotide sequence encoding a soluble receptor for coronavirus. In one embodiment, the disclosure provides a recombinant influenza virus comprising 6, 7 or 8 different viral segments expressing a soluble receptor for coronavirus, comprising: a HEF viral segment, e.g., which encodes an influenza HA, a chimeric HA, or a heterologous cell binding protein, including a HA viral segment, and at least 5 of: a PA viral segment, a PB1 viral segment, a PB2 viral segment, a NP viral segment, a M (M1 and CM2) viral segment, or a NS (NS1 and NS2) viral segment, wherein a viral segment other than the HEF viral segment has a heterologous nucleotide sequence encoding a soluble receptor for coronavirus or wherein an eighth viral segment comprises a heterologous nucleotide sequence encoding a soluble receptor for coronavirus.

In one embodiment, the heterologous nucleotide sequence (encoding ACE2 or a portion thereof, e.g., a soluble portion) replaces or is inserted into influenza virus sequences in one of the viral segments. In one embodiment, the recombinant virus may also have other modifications in one or more viral segments, in one embodiment, the ACE2 expressing virus a M2 knock out, e.g., the virus does not Include sequences that encode a functional M2, e.g., as a result of a deletion of at least some M2 sequences in the M viral segment. In one embodiment, the ACE2 encoding sequences are in a viral segment other than the modified M viral segment, e.g., the NS or NA viral segment. In one embodiment, the ACE2 encoding sequences are In a modified M viral segment. In one embodiment, the ACE2 encoding sequences are in a viral segment other than the modified M virai segment In one embodiment, the AGE2 expressing virus a PB2 knock out, e.g., the virus does not include sequences that encode a functional PB2, e.g., as a result of a deletion of at least some PB2 sequences in the PB2 virai segment. In one embodiment, the ACE2 encoding sequences are in a viral segment other than the modified PB2 viral segment, in one embodiment, the ACE2 encoding sequences are in a modified PB2 viral segment, e.g., one that does not express a functional PB2. In one embodiment, the virus a PBt knock out, e.g., the virus does not include sequences that encode a functional PB1 , e.g., as a result of a deletion of at least some PB1 sequences In the PB1 viral segment. In one embodiment, the heterologous nucleotide sequence replaces at least some PB1 coding sequences in the PB1 viral segment, in one embodiment, the heterologous nucleotide sequence replaces at least some NA coding sequences in the NA viral segment, in one embodiment, the heterologous nucleotide sequence replaces at least some NS coding sequences in the NS viral segment. In one embodiment, the heterologous nucleotide sequence replaces at least some PB2 coding sequences in the PB2 viral segment. In one embodiment, the heterologous nucleotide sequence replaces at least some PA coding sequences in the PA viral segment, in one embodiment, the heterologous nucleotide sequence replaces at least some M coding sequences in the M viral segment, in one embodiment, the heterologous nucleotide sequence replaces at least some NP coding sequences in the NP viral segment. In one embodiment, the heterologous nucleotide sequence is on a ninth virai segment, in one embodiment, the recombinant virus is an influenza A virus. In one embodiment, the recombinant virus Is an influenza B virus. In one embodiment, the soluble receptor has a sequence comprising a portion of one of SEQ ID Nos. 20-24, or a polypeptide with at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence Identity to the extracellular domain in one of SEQ ID Nos. 20-24, that binds coronavirus. The amino acid changes include conservative and/or non-conservative substitutions so long as the activity of the encoded polypeptide has at ieast 50%, 60%, 70%, 80%, 90%, 95% or more of the activity of the reference amino acid sequence. In one embodiment, the coronavirus bound by the soluble receptor is SARS-CoV2.

Also provided is a vaccine comprising an effective amount of the virus, e.g., optionally In combination with a pharmaceutical acceptable carrier. In one embodiment, the vaccine comprises more than one influenza virus isolate, or comprises other anti- microbials, e.g., other non-influenza viruses, bacteria or other microbes for Inducing an Immune response thereto, in one embodiment, a method to prepare an Influenza A or B virus that expresses a soluble receptor for coronavirus is provided, in one embodiment, the method includes contacting a host cell with one or more vectors which include transcription cassettes for RNA production, wherein the transcription cassettes comprise a transcription cassette comprising a Poll promoter operably linked to an influenza virus HA DNA linked to a Poll transcription termination sequence and at Ieast 6 of the following: a transcription cassette comprising a Poii promoter operabiy linked to an Influenza virus PA DNA linked to a Poll transcription termination sequence, a transcription cassette comprising a Poll promoter operabiy linked to an influenza virus PB1 DNA linked to a Poll transcription termination sequence, a transcription cassette comprising a Poll promoter operabiy linked to an influenza virus PB2 DNA linked to a Poll transcription termination sequence, a transcription cassette comprising a Poll promoter operabiy linked to an influenza virus NP DNA linked to a Poll transcription termination sequence, a transcription cassette comprising a Poll promoter operabiy linked to an influenza virus NP DNA linked to a Poll transcription termination sequence, a transcription cassette comprising a Poll promoter operabiy linked to an influenza virus M DNA linked to a Poll transcription termination sequence, a transcription cassette comprising a Poll promoter operabiy linked to an influenza virus NS (NS1 and NS2} DNA linked to a Poll transcription termination sequence, and a transcription cassette comprising a Polll promoter operabiy linked to a DNA coding region for influenza virus PA linked to a PollI transcription termination sequence, a transcription cassette comprising a Poll! promoter operabiy linked to a DNA coding region for influenza virus PB1 linked to a Poll! transcription termination sequence, and a transcription cassette comprising a PollI promoter operabiy linked to a DNA coding region for influenza virus NP linked to a PollI transcription termination sequence, wherein one of the transcription cassettes comprising a Poll promoter other than the transcription cassette comprising a Poii promoter and HA DNA has a heterologous nucleotide sequence encoding a soluble receptor for coronavirus or wherein another transcription cassette comprises a Poll promoter operabiy linked to the heterologous nucleotide sequence encoding a soluble receptor for coronavirus linked to a Poll transcription termination sequence; and isolating the virus from the host cell. In one embodiment, the host cell is an avian cell. In one embodiment, the host cell Is a mammalian cell, e.g., a Vero cell, a canine cell such as a MDCK cell, a human cell, e.g., a 293T cell or PER.C6© cell, a mink cell, e.g.,

MvLu1 cells, or a hamster cell, e.g., CHO cell.

In one embodiment, a method of preventing, Inhibiting or treating coronavirus infection in a mammal is provided that includes administering to the mammal a composition comprising an effective amount of a live (but having reduced pathogenicity), replication-incompetent, knock-out or live, attenuated recombinant Influenza virus comprising a viral segment comprising nucleic acid encoding a soluble form of ACE2. In one embodiment, the mammal is a human, in one embodiment, the composition is systemically administered. In one embodiment, the composition is locally administered. In one embodiment, the composition is administered to the lungs. In one embodiment, the composition is intranasally administered. In one embodiment, the soluble AGE2 has a sequence comprising a portion of one of SEQ ID Nos. 20-23, or a polypeptide having at least 80% amino acid sequence identity to the portion of one of SEQ ID Nos. 20-23 that binds coronavirus. in one embodiment, the recombinant virus has 7, 8 or 9 different viral segments including a HA viral segment, and at least 6 of: a PA viral segment, a PB1 viral segment, a PB2 viral segment, a HA viral segment, a NA viral segment, a NP viral segment, a M (M1 and M2) viral segment, or a NS (NS1 and NS2) viral segment. In one embodiment, a viral segment other than the HA viral segment has a heterologous nucleotide sequence encoding at least the soluble form of ACE2 or wherein a ninth viral segment comprises a hetero!ogous nucleotide sequence encoding at least the soluble form of ACE2. In one embodiment, the recombinant virus has at least 7, 8 or 9 different viral segments including a HA viral segment, and at least 6 of: a PA viral segment, a PB1 viral segment, a PB2 viral segment, a HA viral segment, a NA (NA and NB} viral segment, a NP viral segment, a M (M1 and BM2) viral segment or a NS (NS1 and NS2) viral segment. In one embodiment, the recombinant virus has 6, 7 or 8 different viral segments Including a HEF viral segment, and at least 5 of: a PA viral segment, a PB1 viral segment, a PB2 viral segment, a NP viral segment, a M (M1 and CM2) viral segment, or a NS (NS1 and NS2) viral segment. In one embodiment, a viral segment other than the HEF viral segment has a heterologous nucleotide sequence encoding a soluble receptor for coronavirus or wherein an eighth viral segment comprises a heterologous nucleotide sequence encoding a soluble receptor for coronavirus

The invention thus includes the use of isolated and purified vectors or plasmids, which express or encode influenza virus proteins, or express or encode influenza vRNA or cRNA, both native and recombinant vRNA or cRNA. The vectors comprise influenza cDNA, e.g., influenza A (e.g., any influenza A gene including any of the 18 HA or 11 NA subtypes), B, C or D DNA (see Fields Virology (Fields et al. (eds.), 6 ^th edition, Wolter, Kluwer (2014), which is specifically Incorporated by reference herein). Any suitable promoter or transcription termination sequence may be employed to express a protein or peptide, e.g., a viral protein or peptide, a protein or peptide of a nonviral pathogen, or a therapeutic protein or peptide.

A composition or plurality of vectors comprises a heterologous gene or open reading frame of interest, e.g., a foreign gene encoding an immunogenic peptide or protein useful as a vaccine. When preparing virus, the vector or plasmid comprising the gene or cDNA of interest may substitute for a vector or plasmid for an Influenza viral gene or may be in addition to vectors or plasmids for all Influenza viral genes. Thus, another embodiment comprises a composition or plurality of vectors as described above In which one of the vectors is replaced with, or further comprises, 5' influenza virus sequences optionally including 5' influenza virus coding sequences or a portion thereof, linked to a desired nucleic acid sequence, e.g,, a desired cDNA, linked to 3’ Influenza virus sequences optionally including 3' influenza virus coding sequences or a portion thereof. In one embodiment, the desired nucleic acid sequence such as a cDNA is In an antisense (antigenomic) orientation. The introduction of such a vector In conjunction with the other vectors described above to a host cell permissive for influenza virus replication results In recombinant virus comprising vRNA or cRNA corresponding to the heterologous sequences of the vector. The promoter in a vector for vRNA or cRNA production may be a RNA polymerase I promoter, a RNA polymerase II promoter, a RNA polymerase lll promoter, a T7 promoter, or a T3 promoter, and optionally the vector comprises a transcription termination sequence such as a RNA polymerase ! transcription termination sequence, a RNA polymerase II transcription termination sequence, a RNA polymerase ill transcription termination sequence, or a ribozyme. Ribozymes within the scope of the Invention include, but are not limited to, tetrahymena ribozymes, RNase P, hammerhead ribozymes, hairpin ribozymes, hepatitis ribozyme, as well as synthetic ribozymes. In one embodiment, the RNA polymerase I promoter Is a human RNA polymerase I promoter.

The promoter or transcription termination sequence in a vRNA, cRNA or virus protein expression vector may be the same or different relative to the promoter or any other vector, in one embodiment, the vector or plasmid which expresses influenza vRNA or cRNA comprises a promoter suitable for expression in at least one particular host cell, e.g., avian or mammalian host cells such as canine, feline, equine, bovine, ovine, or primate cells including human cells, or for expression in more than one host. in one embodiment, at least one vector for vRNA or cRNA comprises a RNA polymerase II promoter linked to a ribozyme sequence linked to viral coding sequences linked to another ribozyme sequences, optionally linked to a RNA polymerase li transcription termination sequence, in one embodiment, at least 2, e.g., 3, 4, 5, 6, 7 or 8, vectors for vRNA or cRNA comprise a RNA polymerase li promoter, a first ribozyme sequence, which is 5' to a sequence corresponding to viral sequences including viral coding sequences, which is 5' to a second ribozyme sequence, which is 5' to a transcription termination sequence. Each RNA polymerase II promoter in each vRNA or cRNA vector may be the same or different as the RNA polymerase Il promoter in any other vRNA or cRNA vector. Similarly, each ribozyme sequence in each vRNA or cRNA vector may be the same or different as the ribozyme sequences in any other vRNA or cRNA vector, in one embodiment, the ribozyme sequences in a single vector are not the same. in one embodiment, the invention provides a plurality of influenza virus vectors for a recombinant or reassortant virus comprising a vector for vRNA or cRNA production comprising a promoter operably linked to an influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA or cRNA production comprising a promoter operably linked to an influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA or cRNA production comprising a promoter operably linked to an influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA or cRNA production comprising a promoter operably linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA or cRNA production comprising a promoter operably linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA or cRNA production comprising a promoter operably linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA or cRNA production comprising a promoter operably linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA or cRNA production comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, where, in one embodiment, the DNAs lor PB1 , PB2, PA, NP, NS, and M are from one or more influenza vaccine seed viruses; and a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding influenza virus PB1 , a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding influenza virus PB2, and a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding influenza virus NP, and optionally a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding Influenza virus HA, a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding influenza virus NA, a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding influenza virus M1 , a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding Influenza virus M2, or a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding influenza virus NS2. In one embodiment, at least one vector comprises sequences corresponding to those encoding PB1, PB2, PA, NP, M, or NS, or a portion thereof, having substantially the same activity as a corresponding polypeptide encoded by one of SEQ ID NQs:1 -6 or 10-15, e.g., a sequence encoding a polypeptide with at least 80%, e.g., 85%, 90%, 92%, 95%, 98%, 99% or 100%, Including any Integer between 80 and 100, amino acid identity to a polypeptide encoded by one of SEQ ID NQs:1 -6 or 10-15. Optionally, two vectors may be employed in place of the vector comprising a promoter operabiy linked to an influenza virus M DNA linked to a transcription termination sequence, e.g., a vector comprising a promoter operabiy linked to an influenza virus M1 DNA linked to a transcription termination sequence and a vector comprising a promoter operabiy linked to an influenza virus M2 DNA linked to a transcription termination sequence.

The interna! virai segments of the recombinant virus may include one or more mutations including attenuating mutations, e.g., at least a portion of the M2 or PB2 coding sequence in the corresponding viral segment may be deleted or the coding sequence of a viral protein may encode one or more the following: in PB1 : 51 K or 171V: 156!; 265K, 358E, 521 A, or 686E; 391 K 581G, or 661 T; 265N or 591 l; 317l; or 265S; in PA: 35GK or 327E, 452H, 463A; in PB2: 478V or 265S or 478L or 490R; in NP: 125Y, 1861 or 101 N, 180G or 34G or 3411; in M1 : 144L, 231 D; in NS1 : 23A, 164L. in one embodiment, the coding sequence of a viral protein may encode one or more the following: in FBI : 51 K, 171V, 1561, 265K, 358E, 521A, 686E, 391 K, 581 G, 661 T, 265N, 591 l, 317l or 265S; in PA: 350 K, 327E, 452H, or 463A; in PB2: 478V, 265S, 478L or 490R; in NP: 125Y, 1861, 101 N, 18GG, 34G or 3411; in M1 : 144L or 231 D; in NS1 : 23A or 164L In one embodiment, the coding sequence of a viral protein may encode one or more the foliowing: in PB1 : 51 K and 171 V; 1561, 265K, 358E, 521 A, and 686E; 391 K, 581 G, and 661T; 265S and 591 l, 265N and 591 l; or 317l; in PA: 350K; or 327E, 452H, and 463A; in PB2: 478V; 478L; 265S, or 490R; in NP: 125Y and 186l, 101 N and 180G, 34G, or 341 i; in M1 : 144L and 231 D; in NS1 : 23A and 164L. in one embodiment, the coding sequence of a viral protein may encode one or more the following: in PB2, 265S; in PB1 , 391 E, 581 G, and/or 661 T; or in NP, 34G, ln one embodiment, the coding sequence of a viral protein may encode one or more the following: in PB2, 4781; in PB1, 265N and/or 5911; or in NEP, 1001.

