Recombinant influenza a viruses comprising an NS genomic segment lacking the NS1 coding sequence and uses thereof

Abstract

Provided herein are constructs comprising an NS genomic segment lacking the coding sequence for NS1 and recombinant Influenza A viruses comprising an NS genomic segment lacking the coding sequence for NS1. Also, provided herein are constructs comprising an NS genomic segment lacking the coding sequence for NS 1 and a transgene. Also, provided herein are recombinant Influenza A viruses comprising NS genomic segment lacking the coding sequence for NS 1 and a transgene. Further, provided herein are compositions comprising such recombinant Influenza A viruses and uses of such recombinant Influenza A viruses (e.g., as vaccines).

Description

Attorney Docket No. 6923-426-228RECOMBINANT INFLUENZA A VIRUSES COMPRISING AN NS GENOMIC SEGMENT LACKING THE NS1 CODING SEQUENCE AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 700,537, filed September 27, 2024, the disclosure of which is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under 75N93021C00014 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “06923-426-228_SEQ_LISTING.xml”, was created on September 26, 2025, and is 50,212 bytes in size.1. INTRODUCTION

[0004] Provided herein are constructs comprising an NS genomic segment lacking the coding sequence for NS 1 and recombinant Influenza A viruses comprising an NS genomic segment lacking the coding sequence for NS 1. Also, provided herein are constructs comprising an NS genomic segment lacking the coding sequence for NS1 and a transgene. Also, provided herein are recombinant Influenza A viruses comprising NS genomic segment lacking the coding sequence for NS 1 and a transgene. Further, provided herein are compositions comprising such recombinant Influenza A viruses and uses of such recombinant Influenza A viruses (e.g., as vaccines).2. BACKGROUND

[0005] Influenza A virus (IAV) is a negative strand RNA virus with a segmented genome. Segment 8 of the Influenza A virus genome encodes for non- structural protein 1 (NSl) and nuclear export protein (NEP). The Influenza A virus NSl protein facilitates several functions ranging from inhibition of host mRNA polyadenylation and subsequent inhibition of their nuclear export as well as inhibition of pre-mRNA splicing and inhibition of interferon (IFN) signaling (Fortes, P., Beloso, A. & Ortin, J. Influenza virus NSl protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport. EMBO J. 13, 704-712 (1994); Qiu,1NAI-5003686295vlY. & Krug, R. M. The influenza virus NS1 protein is a poly(A)-binding protein that inhibits nuclear export of mRNAs containing poly(A). J. Virol. 68, 2425-2432 (1994)).3. SUMMARY

[0006] In one aspect, provided herein is an NS nucleotide sequence comprising in 5’ to 3’ order: (a) a nucleotide sequence of a 3’ non-coding region (NCR) of segment 8 of an Influenza A virus, (b) a nucleotide sequence of a partial NS1 open reading frame with the ATG changed to TTG, (c) a nucleotide sequence of a splicing acceptor motif, (d) a nucleotide sequence of a nuclear export protein (NEP) open reading frame of segment 8 of an Influenza A virus, and (e) a nucleotide sequence of 5’ NCR of segment 8 of an Influenza A virus, wherein the partial NS1 open reading frame comprises a splicing donor motif. In some embodiments, the nucleotide sequence of the splicing donor motif comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the nucleotide sequence of the splicing acceptor motif comprises the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides 27 to 64 of segment 8 of an Influenza A virus. In a specific embodiment, the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides of an Influenza A virus segment 8 that correspond to nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934. In a specific embodiment, the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934. In a specific embodiment, the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the NEP open reading frame comprises the NEP open reading frame of Influenza A virus A / Puerto Rico / 8 / 1934 NS1 open reading frame. In some embodiments, the Influenza A virus segment 8 is the segment 8 of Influenza A virus A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interi or Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In a specific embodiment, the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of2NAI-5003686295vlsegment 8 of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, A / Hong Kong / 1 / 68, or A / PR / 8 / 34. In some embodiments, the NEP open reading frame comprises the NEP open reading of Influenza A virus A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the nucleotide sequence of the NEP open reading frame comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the nucleotide sequence of the NEP open reading frame comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the NS nucleotide sequence further comprises a transgene. In some embodiments, the transgene comprises a nucleotide sequence encoding a biomolecule. In some embodiments, the biomolecule comprises a secretory protein. In some embodiments, the biomolecule comprises a cytokine, a chemokine, or an antigen. In some embodiments, the NS nucleotide sequence comprises cDNA. In some embodiments, the NS nucleotide sequence is isolated.