A plurality of the vectors of the invention may be physically linked or each vector may be present on an individual plasmid or other, e,g,, linear, nucleic acid delivery- vehicle. In one embodiment, each vRNA or cRNA production vector is on a separate plasmid, in one embodiment, each mRNA production vector is on a separate plasmid.

The invention also provides a method to prepare influenza virus. The method comprises contacting a cell with a plurality of the vectors of the invention, e.g., sequentially or simultaneously, in an amount effective to yield infectious influenza virus. The invention also Includes isolating virus from a cell contacted with the plurality of vectors. Thus, the invention further provides isolated virus, as weii as a host cell contacted with the plurality of vectors or virus of the invention, in another embodiment, the invention includes contacting the cell with one or more vectors, either vRNA, cRNA or protein production vectors, prior to other vectors, either vRNA or protein production vectors, in one embodiment, the promoter for vRNA or cRNA vectors employed in the method is a RNA polymerase l promoter, a RNA polymerase ll promoter, a RNA polymerase III promoter, a T3 promoter or a T7 promoter, in one embodiment, the RNA polymerase i promoter is a human RNA polymerase i promoter, in one embodiment, each vRNA or cRNA vector employed in the method is on a separate plasmid. In one embodiment, the vRNA or cRNA vectors employed in the method are on one plasmid or on two or three different plasmids, in one embodiment, each mRNA vector employed in the method is on a separate plasmid, in one embodiment, the mRNA vectors for PA, PB1 , PB2 and NP employed in the method are on one plasmid or on two or three different plasmids.

The methods of producing virus described herein, which do not require helper virus Infection, are useful in the production of vaccines (e.g., for coronavirus).

The disclosure also provides isolated viral polypeptides, and methods of preparing and using a recombinant virus of the invention. The methods include administering to a host organism, e.g., a mammal an effective amount of the influenza virus of the invention, e.g., a live preparation or an inactivated virus preparation, optionally In combination with an adjuvant and/or a carrier, e.g., in an amount effective to prevent or ameliorate infection of an animal such as a mammal by SARS-CoV-2 or an antigenically closely related virus, or any virus that employs AGE2 as a receptor. In one embodiment, the virus is administered intramuscularly while in another embodiment, the virus is administered intranasaiiy. in some dosing protocols, all doses may be administered intramuscularly or intranasaiiy, while in others a combination of intramuscular and intranasal administration is employed. The vaccine may further contain other Isolates of influenza virus including recombinant Influenza virus, other pathogen(s), additional biological agents or microbial components, e.g., to form a multivalent vaccine, in one embodiment, intranasal vaccination, for instance containing with live attenuated or single cycle, e.g., knock-out, influenza virus, and optionally a mucosal adjuvant, may induce virus-specific IgA and neutralizing antibody in the nasopharynx as well as serum IgG.

The influenza virus of the invention may be employed with other compounds, e.g., protease or polymerase inhibitors, for instance, remdesivir, anti-malarials, e.g., ch!oroquine, amantadine, rimantadine, and/or neuraminidase inhibitors, e.g., may be administered separately In conjunction with those compounds, for instance, administered before, during and/or after.

Detailed Description

The present disclosure relates to a composition having a recombinant influenza virus, methods of making the virus, and a therapy using the virus to mitigate, for example, lung damage associated with coronavirus infection, and/or to prevent Infections, e.g., in exposed individuals. In one embodiment, an influenza virus that expresses soluble angiotensin-converting enzyme 2 (ACE2) is provided. The influenza virus backbone may be live-αttenuated or have only single-replication capacity, e.g., knock-out viruses. The composition is administered to an avian or a mammal. In one embodiment, the composition is administered intranasaiiy so that it enters the respiratory system and delivers the virus directly to the cells of the respiratory tract. Overexpression of soluble ACE2 neutralizes SARS-CoV2 by binding to it and preventing virus binding to ACE2 on host cells. In a second mode of action, increasing ACE2 levels protects the lungs from SARS-CoV-2-mediated lung injury. ACE2 is produced in two forms: a membrane-bound full-length form expressed widely In organs, and a soluble form that circulates in small amounts in the blood. in one embodiment, a single cycle or live, attenuated influenza virus elicits both systemic and mucosa! immunity at the primary portal of infection. In one embodiment, the live, single cycle or attenuated influenza virus has reduced replication in lung compared to wild-type influenza virus, e.g., the live, single cycle or attenuated influenza virus has titers in lung that are at least one to two logs less, and in one embodiment, replication in nasai turbinates Is not detectable. The live, single cycle or attenuated virus may be employed In a vaccine or Immunogenic composition, and so is useful to Immunize a vertebrate, e.g., an avian or a mammal, or induce an immune response in a vertebrate, respectively. in one embodiment, the heterologous gene (AGE2) replaces influenza virus protein coding sequences (e.g., there Is a deletion of influenza virus coding sequenceswithout deleting encapsidation (incorporation) sequences In coding sequences that are linked to encapsidation sequences in non-coding sequences at one or both ends of the viral segment). In one embodiment, interna! viral protein coding sequences are deleted and replaced with the heterologous gene, in one embodiment, the heterologous gene (ACE2) is inserted into influenza virus protein coding sequences. In one embodiment, the heterologous gene sequence is in genomic orientation, in one embodiment, the heteroiogous gene sequence is fused in frame to N-terminal influenza virus protein coding sequences. In one embodiment, the heterologous gene sequence Is fused in frame to C-terminal influenza virus protein coding sequences. A recombinant influenza virus of the disclosure having a heterologous gene sequence in one of the influenza virus viral segments that also lacks a viral segment (e.g., a “7 segment” virus for Influenza A virus or Influenza B virus) or has an additional viral segment (e.g., a “9 segment” virus for Influenza A virus or influenza B virus) is envisioned, in one embodiment, the heterologous gene (ACE2) replaces influenza virus protein coding sequences (e.g,, there is a deletion of influenza virus coding sequences without deleting encapsidation (incorporation) sequences In coding sequences that are linked to encapsidation sequences In non-coding sequences at one or both ends of the viral segment), in one embodiment, the heteroiogous gene sequence is in genomic orientation, in one embodiment, the heteroiogous gene sequence Is fused in frame to N- terminal influenza virus protein coding sequences, in one embodiment, the heteroiogous gene sequence is fused in frame to C-terminal influenza virus protein coding sequences.

The heterologous gene sequence may be inserted into any viral segment. The heterologous gene sequence may be of length that results in the viral segment with that heteroiogous gene sequence having a length that is up to 4 kb, 4.2 kb, 4.5 kb, 4.7 kb, 5 kb, 5.2 kb, 5,5 kb, 5.7 kb or 6 kb in length. In one embodiment, the heterologous gene replaces iniernai influenza virus sequences in the viral segment. In one embodiment, the insertion of a heterologous gene sequence may result in a ‘‘knock-out” of the respective influenza virus gene product and to prepare such a virus, influenza virus proteln(s) may be provided in trans to complement that type of mutation, in one embodiment, the heterologous gene sequences are in addition to Influenza virus coding sequences in the viral segment. In one embodiment, the heterologous gene sequence Is fused in frame to N-terminal Influenza virus protein coding sequences, in one embodiment, the heterologous gene in is fused in frame to C-terminal influenza virus protein coding sequences. in one embodiment, the heterologous gene sequence is in the NA viral segment. In one embodiment, the heteroiogous gene sequence Is in the HA viral segment. In one embodiment, the heteroiogous gene sequence Is in the M viral segment. In one embodiment, the heteroiogous gene sequence Is in the NS viral segment. In one embodiment, the heteroiogous gene sequence Is in the NR viral segment. In one embodiment, the heteroiogous gene sequence Is in the PA viral segment. In one embodiment, the heteroiogous gene sequence Is in the PB1 viral segment. In one embodiment, the heterologous gene sequence is in the PB2 viral segment. In one embodiment, the heterologous gene sequence Is 5' or 3’ to, replaces at least some of or is inserted into, the PA coding sequence In the PA viral segment. In one embodiment, the heterologous gene sequence is 5’ or 3' to, replaces at least some of or Is inserted Into, the FBI coding sequence in the FBI viral segment. In one embodiment, the heterologous gene sequence is 5' or 3' to, replaces at least some of or Is inserted into, the PB2 coding sequence In the PB2 viral segment, in one embodiment, the heterologous gene sequence is 5 _' or 3' to, replaces at least some of or is Inserted Into, the NS coding sequence in the NS vlra! segment. In one embodiment, the heterologous gene sequence is 5' or 3' to, replaces at least some of or is Inserted into, the NS1 coding sequence In the NS viral segment. In one embodiment, the heterologous gene sequence is 5’ or 3‘ to, replaces at least some of or is inserted into, the NS2 coding sequence In the NS viral segment. In one embodiment, the heterologous gene sequence is 5' or 3' to, replaces at least seme of or is inserted into, the HA coding sequence in the HA viral segment. In one embodiment, the heterologous gene sequence is 5' or 3’ to, replaces at least some of or is inserted into, the NA coding sequence In the NA viral segment (see, e.g., Perez et al. 2004). in one embodiment, the heterologous gene sequence is 5' or 3' to, replaces at least some of or is Inserted into, the M1 coding sequence in the M virai segment. In one embodiment, the heterologous gene sequence is 5' or 3' to, replaces at least some of or Is inserted into, the M2 coding sequence in the M viral segment.

Definitions

As used herein, the term “Isolated” refers to in vitro preparation and/or isolation of a nucleic acid molecule, e.g., vector or plasmid, peptide or polypeptide (protein), or virus of the invention, so that it is not associated with in vivo substances, or is substantially purified from in vitro substances. An isolated virus preparation is generally obtained by in vitro culture and propagation, and/or via passage in eggs, and is substantially free from other infectious agents.

As used herein, “substantially purified” means the object species is the predominant species, e.g., on a molar basis It is more abundant than any other individual species in a composition, and preferably is at least about 80% of the species present, and optionally 90% or greater, e.g., 95%, 98%, 99% or more, of the species present in the composition.

As used herein, “substantially free” means beiow the level of detection for a particular Infectious agent using standard detection methods for that agent.

A “recombinant” virus is one which has been manipulated in vitro, e.g., using recombinant DNA techniques, to introduce changes to the virai genome. Reassortant viruses can be prepared by recombinant or nonrecombinant techniques.

As used herein, the term "recombinant nucleic acid" or "recombinant DNA sequence or segment” refers to a nucleic acid, e.g., to DNA, that has been derived or isolated from a source, that may be subsequently chemically altered in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in the native genome. An example of DNA "derived" from a source, would be a DNA sequence that is identified as a usefui fragment, and which is then chemically synthesized in essentially pure form. An example of such DNA "isolated" from a source would be a useful DNA sequence that Is excised or removed from said source by chemical means, e.g., by the use of restriction endonucleases, so that It can be further manipulated, e.g., amplified, for use in the Invention, by the methodology of genetic engineering.

As used herein, a "heterologous" influenza virus gene or viral segment is from an influenza virus source that is different than a majority of the other influenza viral genes or viral segments in a recombinant, e.g., reassortant, influenza virus.

The terms "isolated polypeptide", "isolated peptide" or "isolated protein" include a polypeptide, peptide or protein encoded by cDNA or recombinant RNA including one of synthetic origin, or some combination thereof.

The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule expressed from a recombinant DNA molecule. In contrast, the term "native protein" is used herein to indicate a protein Isolated from a naturally occurring (i.e., a nonrecombinant) source. Molecular biological techniques may be used to produce a recombinant form of a protein with identical properties as compared to the native form of the protein.

"Conservative” amino acid substitutions are, for example, aspartic-glutamic as polar acidic amino acids; lysine/arginine/histidine as polar basic amino acids; leucine/isoleucine/methionine/vaiine/aianine/g!ycine/proline as non-polar or hydrophobic amino acids; serine/ threonine as polar or uncharged hydrophilic amino acids. Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and lscleueine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine: a group of amino acids having aromatic side chains Is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoieuclne or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying the specific activity of the polypeptide. Naturally occurring residues are divided into groups based on common side-chain properties: (1 ) hydrophobic: norieuclne, met, ala, val, leu, lie; (2) neutral hydrophilic: eys, ser, thr; (3) acidic: asp, giu; (4) basic: asn, gin, his, !ys, arg; (5) residues that Influence chain orientation: g!y, pro; and (6) aromatic: trp, tyr, phe. The disclosure also envisions polypeptides with non-conservative substitutions. Non-conservative substitutions entai! exchanging a member of one o! the classes described above for another.

Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any too sequences can be accomplished using a mathematical algorithm.

Computer Implementations of these mathematical algorithms can be utilized !or comparison of sequences to determine sequence identity. Alignments using these programs can be performed using the default parameters. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (htp://www.rKbi.nlm.nih.gov/). The algorithm may involve first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended In both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the ^• word hits In each direction are halted 'when the cumulative alignment score fails off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative- scoring residue alignments, or the end of either sequence is reached. in addition to calculating percent sequence identity, the BLAST algorithm may also perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm may be the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

The BLASTN program (for nucleotide sequences) may use as defaults a wordlength (W) of 11 , an expectation (E) of 10, a cutoff of 100, M=5. N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program may use as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See http://www.ncbi.n1 m.nih.gov. Alignment may also be performed manually by Inspection. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence Identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. influenza Virus Structure and Propagation

Influenza A viruses possess a genome of eight single-stranded negative-sense viral RNAs (vRNAs) that encode at least ten proteins. The influenza virus life cycle begins with binding of the hemagglutinin (HA) to sia!ic acid-containing receptors on the surface of the host cell, followed by receptor-mediated endocytosis. The low pH in late endosomes triggers a conformational shift in the HA, thereby exposing the N-terminus of the HA2 subunit (the so-called fusion peptide). The fusion peptide initiates the fusion of the viral and endosomal membrane, and the matrix protein (M1 ) and RNP complexes are released into the cytoplasm. RNPs consist of the nucleoprotein (NP), which encapsidates vRNA, and the viral polymerase complex, which Is formed by the PA,

PB1, and PB2 proteins. RNPs are transported into the nucleus, where transcription and replication take place. The RNA polymerase complex catalyzes three different reactions: synthesis of an mRNA with a 5' cap and 3‘ poly A structure, of a full-length complementary RNA (cRNA), and of genomic vRNA using the cRNA as a template. Newly synthesized vRNAs, NP, and polymerase proteins are then assembled into RNPs, exported from the nucleus, and transported to the plasma membrane, where budding of progeny virus particles occurs. The neuraminidase (NA) protein plays a crucial role late In Infection by removing sialic acid from siaiyioligcsaccharides, thus releasing newly assembled virions from the cell surface and preventing the self aggregation of virus particles. Although virus assembly involves protein-protein and proteln-vRNA interactions, the nature of these interactions is largely unknown.

Although influenza B and C viruses are structurally and functionally similar to influenza A virus, there are some differences. For example, influenza R virus does not have a M2 protein but has BM2 and has a viral segment with both NA and NB sequences. Influenza C virus and influenza D virus have only seven viral segments. Cell Lines That Can Be Used to Produce Influenza Viruses

Any cell, e.g., any avian or mammalian cell, such as a human, e.g., 293T or PER.C6© cells, or canine, e.g,, MDCK, e.g., humanized MDCK cells (see US application No. 16/785,449, filed on February 7, 2020, which is incorporated herein by reference) or M2 expressing cell line (see ltwasuki et al., J. Virol.. 80:5233 (2006), the disclosure of which is incorporated by reference herein), bovine, equine, feline, swine, ovine, rodent, for instance mink, e.g., MvLu1 cells, or hamster, e.g., CHO cells, or non- human primate, e.g., Vero cells, including mutant cells, which supports efficient replication of influenza virus can be employed to isolate and/or propagate influenza viruses, isolated viruses can be used to prepare a reassortant virus, in one embodiment, host cells for vaccine production are continuous mammalian or avian cell lines or cell strains. A complete characterization of the cells to be used, may be conducted so that appropriate tests for purity of the final product can be included. Data that can be used for the characterization of a cell includes (a) information on its origin, derivation, and passage history; (b) information on its growth and morphological characteristics; (c) results of tests of adventitious agents; (d) distinguishing features, such as biochemical, immunological, and cytogenetic patterns which allow the cells to be clearly recognized among other cell lines; and (e) results of tests for tumorigenicity. in one embodiment, the passage level, or population doubling, of the host cell used is as low as possible. in one embodiment, the cells are WHO certified, or certifiable, continuous cell lines. The requirements for certifying such cell lines include characterization with respect to at least one of genealogy, growth characteristics, immunological markers, virus susceptibility tumorigenicity and storage conditions, as well as by testing In animals, eggs, and cell culture. Such characterization is used to confirm that the cells are free from detectable adventitious agents. In some countries, karyology may also be required, in addition, tumorigenicity may be tested in cells that are at the same passage level as those used for vaccine production. The virus may be purified by a process that has been shown to give consistent results, before vaccine production (see, e.g., World Health Organization, 1982).

Virus produced by the host cell may be highly purified prior to vaccine or gene therapy formulation. Generally, the purification procedures result in extensive removal of cellular DNA and other cellular components, and adventitious agents. Procedures that extensively degrade or denature DNA may also be used.

Influenza Vaccines

A vaccine of the invention includes an isolated recombinant influenza virus of the invention, and optionally one or more other isolated viruses including other isolated Influenza viruses, one or more immunogenic proteins or glycoproteins of one or more isolated influenza viruses or one or more other pathogens, e.g., an Immunogenic protein from one or more bacteria, non-influenza viruses, yeast or fungi, or Isolated nucleic acid encoding one or more viral proteins (e.g., DNA vaccines) including one or more Immunogenic proteins of the isolated influenza virus of the invention, in one embodiment, the influenza viruses of the invention may be vaccine vectors for influenza virus or other pathogens.

A complete virion vaccine may be concentrated by ultrafiltration and then purified by zonal centrifugation or by chromatography. Viruses other than the virus of the invention, such as those Included In a multivalent vaccine, may be inactivated before or after purification using formalin or beta-propiolactone, for instance.

A subunit vaccine comprises purified glycoproteins. Such a vaccine may be prepared as follows: using viral suspensions fragmented by treatment with detergent, the surface antigens are purified, by ultracentrifugation for example. The subunit vaccines thus contain mainly HA protein, and also NA, The detergent used may be cationic detergent for example, such as hexadecyl trimethyl ammonium bromide (Bachmeyer, 1975), an anionic detergent such as ammonium deoxycho!ate (Laver & Webster, 1976); or a nonionic detergent such as that commercialized under the name TRITON X100. The hemagglutinin may also be isolated after treatment of the virions with a protease such as bromelin, and then purified. The subunit vaccine may be combined with a virus of the invention in a multivalent vaccine.

A split vaccine comprises virions which have been subjected to treatment with agents that dissolve lipids. A split vaccine can be prepared as follows: an aqueous suspension of the purified virus obtained as above, inactivated or not, is treated, under stirring, by lipid solvents such as ethyl ether or chloroform, associated with detergents. The dissolution of the viral envelope lipids results in fragmentation of the viral particles. The aqueous phase is recuperated containing the split vaccine, constituted mainly of hemagglutinin and neuraminidase with their original lipid environment removed, and the core or its degradation products. Then the residua! infectious particles are inactivated If this has not already been done. The split vaccine may be combined with a virus of the Invention in a multivalent vaccine. inactivated Vaccines. Inactivated influenza virus vaccines are provided by inactivating replicated virus using known methods, such as, but not limited to, formalin or b-propio!actone treatment, inactivated vaccine types that can be used in the invention can Include whole-virus (WV) vaccines or subvirion (SV) (split) vaccines. The WV vaccine contains intact, inactivated virus, while the SV vaccine contains purified virus disrupted with detergents that solubilize the lipid-containing viral envelope, followed by chemical inactivation of residual virus. in addition, vaccines that can be used include those containing the Isolated HA and NA surface proteins, which are referred to as surface antigen or subunit vaccines.

Live Attenuated Virus Vaccines. Live, attenuated influenza virus vaccines, can be used for preventing or treating influenza virus infection. In one embodiment, attenuation may be achieved in a single step by transfer of attenuated genes from an attenuated donor virus to a replicated Isolate or reasserted virus according to known methods. Since resistance to Influenza A virus is mediated primarily by the development of an immune response to the HA and/or NA glycoproteins, the genes coding for these surface antigens come from the reassorted viruses or clinical isolates. The attenuated genes may be derived from an attenuated parent. In this approach, genes that confer attenuation generally do not code for the HA and NA glycoproteins.

Viruses (donor Influenza viruses) are available that are capable of reproducibly attenuating Influenza viruses, e.g., a cold adapted (ca) donor virus can be used for attenuated vaccine production. Live, attenuated reassortant virus vaccines can be generated by mating the ca donor virus with a virulent replicated virus. Reassortant progeny are then selected at 25°C (restrictive for replication of virulent virus), in the presence of an appropriate antiserum, which inhibits replication of the viruses bearing the surface antigens of the attenuated ca donor virus. Useful reassortants are: (a) infectious, (b) attenuated for seronegative non-αdult mammals and immunologicaiiy primed adult mammals, (c) immunogenic and (d) genetically stable. The immunogenicity of the ca reassortants parallels their level of replication. Thus, the acquisition of the six transferable genes of the ca donor virus by new wild-type viruses has reproducibly attenuated these viruses for use In vaccinating susceptible mammals both adults and non-αdult.

Other attenuating mutations can be introduced Into Influenza virus genes by site-directed mutagenesis to rescue infectious viruses bearing these mutant genes. Attenuating mutations can be introduced into non-coding regions of the genome, as well as Into coding regions. Such attenuating mutations can also be Introduced Into genes other than the HA or NA, e.g., the PB2 polymerase gene. Thus, new donor viruses can also be generated bearing attenuating mutations introduced by site-directed mutagenesis, and such new donor viruses can be used in the production of live atenuated reassortants vaccine candidates in a manner analogous to that described above for the ca donor virus. Similarly, other known and suitable attenuated donor strains can be reasserted with influenza virus to obtain attenuated vaccines suitable for use In the vaccination of mammals.

In one embodiment, such attenuated viruses maintain the genes from the virus that encode antigenic determinants substantially similar to those of the original clinical Isolates. This is because the purpose of the attenuated vaccine is to provide substantially the same antigenicity as the original clinical isolate of the virus, while at the same time lacking pathogenicity to the degree that the vaccine causes minimal chance of inducing a serious disease condition in the vaccinated mammal.

The viruses in a multivalent vaccine can thus be attenuated, single cycle (live) or inactivated, formulated and administered, according to known methods, as a vaccine to induce an immune response in an animal, e.g., a mammal Methods are well-known In the art for determining whether such attenuated, live single cycle or inactivated vaccines have maintained similar antigenicity to that of the clinical isolate or high growth strain derived therefrom. Such known methods include the use of antisera or antibodies to eliminate viruses expressing antigenic determinants of the donor virus; chemical selection (e.g., amantadine or rimantidine); HA and NA activity and Inhibition; and nucleic acid screening (such as probe hybridization or PCR) to confirm that donor genes encoding the antigenic determinants (e.g., HA or NA genes) are not present in attenuated viruses.

Pharmaceutical Compositions

Pharmaceutical compositions of the present Invention, suitable for inoculation, e.g., nasal, parenteral or oral administration, comprise one or more influenza virus Isolates, e.g., one or more attenuated, live single cycle or Inactivated influenza viruses, a subunit thereof, isolated protein(s) thereof, and/or isolated nucleic acid encoding one or more proteins thereof, optionally further comprising sterile aqueous or mon-αqueous solutions, suspensions, and emulsions. The compositions can further comprise auxiliary agents or excipients, as known in the art. The composition of the invention is generally presented in the form of individual doses (unit doses).

Conventional vaccines generally contain about 0.1 to 200 μg, e.g., 30 to 100 μg, of HA from each of the strains entering Into their composition. The vaccine forming the main constituent of the vaooine composition of the invention may comprise a single Influenza virus, or a combination of Influenza viruses, for example, at least two or three Influenza viruses, including one or more reassortant(s).

Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and/or emulsions, which may contain auxiliary agents or excipients known in the art. Examples of non-αqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl cleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration may general!y comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes Include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used In the art, such as purified water. Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.

When a composition of the present invention Is used for administration to an Individual, it can further comprise salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition. For vaccines, adjuvants, substances which can augment a specific immune response, can be used. Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the organism being immunized.

Heterogeneity in a vaccine may be provided by mixing replicated influenza viruses for at least two influenza virus strains, such as 2-20 strains or any range or value therein. Vaccines can be provided for variations in a single strain of an influenza virus, using techniques known in the art.

A pharmaceutical composition according to the present Invention may further or addltionaily comprise at least one chemotherapeutic compound, for example, for gene therapy, immunosuppressants, anti-inflammatory agents or immune enhancers, and for vaccines, chemotherapeutics including, but not limited to, gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon-α, interferon-β, interferon-y, tumor necrosis factor-αlpha, thiosemicarbarzones, methisazone, rifampin, ribavirin, a pyrimidine analog, a purine analog, fcscarnet, phosphonoacetic acid, acyclovir, dideoxynueleosides, a protease Inhibitor, or ganciclovir. The composition can also contain variable but small quantities of endotoxin-free formaldehyde, and preservatives, which have been found safe and riot contributing to undesirable effects in the organism to which the composition is administered. Pharmaceutical Purposes

The administration of the composition (or the antisera that it elicits) may be for either a “prophylactic” or "therapeutic” purpose. When provided prophylactically, the compositions of the invention which are vaccines are provided before any symptom or clinical sign of a pathogen infection becomes manifest. The prophylactic administration of the composition serves to prevent or attenuate any subsequent infection. When provided prophylactically, the gene therapy compositions of the invention, are provided before any symptom or clinical sign of a disease becomes manifest. The prophylactic administration of the composition serves to prevent or attenuate one or more symptoms or cilnicai signs associated with the disease.

When provided therapeutically, a viral vaccine Is provided upon the detection of a symptom or clinical sign of actual infection. The therapeutic administration of the compound(s) serves to attenuate any actual infection. When provided therapeutically, a gene therapy composition Is provided upon the detection of a symptom or clinical sign of the disease. The therapeutic administration of the compound(s) serves to attenuate a symptom or clinical sign of that disease.

Thus, a vaccine composition of the present invention may be provided either before the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection. Similarly, for gene therapy, the composition may be provided before any symptom or clinical sign of a disorder or disease Is manifested or after one or more symptoms are detected.

A composition Is said to be “pharmacologically acceptable” if its administration can be tolerated by a recipient mammal. Such an agent Is said to be administered in a “therapeutically effective amount” if the amount administered Is physiologically significant. A composition of the present Invention is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, e.g., enhances at least one primary or secondary humoral or cellular Immune response against at least one strain of an infectious Influenza virus.

The “protection” provided need not be absolute, i.e., the influenza infection need not be totally prevented or eradicated, if there is a statistically significant improvement compared with a control population or set of mammals. Protection may be limited to mitigating the severity or rapidity of onset of symptoms or clinical signs of the influenza virus Infection.

Pharmaceutical Administration

A composition of the present Invention may confer resistance to one or more pathogens, e.g., one or more influenza virus strains, by either passive immunization or active immunization, in active immunization, an atenuated or single cycle live vaccine composition is administered prophylactically to a host (e.g., a mammal), and the host’s immune response to the administration protects against infection and/or disease. For passive immunization, the elicited antisera can he recovered and administered to a recipient suspected of having an infection caused by at least one influenza virus strain.

A gene therapy composition of the present invention may yield prophylactic or therapeutic ievels of the desired gene product by active immunization. in one embodiment, the vaccine is provided to a mammalian female (at or prior to pregnancy or parturition), under conditions of time and amount sufficient to cause the production of an immune response which serves to protect both the female and the fetus or newborn (via passive incorporation of the antibodies across the placenta or in the mother’s milk).

The present Invention thus includes methods for preventing or atenuating a disorder or disease, e.g., an Infection by at least one strain of pathogen such as a coronavirus or other pathogen that binds to ACE2. As used herein, a vaccine is said to prevent or attenuate a disease if Its administration results either in the total or partial attenuation (l.e., suppression) of a clinical sign or condition of the disease, or in the total or partial Immunity of the individual to the disease. As used herein, a gene therapy composition is said to prevent or attenuate a disease if its administration results either In the total or partial attenuation (i.e., suppression) of a clinicai sign or condition of the disease, or in the total or partial Immunity of the Individual to the disease.

A composition having at least one influenza virus of the present invention, including one which is single cycle, attenuated or inactivated and one or more other Isolated viruses, one or more Isolated viral proteins thereof, one or more Isolated nucleic acid molecules encoding one or more viral proteins thereof, or a combination thereof, may be administered by any means that achieve the intended purposes.

For example, administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, Intraperitoneal, intranasal, oral or transderma! routes. Parenteral administration can be accomplished by bolus injection or by gradual perfusion over time.

A typical regimen tor preventing, suppressing, or treating an influenza virus related pathology, comprises administration of an effective amount of a vaccine composition as described herein, administered as a single treatment, or repeated as enhancing or booster dosages, over a period up to and including between one week and about 24 months, or any range or value therein.

According to the present invention, an “effective amount” of a composition Is one that is sufficient to achieve a desired effect, it is understood that the effective dosage may be dependent upon the species, age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect wanted. The ranges of effective doses provided below are not Intended to limit the invention and represent dose ranges.

The dosage of a live, attenuated or killed virus vaccine for an animal such as a mammalian aduit organism may be from about 10 ² -10 ¹⁵ , e.g., 10 ³ -10 ¹² , plaque forming units (PFU)/kg, or any range or value therein. The dose of Inactivated vaccine may range from about 0.1 to 1000, e.g., 30 to 100 μg, of HA protein. However, the dosage should be a safe and effective amount as determined by conventional methods, using existing vaccines as a starting point.

The dosage of immunoreactive HA in each dose of replicated virus vaccine may be standardized to contain a suitable amount, e.g., 30 to 100 μg or any range or value therein, or the amount recommended by government agencies or recognized professional organizations. The quantity of NA can also be standardized, however, this glycoprotein may be labile during purification and storage.

The dosage of Immunoreactive HA in each dose of replicated virus vaccine can be standardized to contain a suitable amount, e.g., 1 -50 μg or any range or value therein, or the amount recommended by the U.S. Public Health Service (PHS), which is usually 15 μg per component for older children (greater than or equal to 3 years of age), and 7.5 μg per component for children less than 3 years of age. The quantity of NA can also be standardized, however, this glycoprotein can be labile during the processor purification and storage (Kendal et a!., 1980; Kerr et al,, 1975). Each 0.5-m! dose of vaccine may contains approximately 1 -50 billion virus particles, and preferably 10 billion particles.

In one embodiment, the vaccine generally contains about 0.1 to 200 μg, e.g., 30 to 100 μg, 0.1 to 2 μg, 0.5 to 5 μg, 1 to 10 μg, 10 μg to 20 μg , 15 μg to 30 μg, or 10 to 30 μg, of HA from each of the strains entering into their composition. The vaccine forming the main constituent of the vaccine composition of the invention may comprise a single influenza virus, or a combination of influenza viruses, for example, at least two or three influenza viruses, including one or more reassortant(s). in one embodiment, ,the dosage of a live, attenuated or ki!!ed virus vaccine for an animal such as a mammalian adult organism may be from about 10 ² -10 ^so , e.g., 10 ³ - 10 ¹² , 10 ^S -10 ¹⁰ , 10 ⁶ -10 ¹¹ , 10 ⁶ -10 ¹⁵ , 10 ² -10 ^to , or 10 ¹⁵ -10 ² ° plaque forming units (PFU)/kg, or any range or value therein. The dose of one viral isolate vaccine, e.g., in an Inactivated vaccine, may range from about G.1 to 1000, e.g., 0.1 to 10 μg, 1 to 20 μg, 30 to 100 μg, 10 to 50 μg, 50 to 200 μg, or 150 to 300 μg, of HA protein. However, the dosage should be a safe and effective amount as determined by conventional methods, using existing vaccines as a starting point.

In one embodiment, the dosage of Immunoreactive HA in each dose of replicated virus vaccine may be standardized to contain a suitable amount, e.g., 0.1 μg to 1 μg, 0.5 μg to 5 μg, 1 μg to 10 μg, 10 μg to 20 μg, 15 μg to 30 μg, or 30 μg to 100 μg or any range or value therein, or the amount recommended by government agencies or recognized professional organizations. The quantity of NA can also be standardized, however, this glycoprotein may be labile during purif ication and storage.

The dosage of immunoreactive HA in each dose of replicated virus vaccine can be standardized to contain a suitable amount, e.g., 1 -50 μg or any range or value therein, or the amount recommended by the U.S. Public Health Service (PHS), which is usually 15 μg, per component for older children >3 years of age, and 7.5 μg per component for children <3 years of age. The quantity of NA can also be standardized, however, this glycoprotein can be labile during the processor purification and storage (Kendal ef a!., 1980; Kerr et al., 1975). Each 0.5-mi dose of vaccine may contain approximately 0.1 to 0.5 billion viral particles, 0.5 to 2 billion viral particles, 1 to 50 billion virus particles, 1 to 10 billion viral particles, 20 to 40 billion viral particles, 1 to 5 billion viral particles, or 40 to 80 billion viral particles.

Exemplary M Viral Segments

Wild-type influenza A virus M2 protein consists of three structural domains: a 24-αmino-αcid extracellular domain, a 19-αmino-αcid transmembrane domain, and a 54- amino-αcid cytoplasmic tail domain. The M2 transmembrane domain has ion channel activity, which functions at an early stage of the viral life cycle between the steps of virus penetration and uncoating. The M2 cytoplasmic tail domain may also have an Important role in viral assembly and morphogenesis. M1 protein and M2 protein share N-terminal sequences. The M2 protein Is encoded by a spliced transcript and RNAs encoding the M1 protein and the M2 protein share 3’ sequences, although the coding sequences for M1 and M2 in those 3' sequences are in different reading frames. The C-terminal residues of M1 and C-terminal portion of the extracellular domain of M2 are encoded by the overlapping 3’ coding sequences.

A "functional" M1 protein provides for export of viral nucleic acid from the host cell nucleus, a viral coat, and/or virus assembly and budding. Thus, the M1 protein in the recombinant influenza viruses of the invention has substantially the same function (e.g., at least 10%, 20%, 50% or greater) as a wild-type M1 protein. Thus, any alteration In the M1 coding region in a mutant M viral segment In a recombinant influenza virus does not substantially alter the replication of that virus, e.g., in vitro, for Instance, viral titers are not reduced more than about 1 to 2 logs in a host cell that supplies M2 in trans .

In one embodiment, an Isolated recombinant influenza virus comprises a mutant M2 protein having a deletion of one or more residues of the cytoplasmic tail of M2, which virus replicates in vitro, e.g., producing titers that are substantially the same or at most 10, 100 or 1 ,000 fold less than a corresponding wild-type influenza virus, but wherein the replication of the recombinant virus in vivo is limited to a single cycle (e.g., no progeny viruses are produced). In one embodiment, the deletion Includes 2 or more residues and up to 21 residues of the cytoplasmic tail of M2. In one embodiment, the M viral segment for the mutant M2 has one to two stop codons near the splice donor or splice acceptor site for the M2 transcript. In one embodiment, the coding region for the transmembrane and/or cytoplasmic domain of M2 Is also deleted.

In one embodiment, the deletion of M2 Includes 21 or more residues and up to 54 residues, i.e., the entire cytoplasmic tail, of the cytoplasmic tail of M2. In one embodiment, the mutant M2 protein may also comprise at least one amino acid substitution relative to a corresponding wild-type M2 protein. The substitution(s) in the M2 protein may be in the extracellular domain, the transmembrane (TM) domain, or the cytoplasmic domain, or any combination thereof. For example, substitutions in the TM domain may be at residues 25 to 43 of M2, e.g., positions 27, 30, 31 , 34, 38, and/or 41 of the TM domain of M2. In another embodiment, the mutant M2 protein may also comprise a deletion in at least a portion of the extracellular domain and/or the TM domain, e.g., a deletion of residues 29 to 31 , relative to a corresponding wild-type M2 protein. In yet another embodiment, the mutant M2 protein further comprises a heterologous protein, e.g., the cytoplasmic domain of a heterologous protein (a non- influenza viral protein), which may have a detectable phenotype, fused to the cytoplasmic tail or extracellular domain of M2, forming a chimeric protein, in one embodiment, a cytoplasmic domain of a heterologous protein Is fused to the remaining residues of the cytoplasmic tail of the deleted M2 protein. In one embodiment, the presence of one or more substitutions, deletions, or insertions of hetero!ogous sequences, or any combination thereof, does not substantially alter the properties of the recombinant influenza virus, e.g., the presence of one or more substitutions, deletions, or insertions of heterologous sequences does not result in virus titers in vitro that are more than about 1 .5 to 2 logs lower, but allows for a single cycle of replication in vivo (e.g., no progeny viruses are produced) for a recombinant influenza virus comprising a mutant M2 protein having a deletion of one or more residues of the cytoplasmic tail of M2. in one embodiment, the deletion in the cytoplasmic domain of M2 includes 2, 3, 4, 5 or more, e.g., 11 , 12, 13, 14, or 15 residues, but less than 22 residues, of the C- terminus of the cytoplasmic tail of M2. In one embodiment, the deletion is 2 up to 10 residues, including any integer in between, in one embodiment, the deletion Is from 1 up to less than 8 residues, including any integer In between. In one embodiment, the deletion Is from 5 up to 21 residues, Including any Integer in between. In one embodiment, the deletion is from 5 up to less than 28 residues, including any integer in between. In one embodiment, the deletion is from 9 up to 15 residues, including any Integer In between. In one embodiment, the deletion is from 9 up to 23 residues, including any Integer In between, in one embodiment, the deletion in the cytoplasmic domain of M2 Includes 22, 23, 24, 25 or more, e.g., 41 , 42, 43, 44, or 45 residues, but less than 54 residues, of the C-terminus of the cytoplasmic tail of M2. In one embodiment, the deletion is from 22 up to 35 residues, including any integer in between. In one embodiment, the deletion is from 29 up to 35 residues, including any integer in between. In one embodiment, the deletion Is from 35 up to 45 residues, Including any integer In between. In one embodiment, the deletion is from 9 to less than 28 residues, including any integer in between. in one embodiment, an isolated recombinant influenza virus is provided comprising a mutant M viral segment that Is mutated so that upon viral replication, the mutant M gene expresses a functional M1 protein and a mutant M2 protein with a deletion of the cytoplasmic taii and a deietion of at least a portion of the transmembrane domain, e,g,, Internal or C-terminal deletions, and/or includes one or more substitutions In the transmembrane domain. In one embodiment, the mutant M2 protein has a deletion that includes the entire cytoplasmic tall and transmembrane domain of M2, and has one or more residues of the extracellular domain, e.g., has the first 9 to 15 residues of the extracellular domain. The replication of the recombinant virus Is, in one embodiment, a single cycle in vivo relative to a corresponding virus without a mutant M viral segment. The recombinant influenza virus replicates in vitro In the presence of M2 supplied in trans, e.g., producing titers that are substantially the same or at most 10,

100 or 1 ,000 fold less than a corresponding wild-type influenza virus. in one embodiment, the mutations in the M2 gene result in a mutant M2 protein with a deletion of the entire cytoplasmic tali and deletion or substitution of one or more residues in the transmembrane (TM) domain of M2 and may also comprise at least one amino acid substitution in the extracellular domain, or a combination thereof, relative to a corresponding wild-type M2 protein encoded by a M viral segment. For example, substitutions in the TM domain may include those at residues 25 to 43 of M2, e.g., positions 27, 30, 31, 34, 38, and/or 41 of the TM domain of M2. Substitutions and/or deletions in the TM domain may result in a truncated M2 protein that is not embedded in the viral envelope. For example, a deletion of 10 residues at the C-terminus of the transmembrane domain may result in a truncated M2 protein that is not embedded in the viral envelope. In another embodiment, the mutant M2 protein may also comprise a deletion In at least a portion of the extracellular domain in addition to deletion of the cytoplasmic domain and a deietion In the TM domain, in one embodiment, the mutant M2 protein has a deietion of the entire cytoplasmic tail and the TM domain and at least one residue of the extracellular domain, e.g., 1 to 15 residues, or any Integer in between, of the C-terminal portion of the extracellular domain. In yet another embodiment, the mutant M2 protein having at least a portion of the extracellular domain further comprises a heterologous protein, e.g., the cytoplasmic and/or TM domain of a heterologous protein (a non-influenza viral protein), which may have a detectable phenotype, that is fused to the C-terminus of at least the extracellular domain of M2, forming a chimeric protein. In one embodiment, the presence of one or more substitutions, deletions, or Insertions of heterologous sequences, or any combination thereof, in the M2 gene does not substantially alter the properties of the recombinant Influenza virus, e.g., the presence of one or more substitutions, deletions, or insertions of heterologous sequences does not result in virus titers in vitro that are more than about 1 .5 to 2 logs lower, and/or but allows for a single cycle (e.g., no progeny viruses are produced) of replication in vivo for the recombinant influenza virus with a mutant M2 protein gene having a deletion of the cytoplasmic fail and TM domain of M2. in one embodiment, the deietion in the TM domain of M2 includes 1 , 2, 3, 4, 5 or more, e.g., 11 , 12, 13, 14, or 15 residues, up to 19 residues. In one embodiment, the deletion Is from 2 up to 9 residues, including any integer in between. In one embodiment, the deletion is from 15 up to 19 residues, including any integer in between, in one embodiment, the deletion Is from 10 up to 19 residues, including any integer in between, in one embodiment, the deletion is the result of at least one substitution of a codon for an amino acid to a stop codon, in one embodiment, the deletion Is the result of deletion o! at least one codon for an amino acid. In one embodiment, the TM domain of M2 has one or more substitutions, e.g., includes 1, 2, 3, 4, 5 or more, e.g., 11 , 12, 13, 14, or 15 substitutions, up to 19 residues of the TM domain, in one embodiment, the one or more amino acid deietions and/or substitutions in the TM domain in a mutant M2 protein that also lacks the cytoplasmic tail of M2, provides for a mutant M2 protein that lacks M2 activity and/or when expressed in a virus yields a live, single cycle virus. in one embodiment, a deletion in the extracellular (ectodomain) domain of M2 may Include 1 , 2, 3, 4 or more, e.g., 5, 10, 15, or 20 residues, up to 24 residues of the extracellular domain, in one embodiment, the deletion in the extracellular domain Is from 1 up to 15 residues, including any integer in between. In one embodiment, the deletion Is the result of at least one substitution of a codon for an amino acid to a stop codon, in one embodiment, the deletion is the result of deletion of at least codon for an amino acid. In one embodiment, the extracellular domain of M2 may also include one or more substitutions. In one embodiment, the mutations In the M2 gene of a M viral segment that result in deletion(s) or substitution(s) in the extracellular domain of M2 do not substantially alter the function of the protein encoded by the Mi gene. in one embodiment, fewer than 20%, e.g., 10% or 5%, of the residues In the TM domain or extracellular domain are substituted. In one embodiment, fewer than 60%, e.g., 50%, 40%, 30%, 20%, 10%, or 5% of the residues in the extracellular domain are deleted, in one embodiment, more than 20%, e.g., 30%, 40%, 50%, 80% or more, of the residues in the TM domain are deleted.

Exemplary PB2 Viral Segments in one embodiment, the recombinant influenza virus has a viral gene segment that does not comprise contiguous nucleic acid sequences corresponding to those encoding PB2 (a mutant PB2 viral gene segment), a protein which Is one of the viral RNA polymerase subunits and is essential for virus replication. To prepare such a virus in cell culture, a cel! line is employed that expresses PB2 in trans in combination with vectors for influenza virus vRNA production, but not one for a wild-type PB2 viral gene segment, and in one embodiment vectors for influenza virus mRNA protein production. The resulting virus is not competent to express PB2 after infection of cells that do not express PB2 in trans or are not infected with helper virus. However, virions produced from cells that express PB2 in trans contain PB2. Such an infectious influenza virus with a mutant PB2 viral gene segment can be generated in muitiple cel! lines that express PB2 in trans, such as PB2 -expressing 293 human embryonic kidney (293), human lung adenocarcinoma epithelial (A549), or 2,6-linked siaiyltransferase- overexpress!ng Madin-Darby canine kidney (MOCK) cells (AX4 cells), resulting In high virus titers of at least 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ or 10 ⁸ PFU/mL ,or more. For example, a PB2-KO virus replicated to high titers (>10 ⁸ PFU/mL) in PB2- expressing but not in norma! uninfected cells (cells that do not express PB2 in trans), accommodated HA and NA genes of a heterologous Influenza virus), stably incorporated a reporter gene into progeny PB2-KO virions that was retained through sequential passages, and was attenuated in mice. Significantly higher levels of IgG and IgA antibodies were induced in sera, nasal washes and broncho-αlveolar lavage samples from mice immunized with only one dose of PB2-KO (GFP) virus compared to inactivated influenza vaccine. Ail PB2-KO virus-treated mice survived challenge with various lethal doses of PR8. in one embodiment, limited replication of that virus occurs in vivo as the virus produced in cells that express PB2 in trans carries along a small amount of PB2 protein into the host cell which is subsequently infected (such as a host cell which does not itself express or comprise PB2 or comprise wild-type PB2 vRNA), thereby allowing for a limited amount (e.g., a round or so) of replication to occur but without a significant infectious process (for Instance, amplification of virus titers of over about 1000). The limited replication of the KQ virus in vivo allows for an immune response that provides for a more robust immune response than induced by conventional inactivated influenza vaccines. The PB2-KO exhibited similar or better safety and efficacy profiles when compared to controls.

In one embodiment, the disclosure provides an isolated recombinant Influenza virus comprising 8 gene segments including a PA viral gene segment, a PB1 viral gene segment, a mutant PB2 viral gene segment, a HA viral gene segment, a NA viral gene segment, a NP viral gene segment, a M (M1 and M2) viral gene segment, and a NS (NS1 and NS2) viral gene segment. In another embodiment, the disclosure provides an Isolated recombinant influenza virus comprising 8 gene segments including a PA viral gene segment, a PB1 viral gene segment, a mutant PB2 viral gene segment, a HA viral gene segment, a NA (NA and NB) viral gene segment, a NP viral gene segment, a M (Mi and BM2) viral gene segment, and a NS (NS1 and NS2) viral gene segment, in one embodiment, the recombinant influenza virus has a M viral gene segment for M1 and M2, in one embodiment, the recombinant influenza virus has a NA viral gene segment for NB and NA. in one embodiment, the recombinant influenza virus has a HEF gene segment.

In yet another embodiment, the disclosure provides an isolated recombinant influenza virus comprising gene segments including a PA viral gene segment, a PB1 viral gene segment, a mutant PB2 viral gene segment, a NP viral gene segment, a M viral gene segment, a NS viral gene segment (for NS1 and NS2), and a HEF viral gene segment, in one embodiment, the mutant PB2 viral gene segment includes 5' and/or 3‘ PB2 viral non-coding and coding incorporation sequences, optionally flanking a heterologous nucleotide sequence, and does not include contiguous sequences corresponding to sequences encoding a functional PB2. The PB2 open reading frame In the mutant PB2 viral gene segment may be replaced with or disrupted by a heterologous nucleotide sequence, such as one that is readily detectable after transfection or infection, e.g., a reporter gene such as a GFP gene or a luciferase gene, e.g., a Renilla luciferase gene, or a gene encoding ACE2. in one embodiment, the PB2 coding region in the mutant PB2 viral gene segment may inciude mutations such as insertions or deletions of one or more nucleotides or those that result in one or more amino acid substitutions or a stop codon, or any combination thereof, that yields a non- functional PB2 coding sequence. In one embodiment, the heterologous nucleotide sequence Is about 30 to about 5,000, e.g., about 100 to about 4,500 or about 500 to about 4,000, nucleotides in length.

The viruses may thus be used as Influenza vaccines to induce an immunogenic response in a host, without the risk of symptoms associated with an infection or genetic reversion to a fully infectious form. The viruses may elicit a better Immune response than chemically inactivated viruses because they are live viruses, yet because they are biologically contained, the viruses of the invention likely do not cause symptoms of the disease, which is often an issue with live attenuated vaccines. And In contrast to the use of virus-!ike particles (VLPs), which are non-repiicative, the KO viruses contain RNA, which Is an adjuvant that enhances the host's immune response against the virus. in one embodiment, the invention provides an isolated recombinant influenza virus comprising 7 gene segments including a PA viral gene segment, a PB1 viral gene segment, a HA viral gene segment, a NA viral gene segment, a NP viral gene segment, a M viral gene segment, and a NS1 and NS2 viral gene segment, i.e., the virus lacks a PB2 viral gene segment. in one embodiment, for the 8 segment PB2-KO influenza virus having a mutant PB2 viral gene segment, the mutant PB2 viral gene segment has a deletion of PB2 coding sequences, a deletion of PB2 coding sequences and an insertion of heterologous nucleotide sequences, or an insertion of heterologous nucleotide sequences which disrupts PB2 coding sequences. That virus replicates in vitro when PB2 is supplied in trans to titers that are substantially the same or at most 10, 100 or 1 ,000 fold less than a corresponding wild-type influenza virus, but Is attenuated in vivo or in vitro in the absence of PB2 supplied in trans. In one embodiment, the deletion of PB2 ceding sequences includes 1 or more contiguous or noncontiguous nucleotides of PB2 and may include a deletion of the entire coding region, e.g., a region encoding 759 amino acids. In one embodiment, the deletion includes at least 10%, 30%, 40%, 50%, 70%, 80%, 85%, 90%, 93%, 95% and up to 99%, or a percent numerical value that is any integer between 10 and 99, but not all, of the PB2 coding region. In one embodiment, the deletion of PB2 coding sequences does not include the deletion of 5’ or 3' coding sequences that enhance incorporation of the resulting viral gene segment Into virions, e.g., sequences that are contiguous to 3' or 5‘ non-coding PB2 sequences, relative to a recombinant viral gene segment with cnly non-coding PB2 Incorporation sequences. in one embodiment, the mutant PB2 gene segment may comprise an insertion of one or more nucleotides, e.g., those that result in a frame-shift, so that functional PB2 cannot be expressed. In one embodiment, the insertion does not include the alteration of 5’ or 3’ coding sequences that enhance incorporation of the gene segment into virions relative to a recombinant gene segment with only non-coding PB2 incorporation sequences.

In one embodiment, the mutant PB2 viral gene segment may comprise at least one mutation that results in at least one amino acid substitution re!ative to a corresponding wild- type PB2 protein, e.g., a mutation that removes or replaces the Initiator codon, or that introduces one or more stop codons into the coding region, so that functional PB2 cannot be expressed from that viral gene segment after infection. In one embodiment, the substitution, removal or replacement of the Initiator codon, or Introduction of the one or more stop codons in the reading frame for PB2, does not include the alteration of 5’ or 3’ coding sequences that enhance incorporation of the gene segment into virions relative to a recombinant gene segment with only non-coding PB2 Incorporation sequences.

Exemplary NS Viral Segments in one embodiment, the coding region for the soluble portion of ACE2 is fused to the coding region for NS1 in the NS viral segment. For example, the open reading frame

(ORF) of the NS1 gene without the stop codon is fused with the open reading frame for soluble ACE2 via a linker that includes a protease site, in one embodiment, the linker may Include amino acid residues that do not form a protease site, e.g., GSGG.

In one embodiment, an open reading frame encoding a foot-αnd-mouth virus protease 2A autoproteolytic site optionally with 57 nucleotides from porcine teschovirus-1 is employed, in one embodiment, a second open reading frame encoding a protease site is at the C-terminus of the soluble ACE2 open reading frame, so that the open reading frame tor nuclear export protein (NEP: NS2) can be added to the fusion. In addition, silent mutations may be introduced Into the endogenous splice acceptor site of the NS1 gene to abrogate splicing.

Exemplary PR8 Viral Segments for internal Viral Proteins

Exemplary viral sequences for a backbone master vaccine strain (PR.8UW) are as follows:

PA

AGCG.A.AAGCA GGTACTGATC CAAAATGGAA GATTTTGTGC GACAATGCTT CAATCCGATG ATTGTCGAGC TTGCGGAAAA AACAATGAAA GAGTATGGGG AGGACCTGAA AATCGAAACA AACAAATTTG CAGCAATATG CACTCACTTG GAAGTATGCT TCATGTATTC AGATTTTCAC TTCATCAATG AGCAAGGCGA GTCAATAATC GTAGAACTTG GTG ATCCAAA T GCACTTTT G AAGCACAGAT TIG AAAI A AT CGAGGGAAGA GATCGCACAA TGGCCTGGAC AGTAGTAAAC AGTATTTGCA ACACTACAGG GGCTGAGAAA CCAAAGTTTC TACCAGATTT GTATGATTAC AAGGAGAATA GATTCATCGA AATTGGAGTA ACAAGGAGAG AAGTTCACAT ATACTATCTG GAAAAGGCCA ATAAAATTAA ATCTGAGAAA ACACACATCC ACATTTTCTC GTTC.ACTGGG GAAGAAATGG CCACAAAGGC AGACTACACT CTCGATGAAG AAAGCAGGGC TAGGATCAAA ACCAGACTAT TCACCATAAG ACAAGAAATG GCCAGCAGAG GCCTCTGGGA TTCCTTTCGT CAGTCCGAGA GAGGAGAAGA GACAATTGAA GAAAGGTTTG AAATCACAGG AACAATGCGC AAGCTTGCCG ACCAAAGTCT CCCGCCGAAC TTCTCCAGCC TTGAAAATTT TAGAGCCTAT GTGGATGGAT TCGAACCGAA CGGCTACATT GAGGGCAAGC TGTCTCAAAT GTCCAAAGAA GTAAATGCTA GAATTGAACC TTTTTTGAAA ACAACACCAC GACCACTTAG ACTTCCGAAT GGGCCTCCCT GTTCTCAGCG GTCCAAATTC CTGCTGATGG ATGCCTTAAA ATTAAGCATT GAGGACCCAA GTCATGAAGG AGAGGGAATA CCGCTATATG ATGCAATCAA ATGCATGAGA ACATTCTTTG GATGGAAGGA ACCCAATGTT GTTAAACCAC ACGAAAAGGG AATAAATCCA AATTATCTTC TGTCATGGAA GCAAGTACTG GCAGAACTGC AGGACATTGA GAATGAGGAG AAAATTCCAA AGACTAAAAA TATGAAGAAA ACAAGTCAGC TAAAGTGGGC ACTTGGTGAG AACATGGCAC CAGAAAAGGT AGACTTTGAC GACTGTAAAG ATGTAGGTGA TTTGAAGCAA TATGATAGTG ATGAACCAGA ATTGAGGTCG CTTGCAAGTT GGATTCAGAA TGAGTTTAAC AAGGCATGCG AACTGACAGA TTCAAGCTGG ATAGAGCTCG ATGAGATTGG AGAAGATGTG GCTCCAATTG AACACATTGC AAGCATGAGA AGGAATTATT TCACATCAGA GGTGTCTCAC TGCAGAGCCA CAGAATACAT AATGAAGGGA GTGTACATCA ATACTGCCTT GCTTAATGCA TCTTGTGCAG CAATGGATGA TTTCCAATTA ATTCCAATGA TAAGCAAGTG TAGAACTAAG GAGGGAAGGC GAAAGACCAA CTTGTATGGT TTCATCATAA AAGGAAGATC CCACTTAAGG AATGACACCG ACGTGGTAAA CTTTGTGAGC ATGGAGTTTT CTCTCACTGA CCCAAGACTT GAACCACATA AATGGGAGAA GTACTGTGTT CTTGAGATAG GAGATATGCT TATAAGAAGT GCCATAGGCC AGGTTTCAAG GCCCATGTTC TTGTATGTGA GAACAAATGG AACCTCAAAA ATTAAAATGA AATGGGGAAT GGAGATGAGG CGTTGCCTCC TCCAGTCACT TCAACAAATT GAGAGTATGA TTGAAGCTGA GTCCTCTGTC AAAGAGAAAG ACATGACCAA AGAGTTCTTT GAGAACAAAT CAGAAACATG GCCCATTGGA GAGTCCCCCA AAGGAGTGGA GGAAAGTTCC ATTGGGAAGG TCTGCAGGAC TTTATTAGCA AAGTCGGTAT TCAACAGCTT GTATGCATCT CCACAACTAG AAGGATTTTC AGCTGAATCA AGAAAACTGC TTCTTATCGT TCAGGCTCTT AGGGACAACC TGGAACCTGG G ACCTTT GAT CTTGGGGGGC TATATGAAGC AATTGAGGAG TGCCTGATTA ATGATCCCTG GGTTTTGCTT AATGCTTCTT GGTTCAACTC CTTCCTTACA CATGCATTGA GTTAGTTGTG GCAGTGCTAC TATTTGCTAT CCATACTGTC CAAAAAAGTA CCTTGTTTCT ACT (SEQ, ID NQ:3)

PB1

AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACCTTACTTTTCTTAAAA GTGCCA

GCACAAAATGCTATAAGCACAACTTTCCCTTATACTGGAGACCCTCCTTACAGCCAT GGGACAG

GAACAGGATACACCATGGATACTGTCAACAGGACACATCAGTACTCAGAAAAGGGAA GATGG

ACAACAAACACCG AAACTG GAG CACCGCAACTCAACCCG ATTG ATG GG CCACTGCCAG AAGA

CAATGAACCAAGTGGTTATGCCCAAACAGATTGTGTATTGGAGGCGATGGCT7TCCT TGAGGA

ATCCCATCCTGGTATTTTTGAAAACTCGTGTATTGAAACGATGGAGGTTGTTCAGCA AACACG

AGTAGACAAGCTGACACAAGGCCGACAGACCTATGACTGGACTCTAAATAGAAACCA ACCTG

CTGCAACAGCATTGGCCAACACAATAGAAGTGTTCAGATCAAATGGCCTCACGGCCA ATGAGT

CTGGAAGGCTCATAGACTTCCTTAAGGATGTAATGGAGTCAATGAACAAAGAAGAAA TGGGG

ATCACAACTCATTTTCAGAGAAAGAGACGGGTGAGAGACAATATGACTAAGAAAATG ATAAC

ACAGAGAACAATGGGTAAAAAGAAGCAGAGATTGAACAAAAGGAGTTATCTAATTAG AGCAT

TGACCCTGAACACAATGACCAAAGATGCTGAGAGAGGGAAGCTAAAACGGAGAGCAA TTGC

AACCCCAGGGATGCAAATAAGGGGGTTTGTATACTTTGTTGAGACACTGGCAAGGAG TATAT

GTGAGAAACTTGAACAATCAGGGTTGCCAGTTGGAGGCAATGAGAAGAAAGCAAAGT TGGC

A AAT GTTGT AAG G AAG ATG ATG ACC AATTCT CAG G AC ACCG AACTTT CTTT CACCAT CACT G G A

GATAACACCAAATGGAACGAAAATCAGAATCCTCGGATGTTTTTGGCCATGATCACA TATATG

ACCAGAAATCAGCCCGAATGGTTCAGAAATGTTCTAAGTATTGCTCCAATAATGTTC TCAAACA

AAATGGCGAGACTGGGAAAAGGGTATATGTTTGAGAGCAAGAGTATGAAACTTAGAA CTCAA

ATACCTGCAGAAATGCTAGCAAGCATCGATTTGAAATATTTCAATGATTCAACAAGA AAGAAG ATTGAAAAAATCCGACCGCTCTTAATAGAGGGGACTGCATCATTGAGCCCTGGAATGATG ATG

GGCATGTTCAATATGTTAAGCACTGTATTAGGCGTCTCCATCCTGAATCTTGGACAA AAGAGA

TACACCAAGACTACTTACTGGTGGGATGGTCTTCAATCCTCTGACGATTTTGCTCTG ATTGTGA

ATGCACCCAATCATGAAGGGATTCAAGCCGGAGTCGACAGGTTTTATCGAACCTGTA AGCTAC

TTGGAATCAATATGAGCAAGAAAAAGTCTTACATAAACAGAACAGGTACATTTGAAT TCACAA

G TTTTTTCTATCGTT ATG G GTTT GTTG CC AATTTC AG CATG G AG C TTCCC AGTTTTG G G G TGT CT

GGGATCAACGAGTCAGCGGACATGAGTATTGGAGTTACTGTCATCAAAAACAATATG ATAAA

CAATGATCTTGGTCCAGCAACAGCTCAAATGGCCCTTCAGTTGTTCATCAAAGATTA CAGGTAC

ACGTACCGATGCCATATAGGTGACACACAAATACAAACCCGAAGATCATTTGAAATA AAGAAA

CTGTGGGAGCAAACCCGTTCCAAAGCTGGACTGCTGGTCTCCGACGGAGGCCCAAAT TTATAC

AACATTAGAAATCTCCACATTCCTGAAGTCTGCCTAAAATGGGAATTGATGGATGAG GATTAC

CAGGGGCGTTTATGCAACCCACTGAACCCATTTGTCAGCCATAAAGAAATTGAATCA ATGAAC

AATGCAGTGATGATGCCAGCACATGGTCCAGCCAAAAACATGGAGTATGATGCTGTT GCAAC

AACACACTCCTGGATCCCCAAAAGAAATCGATCCATCTTGAATACAAGTCAAAGAGG AGTACT

TGAGGATGAACAAATGTACCAAAGGTGCTGCAATTTATTTGAAAAATTCTTCCCCAG CAGTTCA

TACAGAAGACCAGTCGGGATATCCAGTATGGTGGAGGCTATGGTTTCCAGAGCCCGA ATTGA

TGCACGGATTGATTTCGAATCTGGAAGGATAAAGAAAGAAGAGTTCACTGAGATCAT GAAGA

TCTGTTCCACCATTGAAGAGCTCAGACGGCAAAAATAGTGAATTTAGCTTGTCCTTC ATGAAAA

A AT G CCTTGTTTCT ACT (SEQ ID N0:2)

PB2

AGCGAAAGCA GGTCAATTAT ATTCAATATG GAAAGAATAA AAGAACTACG AAATCTAATG TCGCAGTCTC GCACCCGCGA GATACTCACA AAAACCACCG TGGACCATAT GGCCATAATC AAGAAGTACA CATCAGGAAG ACAGGAGAAG AACCCAGCAC TTAGGATGAA ATGGATGATG GCAATGAAAT ATCCAATTAC AGCAGACAAG AGGATAACGG AAATGATTCC TGAGAGAAAT GAGCAAGGAC AAACTTTATG GAGTAAAATG AATGATGCCG GATCAGACCG AGTGATGGTA TCACCTCTGG CTGTGACATG GTGGAATAGG AATGGACCAA TAACAAATAC AGTTCATTAT CCAAAAATCT ACAAAACTTA TTTTGAAAGA GTCGAAAGGC TAAAGCATGG AACCTTTGGC CCTGTCCATT TTAGAAACCA AGTCAAAATA CGTCGGAGAG TTGACATAAA TCCTGGTCAT GCAGATCTCA GTGCCAAGGA GGCACAGGAT GTAATCATGG AAGTTGTTTT CCCTAACGAA GTGGGAGCCA GGATACTAAC ATCGGAATCG CAACTAACGA TAACCAAAGA GAAGAAAGAA GAACTCCAGG ATT G CAAAAT TTCT CCTTT G ATG GTTG CAT ACATGTTGGA GAGAGAACTG GTCCGCAAAA CGAGATTCCT CCCAGTGGCT GGTGGAACAA GCAGTGTGTA CATTGAAGTG TTGCATTTGA CTCAAGGAAC ATGCTGGGAA CAGATGTATA CTCCAGGAGG GGAAGTGAGG AATGATGATG TTGATCAAAG CTT GATT ATT GCTGCTAGGA ACATAGTGAG AAGAGCTGCA GTATCAGCAG ATCCACTAGC ATCTTTATTG GAGATGTGCC ACAGCACACA GATTGGTGGA ATTAGGATGG TAGACATCCT TAGGCAG AAC CCAACAGAAG AGCAAGCCGT GGATATATGC AAGGCTGCAA TGGGACTGAG AATTAGCTCA TCCTTCAGTT TTGGTGGATT CACATTTAAG AGAACAAGCG GATCATCAGT CAAGAGAGAG GAAGAGGTGC TTACGGGCAA TCTTCAAACA TTGAAGATAA GAGTGCATGA GGGATATGAA GAGTTCACAA TGGTTGGGAG AAGAGCAACA GCCATACTCA GAAAAGCAAC CAGGAGATTG ATTCAGCTGA TAGTGAGTGG GAGAGACGAA CAGTCGATTG CCGAAGCAAT AATTGTGGCC ATGGTATTTT CACAAGAGGA TTGTATGATA AAAGCAGTCA GAGGTGATCT GAATTTCGTC AATAGGGCGA ATCAACGATT GAATCCTATG CATCAACTTT TAAGACATTT TCAGAAGGAT GCGAAAGTGC

CCGACATGAC TCCAAGCATC GAGATGTCAA TGAGAGGAGT GAGAATCAGC AAAATGGGTG TAGATGAGTA CTCCAGCACG GAGAGGGTAG TGGTGAGCAT TG ACCGTTTT TTGAGAATCC GGGACCAACG AGGAAATGTA CTACTGTCTC CCGAGGAGGT CAGTGAAACA CAGGGAACAG AGAAACTGAC AATAACTTAC TCATCGTCAA TGATGTGGGA GATTAATGGT CCT GAATCAG TGTTGGTCAA TACCTATCAA TGGATCATCA GAAACTGGGA AACTGTTAAA ATTCAGTGGT CCCAGAACCC TACAATGCTA TACAATAAAA TGGAATTTGA ACCATTTCAG TCTTTAGTAC CTAAGGCCAT TAGAGGCCAA TACAGTGGGT TTGTAAGAAC TCTGTTCCAA CAAATGAGGG ATGTGCTTGG GACATTTGAT ACCGCACAGA TAATAAAACT TCTTCCCTTC GCAGCCGCTC CACCAAAGCA AAGTAGAATG CAGTTCTCCT CATTTACTGT GAATGTGAGG GGATCAGGAA TGAGAATACT TGTAAGGGGC AATTCTCCTG TATTCAACTA TAACAAGGCC ACGAAGAGAC TCACAGTTCT CGGAAAGGAT GCTGGCACTT TAACTGAAGA CCCAGATGAA GGCACAGCTG GAGTGGAGTC CGCTGTTCTG AGGGGATTCC TCATTCTGGG CAAAGAAGAC AAGAGATATG GGCCAGCACT AAGCATCAAT GAACTGAGCA ACCTTGCGAA AGGAGAGAAG GCTAATGTGC TAATTGGGCA AGGAGACGTG GTGTTGGTAA TGAAACGGAA ACGGGACTCT AGCATACTTA CTGACAGCCA GACAGCGACC AAAAGAATTC GGATGGCCAT CAATTAGTGT CGAATAGTTT AAAAACGACC TTGTTTCTAC T (SEQ ID NQ:1) NP

AGCAAAAGCA GGGTAGATAA TCACTCACTG AGTGACATCA AAATCATGGC GTCTCAAGGC ACCAAACGAT CTTACGAACA GATGGAGACT GATGGAGAAC GCCAGAATGC CACTGAAATC AGAGCATCCG TCGGAAAAAT GATTGGTGGA ATTG G ACG AT TCTACATCCA AATGTGCACC GAACTCAAAC TCAGTGATTA TGAGGGACGG TTGATCCAAA ACAGCTTAAC AATAGAGAGA ATGGTGCTCT CTGCTTTTGA CGAAAGGAGA AATAAATACC TTGAAGAACA TCCCAGTGCG GGGAAAGATC CTAAGAAAAC TGGAGGACCT AT AT AC AG G A GAGTAAACGG AAAGTGGATG AGAGAACTCA TCCTTTATGA CAAAGAAGAA ATAAGGCGAA TCTGGCGCCA AGCTAATAAT GGTGACGATG CAACGGCTGG TCTGACTCAC ATGATGATCT GGCATTCCAA TTTGAATGAT GCAACTTATC AGAGGACAAG AGCTCTTGTT CGCACCGGAA TGGATCCCAG GATGTGCTCT CTGATGCAAG GTTCAACTCT CCCTAGGAGG TCTGGAG CCG CAGGTGCTGC AGTCAAAGGA GTTGGAACAA TGGTGATGGA ATTGGTCAGA ATGATCAAAC GTGGGATCAA TGATCGGAAC TTCTGGAGGG GTGAGAATGG ACGAAAAACA AGAATTGCTT ATGAAAGAAT GTGCAACATT CTCAAAGGGA AATTTCAAAC TGCTGCACAA AAAGCAATGA TGGATCAAGT GAGAGAGAGC CGGAACCCAG GGAATGCTGA GTTCGAAGAT CTCACTTTTC TAGCACGGTC TGCACTCATA TTGAGAGGGT CGGTTGCTCA CAAGTCCTGC CTGCCTGCCT GTGTGTATGG ACCTGCCGTA GCCAGTGGGT ACGACTTTGA AAGGGAGGGA TACTCTCTAG TCGGAATAGA CCCTTTCAGA CTGCTTCAAA ACAGCCAAGT GTACAGCCTA ATCAGACCAA ATGAGAATCC AGCACACAAG AGTCAACTGG TGTGGATGGC ATGCCATTCT GCCGCATTTG AAGATCTAAG AGTATTAAGC TTCATCAAAG GGACGAAGGT GCTCCCAAGA GGGAAGCTTT CCACTAGAGG AGTTCAAATT GCTTCCAATG AAAATATGGA GACTATGGAA TCAAGTACAC TTGAACTGAG AAGCAGGTAC TGGGCCATAA GGACCAGAAG TGGAGGAAAC ACCAATCAAC AGAGGGCATC TGCGGGCCAA AT C AG CAT AC AACCTACGTT CTCAGTACAG AGAAATCTCC CTTTTGACAG AACAACCATT ATGGCAGCAT TCAATGGGAA TACAGAGGGG AGAACATCTG ACATGAGGAC CGAAATCATA AGGATGATGG AAAGTGCAAG ACCAGAAGAT GTGTCTTTCC AGGGGCGGGG AGTCTTCGAG CTCTCGGACG AAAAGGCAGC GAGCCCGATC GTGCCTTCCT TTGACATGAG TAATGAAGGA TCTTATTTCT TCGGAG ACAA TGCAGAGGAG TACGACAATT AAAGAAAAAT ACCCTTGTTT CTACT (SEQ ID N0:4)

M

AGCAAAAGCA GGTAGATATT GAAAGATGAG TCTTCTAACC GAGGTCGAAA CGTACGTACT CTCTATCATC CCGTCAGGCC CCCTCAAAGC CGAGATCGCA CAGAGACTTG AAGATGTCTT TGCAGGGAAG AACACCGATC TTGAGGTTCT CATGGAATGG CTAAAGACAA GACCAATCCT GTCACCTCTG ACTAAGGGGA TTTTAGGATT TGTGTTCACG CTCACCGTGC CCAGTGAGCG AGGACTGCAG CGTAGACGCT TTGTCCAAAA TGCCCTTAAT GGGAACGGGG ATCCAAATAA CATGGACAAA GCAGTTAAAC TGTATAGGAA GCTCAAGAGG GAGATAACATTCCATGGGGC CAAAGAAATC TCACTCAGTT ATTCTGCTGG TGCACTTGCC AGTTGTATGG GCCTCATATA CAACAGGATG GGGGCTGTGA CCACTGAAGT GGCATTTGGC CTGGTATGTG CAACCTGTGA ACAGATTGCT GACTCCCAGC ATCGGTCTCA TAGGCAAATG GTGACAACAA CCAATCCACT AATCAGACAT GAGAACAGAA TGGTTTTAGC CAGCACTACA GCTAAGGCTA TGGAGCAAAT GGCTGGATCG AGTGAGCAAG CAGCAGAGGC CATGGAGGTT GCTAGTCAGG CTAGACAAAT GGTGCAAGCG ATGAGAACCA TTGGGACTCA TCCTAGCTCC AGTGCTGGTC TGAAAAATGA TCTTCTTGAA AATTT GCAGG CCTATCAGAA ACGAATGGGG GTGCAGATGC AACGGTTCAA GTGATCCTCT CACTATTGCC GCAAATATCA TTGGGATCTT GCACTTGACA TTGTGGATTC TTGATCGTCT TTTTTTCAAA TGCATTTACC GTCGCTTTAA ATACGGACTG AAAGGAGGGC CTTCTACGGA AGGAGTGCCA AAGTCTATGA GGGAAGAATA TCGAAAGGAA CAGCAGAGTG CTGTGGATGC TGACGATGGT CATTTTGTCA GCATAGAGCT GGAGTAAAAA ACTACC7TGT TTCTACT (SEQ ID N0:5)

NS

AGCAAAAGCA GGGTGACAAA AACATAATGG ATCCAAACAC TGTGTCAAGC TTTCAGGTAG ATTGCTTTCT TTGGCATGTC CGCAAACGAG TTGCAGACCA AGAACTAGGC GATGCCCCAT TCCTTGATCG GCTTCGCCGA GAT CAGAAAT CCCTAAGAGG AAGGGGCAGT ACTCTCGGTC TGGACATCAA GACAGCCACA CGTGCTGGAA AGCAGATAGT GGAGCGGATT CTGAAAGAAG AATCCGATGA GGCACTTAAA ATGACCATGG CCTCTGTACC TGCGTCGCGT TACCTAACTG AC ATG ACTCT TG AG G AAAI G TCAAGGGACT GGTCCATGCT CATACCCAAG CAGAAAGTGG CAGGCCCTCT TTGTATCAGA ATGG ACCAGG CGATCATGGA TAAGAACATC ATACTGAAAG CGAACTTCAG TGTGATTTTT GACCGGCTGG AGACTCTAAT ATTGCTAAGG GCTTTCACCG AAGAGGGAGC AATTGTTGGC GAAATTTCAC CATTGCCTTC TCTTCCAGGA CATACTGCTG AGGATGTCAA AAATGCAGTT GGAGTCCTCA TCGGAGGACT TGAATGG AAT GATAACACAG TTCGAGTCTC TGAAACTCTA CAGAGATTCG CTTGGAGAAG CAGTAATGAG AATGGGAGAC CTCCACTCAC TCCAAAACAG AAACGAGAAA TGGCGGGAAC AATTAGGTCA GAAGTTTGAA GAAATAAGAT GGTTGATTGA AGAAGTGAGA CACAAACTGA AGATAACAGA GAATAGTTTT G AG C AAAI A A CATTTATGCA AGCCTTACAT CTATTGCTTG AAGTGGAGCA AGAGATAAGA ACTTTCTCGT TTCAGCTT.AT TTAGTACTAA AAAACACCCT TGTTTCTACT (SEQ ID N0:6).

Also included are sequences encoding a polypeptide with at least 80%, e.g., 85%, 90%, 92%, 95%, 98%, 99% or 100%, including any integer between 80 and 100, amino acid Identity to a polypeptide encoded by one of SEQ ID NQs:1 -6.

Exemplary Cambridge Sequences for Internal Viral Proteins agcgaaagca ggtcaattat attcaatatg gaaagaataa aagaactaag aaatctaatg tcgcagtctc gcacccgcga gatactcaca aaaaccaccg tggaccatat ggccataatc aagaagtaca catcaggaag aeaggagaag aacceagcac ttaggatgaa atggatgatg gcaatgaaat atccaattac agcagacaag aggataacgg aaatgaticc tgagagaaat gagcaaggac aaactttatg gagtaaaatg aatgatgccg gatcagaccg agtgatggta icacctctgg ctgtgacatg gtggaatagg aatggaccaa tgacaaatac agttcattat ccaaaaatct acaaaactta ttttgaaaga gtcgaaaggc taaagcatgg aacctttggc cctgtccatt ttagaaacca agtcaaaata egtcggagag ttgacataaa tcctggtcat gcagaictca gtgccaagga ggcacaggat giaatcatgg aagttgtttt ccctaacgaa gtgggagcca ggatactaac atcggaatcg caactaacga taaccaaaga gaagaaagaa gaactccagg attgcaaaat ttctcctttg atggttgcat acatgttgga gagagaactg gtccgcaaaa cgagattcct cccagtggct ggtggaacaa gcagtgtgta cattgaagtg itgcattga ctcaaggaac atgctgggaa eagatgtata ctccaggagg ggaagtgaag aatgatgatg ttgatcaaag cttgattatt gctgctagga acatagtgag aagagctgca gtatcagcag acceaciagc aicttattg gagatgtgcc acagcacaca gattggtgga attaggatgg tagacatcct taagcagaac ccaacagaag agcaagccgt ggatatatgc aaggctgcaa tgggactgag aattagctca tccttcagtt ttggtggatt cacatttaag agaacaagcg gatcatcagt caagagagag gaagaggtgc ttacgggcaa icttcaaaca ttgaagataa gagtgcatga gggatcigaa gagttcacaa tggtigggag aagagcaaca gccatactca gaaaagcaac caggagattg attcagctga tagtgagtgg gagagacgaa cagtcgattg ccgaagcaat aattgtggcc atggtatttt cacaagagga ttgtatgata aaagcagtta gaggigatct gaaittcgic aatagggcga atcagcgact gaatcciaig catcaacttt taagacattt tcagaaggat gcgaaagtgc tttttcaaaa ttggggagtt gaacctatcg acaatgtgat gggaatgat gggatattgc ccgacatgac tccaagcatc gagatgtcaa tgagaggagt gagaatcagc aaaatgggtg tagatgagta ctccagcacg gagagggtag tggigagcat tgaccggttc ttgagagtca gggaccaacg aggaaatgta ciactgtctc ccgaggaggt cagtgaaaca cagggaacag agaaactgac aataacttac tcatcgtcaa tgatgtggga gattaatggt cctgaatcag tgtggtcaa tacctatcaa iggatcatca gaaactggga aactgttaaa attcagtggt cccagaaccc tacaatgcta tacaataaaa tggaatttga accatttcag tctttagtac ctaaggccat tagaggccaa iacagtgggi tgiaagaac tcigttccaa caaatgaggg atgtgcitgg gacaiitgat accgcacaga taataaaact tcttcccttc gcagccgctc caccaaagca aagtagaatg cagttctcct catttactgt gaatgtgagg ggatcaggaa tgagaaiact tgtaaggggc aattcicctg tatteaacta caacaaggcc acgaagagac tcaeagttct cggaaaggat gctggcactt taaccgaaga cccagatgaa ggcacagctg gagtggagtc cgctgttctg aggggattcc tcaitciggg caaagaagac aggagaiaig ggccagcat aagcatcaat gaactgagca accttgcgaa aggagagaag gciaatgtgc taattgggca aggagacgtg gtgtiggtaa tgaaacgaaa acgggacict agcatactta ctgacagcca gacagcgacc aaaagaattc ggatggccai caattagtgt cgaatagttt aaaaacgacc tgtttctac t (SEQ ID NO: 10) agcgaaagca ggcaaaccat ttgaatggat gtcaatccga ccitactttt cttaaaagtg ccagcacaaa aigctataag cacaacitic ccttataccg gagaccctcc tacagccat gggacaggaa caggatacac catggatact gtcaacagga cacatcagta ctcagaaaag ggaagatgga caacaaacac cgaaactgga gcaccgcaac tcaacccgat tgatgggcca ctgccagaag acaaigaaec aagtggttat gcccaaacag attgtgtatt ggaagcaatg gctttccttg aggaatccca tcctggtatt tttgaaaact cgtgiaitga aacgatggag gttgttcagc aaacacgagt agacaagctg aeaeaaggec gacagaccta tgactggact ttaaatagaa accagcctgc tgcaacagca ttggccaaca eaatagaagt gitcagaica aatggcctca cggccaatga gtcaggaagg ctcatagact tccttaagga tgtaatggag tcaatgaaaa aagaagaaat ggggatcaca actcattttc agagaaagag acgggtgaga gacaatatga ctaagaaaat gataacacag agaacaatag gtaaaaggaa acagagatig aacaaaaggg gttatctaat tagagcattg acectgaaea caatgaccaa agatgctgag agagggaagc taaaacggag agcaattgca accccaggga tgcaaataag ggggtttgta tactttgttg agacactggc aaggagtata tgtgagaaac ttgaacaatc agggttgcca gtiggaggca aigagaagaa agcaaagitg gcaaaigiig taaggaagat gatgaccaat tctcaggaca ccgaacittc titcaccatc actggagata acaccaaatg gaacgaaaai cagaaicctc ggatgttttt ggccaigatc acaiaiaiga ccagaaatca gcccgaatgg ttcagaaatg ttctaagtat tgctccaata atgttctcaa acaaaatggc gagactggga aaagggtata tgtttgagag caagagtatg aaacttagaa ctcaaatacc tgcagaaatg ciagcaagca ttgatttgaa atattteaat gattcaacaa gaaagaagat tgaaaaaatc cgaccgctct taatagaggg gactgcatca ttgagccctg gaatgatgat gggcatgttc aatatgttaa gcactgtati aggcgtctcc atcctgaatc ttggacaaaa gagatacacc aagactactt actggtggga tggtcttcaa tcctctgacg attttgctct gattgtgaat gcacccaatc aigaagggat tcaagccgga gicgacaggi iitatcgaac etgiaagcta cttggaatca atatgagcaa gaaaaagtct tacataaaca gaacaggtac atttgaattc acaagttttt tctatcgtta tgggtttgtt gccaatttca gcatggagct tcocagtttt ggggtgtctg ggatcaacga gtcagcggac atgagiaiig gagttactgt caicaaaaac aatatgataa acaatgatct tggtccagca acagctcaaa tggcccttca gttgttcatc aaagaitaca ggtacacgta ccgatgccat agaggtgaca cacaaataca aacccgaaga tcatttgaaa taaagaaact gtgggagcaa acccgttcca aagctggact gctggtctcc gacggaggcc caaattaia caacattaga aateiccaea itcctgaagt ctgcciaaaa tgggaattga iggatgagga itaccagggg cgtitatgca acccactgaa cccatttgtc agccataaag aaattgaatc aatgaacaat gcagtgatga tgccagcaea tggtccagcc aaaaacatgg agtatgatgc tgttgcaaca acacactcct ggatccccaa aagaaatega tccatcttga atacaagtca aagaggagta cttgaagatg aacaaatgta ccaaaggtgc tgcaatttat itgaaaaait cttccccagc agtcataca gaagaccagt cgggaiaicc agtatggtgg aggctatggt itccagagcc cgaattgatg cacggattga tttcgaatct ggaaggataa agaaagaaga gttcactgag atcatgaaga tctgttccac cattgaagag ctcagacggc aaaaatagtg aatttagctt gtccttcatg aaaaaatgcc ttgtttctac t (SEQ ID N0:11} agcgaaagca ggiactgait caaaaiggaa gattttgtgc gacaaigcti caaiccgaig attgtcgagc ttgcggaaaa aacaatgaaa gagtatgggg aggacctgaa aatcgaaaca aacaaatttg cagcaatatg cactcacttg gaagtatgct tcatgtattc agatttccac itcatcaatg agcaaggcga gtcaataate gtagaactig gtgatcctaa tgcacttttg aagcacagat ttgaaataat cgagggaaga gatcgcacaa tggcctggac agtagtaaac agtatttgca acactacagg ggctgagaaa ccaaagittc taccagatti gtatgaitac aaggaaaata gattcatcga aattggagta acaaggagag aagttcacat atactatctg gaaaaggcca ataaaattaa ateigagaaa acacacatcc acatittcic gtcactggg gaagaaaigg ccacaagggc cgactacact ctcgatgaag aaagcagggc taggatcaaa accaggctat tcaccataag acaagaaaig gccagcagag gcctciggga ttcctttcgt cagtccgaga gaggagaaga gacaattgaa gaaaggtttg aaatcacagg aacaatgcgc aagcttgccg accaaagtct cccgccgaac ttctccagcc ttgaaaattt tagagcctat gtggatggat tcgaaccgaa cggctacatt gagggcaagc tgtctcaaat gtccaaagaa gtaaatgcta gaattgaacc ttttttgaaa acaacaccac gaccacttag acttccgaat gggcctccct gticteagcg gtecaaatic ctgctgatgg atgcctiaaa attaagcatt gaggacccaa gtcatgaagg agagggaata ccgctatatg atgcaatcaa atgcatgaga acatctttg gatggaagga acccaatgti gttaaaccac acgaaaaggg aataaaicca aattatcttc tgtcatggaa gcaagtactg gcagaactgc aggacattga gaatgaggag aaaattccaa agactaaaaa tatgaaaaaa acaagtcagc taaagtgggc acttggtgag aacatggcac cagaaaaggt agactttgac gactgtaaag atgtaggtga tttgaagcaa tatgatagtg atgaaccaga attgaggtcg cttgcaagtt ggattcagaa tgagttcaac aaggcaigcg aactgacaga itcaagctgg aiagagctg aigagatgg agaagaigtg gctccaattg aacacattgc aagcatgaga aggaattatt tcacatcaga ggtgtctcac igcagagcca cagaatacat aatgaagggg gtgtacatca atactgcctt aetaatgca tcttgtgcag caatggatga ttccaatta attccaatga iaagcaagtg tagaactaag gagggaaggc gaaagaccaa cttgtatggt tteatcataa aaggaagatc ccacttaagg aatgacaccg acgtggtaaa ctttgtgagc atggagtttt ctctcactga cccaagactt gaaccacaca aatgggagaa gtactgtgtt cttgagatag gagatatgct tctaagaagt gccaiaggcc aggittcaag gcecaigite ttgtatgtga ggacaaatgg aaccicaaaa attaaaatga aatggggaat ggagatgagg cgitgtctcc tccagtcact tcaacaaatt gagagtatga ttgaagctga gtcctctgtc aaagagaaag acatgaccaa agagttcttt gagaacaaat cagaaacatg gcccattgga gagtctccca aaggagtgga ggaaagticc attgggaagg tcigcaggac titatiagca aagtcggtat ttaacageit gtatgcatct ccacaactag aaggattttc agctgaatca agaaaactgc ttcttatcgt tcaggctctt agggacaatc tggaacctgg gacctttgat citggggggc tatatgaage aattgaggag tgcctaatta atgatccctg ggttttgctt aatgcttctt ggttcaactc cttccttaca catgcattga gttagttgtg gcagtgctac tatttgctat ccatactgtc caaaaaagta ecttgtttct act (SEQ ID NO: 12) agcaaaagca gggtagataa tcactcactg agtgacatca aaatcatggc gtcccaaggc accaaacggt cttacgaaca gatggagact gatggagaac gccagaatgc cactgaaatc agagcatccg tcggaaaaat gattggtgga atggacgat tctacatcca aatgtgcaca gaacttaaac tcagtgatta tgagggaegg ttgatecaaa acagcttaac aatagagaga atggtgctct ctgcttttga cgaaaggaga aataaatacc tggaagaaca tcccagtgcg gggaaagatc ctaagaaaac tggaggacct atatacagaa gagtaaacgg aaagtggatg agagaactca tcctttatga caaagaagaa ataaggcgaa tctggcgcca agctaataat ggtgacgatg caacggctgg tctgactcac atgatgatct ggcattccaa tttgaatgat gcaacttatc agaggacaag ggctcttgtt cgcaccggaa tggatcccag gatgtgctct ctgatgcaag gttcaacict ccctaggagg ictggagccg caggtgctgc agtcaaagga gttggaacaa tggtgatgga attggtcagg atgatcaaac gtgggatcaa tgatcggaac itctggaggg gtgagaatgg acgaaaaaca agaattgctt atgaaagaat gtgcaacatt ctcaaaggga aatttcaaac tgctgcacaa aaagcaatga tggatcaagt gagagagagc cggaacccag ggaatgctga gttcgaagat ctcacttttc tagcacggtc tgcacicata ttgagagggt cggttgctca caagtcctgc ctgcctgcct gtgtgtatgg acctgccgta gccagtgggt acgactttga aagagaggga tactctctag tcggaataga ccctttcaga cigcttcaaa acagccaagt giacagecta atcagaccaa aigagaatcc agcacacaag agtcaactgg tgtggatggc atgccattct gccgcatttg aagatctaag agtattgagc itcatcaaag ggacgaaggi ggtcccaaga gggaagcttt ccactagagg agticaaatt gcttccaatg aaaatatgga gactatggaa tcaagtacac ttgaactgag aagcaggtac tgggccataa ggaccagaag tggaggaaac aceaateaac agagggcatc tgcgggccaa atcagcatac aacctacgtt ctcagtacag agaaatctcc cttttgacag aacaaccgit atggcagcat tcactgggaa tacagagggg agaacatctg acatgaggac cgaaatcata aggatgatgg aaagtgcaag accagaagai gtgtctttcc aggggcgggg agtcttcgag cictcggacg aaaaggcagc gagcccgatc gtgccttcct ttgacatgag taatgaagga tcttatttct tcggagacaa tgcagaggag tacgacaatt aaagaaaaat acccttgttt ctact (SEQ ID NO: 13) agcaaaagca ggtagatatt gaaagatgag tcttctaacc gaggtcgaaa cgtacgttct cictatcatc ccgtcaggcc ccctcaaagc cgagatcgca cagagacitg aagatgtctt tgcagggaag aacaccgatc ttgaggttct catggaatgg ctaaagacaa gaccaatcct gtcacctctg actaagggga ttttaggatt tgtgttcacg ctcaccgtgc ccagtgagcg aggacigcag cgtagacgct ttgtccaaaa tgcccttaat gggaacgggg atccaaataa catggacaaa gcagtaaac tgtataggaa gctcaagagg gagataacat tccatggggc caaagaaatc tcactcagtt attctgctgg tgcactigec agttgtatgg gcctcatata caacaggaig ggggctgtga ccactgaagi ggcatttggc ctggiaigtg caacctgtga acagattgct gacicccagc atcggtctca taggcaaaig gigacaacaa ccaacccaci aatcagacat gagaacagaa tggttttagc cagcactaca gctaaggcta tggagcaaat ggctggaicg agigagcaag cagcagaggc catggaggtt gctagicagg ctaggeaaai ggtgcaagcg atgagaacca ttgggactca tcctagctcc agtgctggtc tgaaaaatga tcttcttgaa aatttgcagg cctatcagaa acgaatgggg gtgcagatgc aacggttcaa gtgatcctct cgctattgcc gcaaatatca ttgggatctt gcactigata ttgtggattc ttgatcgtct ttttttcaaa tgcatttacc gtcgctttaa atacggactg aaaggagggc cttctacgga aggagtgcca aagtctatga gggaagaata tcgaaaggaa cagcagagtg ctgtggatgc tgacgatggt catttgtca gcatagagct ggagtaaaaa actaccttgt ttctact (SEQ ID NO: 14) agcaaaagca gggtgacaaa gacataatgg atccaaacac tgtgtcaagc tttcaggtag attgctttct itggcatgtc cgcaaacgag ttgcagacca agaactaggt gatgccccat tccttgatcg gcttcgccga gatcagaaat ccctaagagg aaggggcagc actcttggtc tggacatcga gacagccaca cgigctggaa agcagaiagt ggagcggati cigaaagaag aatccgatga ggcacttaaa atgaccatgg cctctgtacc tgcgtcgcgt tacctaaccg acatgactct igaggaaaig tcaagggaat ggtccaigct caiacccaag cagaaagigg caggccctct itgtatcaga atggaccagg cgatcatgga taaaaacatc atactgaaag cgaacttcag tgtgaittit gaccggctgg agactctaat attgctaagg gctttcaccg aagagggagc aattgtiggc gaaaitteae eattgccttc tcttccagga catactgctg aggatgtcaa aaatgcagtt ggagtcctca teggaggact tgaatggaai gataacacag ttcgagtctc tgaaactcta cagagaticg ctiggagaag cagtaatgag aatgggagac ciccactcac tccaaaacag aaacgagaaa tggcgggaac aattaggtca gaagttigaa gaaataagat ggttgattga agaagtgaga cacaaactga aggtaacaga gaatagtttt gagcaaataa catttatgca agccttacat ctattgcttg aagtggagca agagataaga actttctcat ttcagcttat ttaataataa aaaacaccct tgtttctact (SEQ ID N0:15}

Also Included are sequences encoding a polypeptide with at least 80%, e.g., 85%, 90%, 92%, 95%, 98%, 99% or 100%, including any integer between 80 and 100, amino acid identity to a polypeptide encoded by one of SEQ ID NQs:10-15.

Exemplary Residues and Positions for Attenuated Strains

The internal viral segments of the recombinant virus may encode a viral protein that attenuates virus having the viral protein, e.g,, the coding sequence of a viral protein may encode one or more the following: in PB1 : 51 K or 171V; 1561; 265K, 358E, 521A, or 886E: 391 K 581 G, or 661 T; 265N or 591 i; 3171; or 265S; in PA: 350K or 327E, 452H, 463A; in PB2: 478V or 265S or 478L or 490R; in NP: 125Y, 1861 or 101 N, 180G or 34G or 3411; in M1 : 144L, 231 D; in NS1 : 23A, 164L. in one embodiment, the coding sequence of a viral protein may encode one or more the following: In PB1 : 51 K, 171V, 1561, 265 K, 358 E, 521 A, 686E, 391 K, 581 G, 661 T, 265N, 5911, 317! or 265S: in PA: 350K, 327 E, 452H, or 463A; in PB2: 478V, 265S, 4781 or 490R; in NP: 125Y, 1861,

101 N, 180G, 34G or 3411; in M1 : 1441 or 231 D; in NS1 : 23A or 164L. In one embodiment, the coding sequence of a viral protein may encode one or more the following: in FBI : 51 K and 171V; 156I, 265K, 358E, 521A, and 686E; 391 K, 581 G, and 661 T; 265S and 591 !, 265N and 5911; or 3171; in PA: 350K; or 327E, 452H, and 463A; in PB2: 478V; 478L; 265S, cr 490R; in NP: 125Y and 1861, 101 N and 180G, 34G, or 3411: in M1 : 144L and 231 D; In NS1 : 23A and 164L.

Exemplary 5ACE2 Amino Acid Sequences

In general, a full-length ACE2 polypeptide includes a signal peptide, e.g., the endogenous signal peptide or a heterologous signal peptide, an extracellular domain, a transmembrane domain and a cytoplasmic domain. For example, the signal peptide may be from about 15 to 20 amino acids In length, the extracellular domain may be from about 700 to 750 amino acids in length, the transmembrane domain may be from 18 to 27 amino acids In length, and the cytoplasmic domain may be from about 30 to 50 amino acids in length. In one embodiment, the soluble form of ACE2 lacks the cytoplasmic domain and at least a portion of the transmembrane domain (a portion that allows for secretion and not membrane bound ACE2). In one embodiment, the soluble form of ACE2, once secreted, lacks the signal peptide. In one embodiment, the soluble form of AGE2 includes a portion of the extracellular domain th binds to coronavlrus. For example, the extracellular domain that is expressed as soluble ACE2 is about 400 to 500, 500 to 600, 600 to 700, 650 to 700, or 700 to 725 amino acids in length.

Exemplary ACE2 sequences include but are not limited to:

NP 001358344,1

Aiso included are sequences encoding a polypeptide with at least 80%, e.g., 85%, 90%, 92%, 95%, 98%, 99% or 100%, Including any integer between 80 and 100, amino acid Identity to one of SEQ ID NOs:20 to 24.

The soluble ACE2 sequence lacks at least a portion ot the transmembrane domain and the cytoplasmic domain. In one embodiment, the soluble ACE2 has about 1 to about 750 residues, about 50 to about 740 residues, about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 750 or 700 residues, or any range between 100 to 740 residues, in one embodiment, the soluble ACE2 may have a residue that is associated with enhanced binding to the spike protein. In one embodiment the soluble ACE2 may have one or more of 19P, 24T, 25 V, 26D, 27Y, 29F, 31 Y, 31 R, 33!, 33D, 34A, 34R, 35 K, 37K, 38V, 50 F, 51 S, 62V, 68E, 72 V, 83H, 326E, 352V, 355N, 388L, 509Y, 40D, 41 R,

42L, 69V, 72Y, 75K, 76T, 89P, 90Q, 91 P, 92Q, 206G, 324P, 325P, 468V, 219G, 341 R,

211 R, 330Y, 351 F, 386L, 389D, 393K, 518G, 329G, 211 R, 206G, 219C, 219H, 341 R, 468V, 547C, 720D, 26R, 351V, 389H, or 30E. See, e.g., Rakhshandeh et a!., M.

Genetic Evol.. 90:104773 (2021), the disclosure o! which is Incorporated by reference herein.

Exemplary Viral Segments for Expressing ACE2

Any of the eight viral segments from influenza A or Influenza R, or any of the seven viral segments from Influenza C or D, may be modified to incorporate coding sequences for AGE2. in one embodiment, the viral segment with ACE2 is In a NS, PB1 , NA or M viral segment, in one embodiment, the viral segment with ACE2 is in a NS viral segment. In one embodiment, the viral segment with AGE2 Is in a PB1 viral segment. The ACE2 coding sequences may be fused to influenza viral protein coding sequences or a portion thereof, e.g., the Influenza viral protein sequences are at the N-terminus or are at the G-terminus of ACE2 coding sequences. In one embodiment, the ACE coding sequences replace the influenza virus coding sequences, e.g., the only influenza virus sequences are the non-coding sequences at the end of the viral segment, in one embodiment, the ACE coding sequences rep!ace a portion of or are inserted into the influenza virus coding sequences, e.g., at least some viral coding sequences at the 5’ and/or 3’ end of the coding region, for instance, those having incorporation signals, as we!l as the contiguous non-coding sequences, f!ank the ACE2 coding sequences.

The 5’rnost and/or 3’ most coding sequences in influenza virus segments contain signals that increase packaging (encapsidat!on or incorporation) of vRNAs. In genera!, those coding sequences are in the first or last 300 nucleotides of coding sequences. in one embodiment, the 3’NA incorporation sequences correspond to nucleotides 1 to 183, nucleotides 1 to 90, nucleotides 1 to 45, nucleotides 1 to 21 , nucleotides 1 to 19 or any integer between 19 and 183, of the N-term!na! NA coding region, and may include a mutation at the NA initiation codon, in another embodiment, the 5’ NA incorporation sequences correspond to sequences in the C-terminai coding region of NA, sequences corresponding to the 3’ most 39, 78, or 157, or any integer between 1 and 157, nucleotides for C-terminai NA coding region. In one embodiment, the 3’ NA incorporation sequences correspond to nucleotides 1 to 20 up to 185 and/or the 5’ NP incorporation sequences correspond to the last about 35 up to 160 nucleotides of the coding region in another embodiment, the 5’ HA incorporation sequences correspond to sequences in the C-terminai coding region of HA, sequences corresponding to the 3’ most 75, 80, 268, 291 , or 518, or any integer between 1 and 518, nuciectides of the C- terminai HA coding region. The 3’ HA Incorporation sequences correspond to nucleotides 1 to 3, 1 to 6, 1 to 9, 1 to 15, 1 to 216, 1 to 468, or any integer between 1 and 468, of the N-terminal HA coding region. in one embodiment, the 3’ PB1 incorporation sequences correspond to nucleotides 1 to 250, nucleotides 1 to 200, nucleotides 1 to 150, or any integer between 1 and 250, of the N-termina! PB1 coding region. In one embodiment, the 5’ PB1 incorporation sequences correspond to the 3’ most nuciectides, e.g., the 3’ 1 to 250 nucleotides, 1 to 200 nucleotides, nucleotides 1 to 150, or any integer between 1 and 250, of the C-terminai PB1 coding region. In one embodiment, the 3’ PB1 incorporation sequences correspond to nucleotides about 10 or 12 to about 120 and/or the 5’ PB1 incorporation sequences correspond to the last about 10 or 12 up to about 120 nucleotides of the coding region. in one embodiment, the 3’ PB2 incorporation sequences correspond to nucleotides 1 to 250, nucleotides 1 to 200, nudeot!des 1 to 150, or any integer between 1 and 250, of the N-termina! PB2 coding region, in one embodiment, the 5’ PB2 incorporation sequences correspond to the 3’ most nucleotides, e.g., the 3’ 1 to 250 nucleotides, 1 to 200 nucleotides, nucleotides 1 to 150, or any integer between 1 and 250, of the C-terminai PB2 coding region, in one embodiment, the 3’ PB2 incorporation sequences correspond to nucleotides about 30 to about 120 and/or the 5’ PB2 incorporation sequences correspond to the last about 30 up to about 120 nucleotides of the coding region. in one embodiment, the 3’ PA incorporation sequences correspond to nucleotides 1 to 250, nucleotides 1 to 200, nucleotides 1 to 150, or any integer between 1 and 250, of the N-termina! PA coding region. In one embodiment, the 5’ PA Incorporation sequences correspond to the 3’ most nucleotides, e.g., the 3’ 1 to 250 nucleotides, 1 to 200 nucleotides, nucleotides 1 to 150, or any integer between 1 and 250, of the C-terminai PA coding region, in one embodiment, the 3' PA incorporation sequences correspond to nucleotides 0, 12 or 21 up to about 120 and/or the 5’ PA incorporation sequences correspond to the last about 0, 12, 21 , or up to about 120 nucleotides of the coding region.

In another embodiment, the 3’ M incorporation sequences correspond to nucleotides 1 to 250, nucleotides 1 to 242, nucleotides 1 to 240, or any integer between 1 and 250, of the N-termina! M coding region, and may include a mutation at the M initiation codon, in another embodiment, the 5’ M incorporation sequences correspond to sequences in the C-terminal ceding region o! M, sequences corresponding to the 3' most 50, 100, or 220, or any integer between 1 and 250, nucleotides for C-terminai M coding region. In one embodiment, the 3’ M incorporation sequences correspond to nucleotides about 100 to about 225 and/or the 5’ M Incorporation sequences correspond to the last about 100 to 225 nucleotides of the coding region in another embodiment, the 3’ NS incorporation sequences correspond to nucleotides 1 to 250, nucleotides 1 to 200, nucleotides 1 to 150, nucleotides 1 to 30, nucleotides 1 to 20 or any integer between 1 and 250, of the N-terminai NS coding region, and may include a mutation at the NS initiation codon. In another embodiment, the 5’ NS incorporation sequences correspond to sequences in the C-terminai coding region of NS, sequences corresponding to the 3’ most 10, 30, 150, 200 or 250, or any integer between 1 and 250, nucleotides for the C-terminai NS coding region. In one embodiment, the 3’ NS incorporation sequences correspond to nucleotides about 30 to about 150 and/or the 5’ NS incorporation sequences correspond to the last about 30 to 150 nucleotides of the coding region. in another embodiment, the 3' NP incorporation sequences correspond to nucleotides 1 to 250, nucleotides 1 to 200, nucleotides 1 to 150, nucleotides 1 to 30, nucleotides 1 to 20 or any integer between 1 and 250, of the N-terminai NP coding region, in another embodiment, the 5’ NP Incorporation sequences correspond to sequences in the C-terminai coding region of NP, sequences corresponding to the 3’ most 10, 30, 150, 200 or 250, or any Integer between 1 and 250, nucleotides for the C- terminal NP coding region, in one embodiment, the 3 ^’ NP Incorporation sequences correspond to nucleotides 1 to about 120 and/or the 5’ NP incorporation sequences correspond to the last about 120 nucleotides of the coding region. All publications, patents and patent applications are incorporated herein by reference. While In the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention Is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.