[0007] In some embodiments, proved herein is an NS nucleotide sequence comprising in 5’ to 3’ order: (a) a nucleotide sequence of a 3’ non-coding region (NCR) of segment 8 of an Influenza A virus, (b) a nucleotide sequence of a partial NS1 open reading frame with the ATG changed to TTG, (c) a transgene, (d) a nucleotide sequence of a splicing acceptor motif, (e) a nucleotide sequence of a nuclear export protein (NEP) open reading frame of segment 8 of an Influenza A virus, and (f) a nucleotide sequence of 5’ NCR of segment 8 of an Influenza A virus, wherein the partial NS1 open reading frame comprises a splicing donor motif. In some embodiments, the transgene comprises a nucleotide sequence encoding a biomolecule. In some embodiments, the biomolecule comprises a secretory protein. In some embodiments, the biomolecule comprises a cytokine, a chemokine, or an antigen. In some embodiments, the transgene comprises a nucleotide sequence encoding interleukin (IL)-2 or IL-15. In some embodiments, the nucleotide sequence of the splicing donor motif comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the nucleotide sequence of the splicing acceptor motif comprises the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the nucleotide sequence of the partial NS1 open reading frame with the ATG3NAI-5003686295vlchanged to TTG comprises nucleotides 27 to 64 of segment 8 of an Influenza A virus. In some embodiments, the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides of an Influenza A virus segment 8 that correspond to nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934. In some embodiments, the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934. In some embodiments, the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the NEP open reading comprises the NEP open reading of Influenza A virus A / Puerto Rico / 8 / 1934 NS1 open reading frame. In some embodiments, the Influenza A virus segment 8 is the segment 8 of Influenza A virus A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interi or Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, A / Hong Kong / 1 / 68, or A / PR / 8 / 34. In some embodiments, the NEP open reading frame comprises the NEP open reading frame of Influenza A virus A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the nucleotide sequence of the NEP open reading frame comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the nucleotide sequence of the NEP open reading frame comprises the nucleotide sequence of SEQ ID NO: 8. In a specific embodiment, the4NAI-5003686295vltransgene comprises a nucleotide sequence encoding IL-2. In some embodiments, the IL-2 comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the IL-2 comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding IL-2 comprises the nucleotide sequence of SEQ ID NO: 20. In some embodiments, the nucleotide sequence encoding IL-2 comprises the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the transgene comprises a nucleotide sequence encoding IL-15. In some embodiments, the IL- 15 comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the IL- 15 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 comprises the amino acid sequence of SEQ ID NO: 21, and wherein the IL-15 is fused to the amino acid sequence of signal peptide of IL-2. In some embodiments, the signal peptide of IL-2 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the nucleotide sequence encoding IL-15 comprises the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the nucleotide sequence encoding IL-15 comprises the nucleotide sequence of SEQ ID NO: 14 or 15. In some embodiments, the NS nucleotide sequence comprises cDNA. In some embodiments, the NS nucleotide sequence is isolated.

[0008] In another aspect, provided herein is a vector comprising of an NS nucleotide sequence described herein. In some embodiments, the vector is plasmid.

[0009] In another aspect, provided herein is an NS genomic segment comprising the corresponding negative-sense RNA sequence of an NS nucleotide sequence described herein. In some embodiments, provided herein is a vector comprising the NS genomic segment.

[0010] In another aspect, provided herein is a recombinant Influenza A virus comprising an NS genomic segment described herein. In some embodiments, the recombinant Influenza A virus comprises PB1, PB2, PA, NP, M, HA and NA genomic segments. In some embodiments, the HA and NA genomic segments are from a seasonal Influenza A virus. In some embodiments, the PB1, PB2, PA, NP, M, HA and NA genomic segments are from the same Influenza A virus. In some embodiments, the PB1, PB2, PA, NP, and M genomic segments are from the same Influenza A virus. In specific embodiments, the recombinant Influenza A virus described herein provides efficient secreted protein. In some embodiments, the recombinant Influenza A virus described herein does not contain or contains negligible contaminant peptides.

[0011] In another aspect, provided herein is a pharmaceutical composition comprising a recombinant Influenza A virus described herein, and a pharmaceutically acceptable carrier.5NAI-5003686295vl

[0012] In another aspect, provided herein are methods for delivering a biomolecule to a cell(s), comprising contacting the cell(s) with a recombinant Influenza A virus described herein, or a composition described herein. In some embodiments, provided herein are methods for delivering IL-2 or IL-15 to a cell(s), comprising contacting the cell(s) with a recombinant Influenza A virus described herein, or a composition described herein. In some embodiments, the cell(s) is a cell line. In some embodiments, the cell(s) is in vitro or ex vivo.

[0013] In another aspect, provided herein are methods for delivering a biomolecule to a subject, comprising administering to the subject a recombinant Influenza A virus described herein, or a composition described herein. In some embodiments, provided herein are methods for delivering IL-2 or IL- 15 to a subject, comprising administering to the subject a recombinant Influenza A virus described herein, or a composition described herein. In some embodiments, the subject is a human.

[0014] In another aspect, provided herein are methods of inducing an immune response to Influenza A virus in a subject, comprising administering to the subject a recombinant Influenza A virus described herein, or a composition described herein. In another aspect, provided herein are methods of inducing an immune response to an antigen in a subject, comprising administering to the subject a recombinant Influenza A virus described herein, or a composition described herein. In some embodiments, the subject is a human.

[0015] In another aspect, provided herein are methods of immunizing a subject against Influenza A virus, comprising administering to the subject a recombinant Influenza A virus described herein, or a composition described herein. In another aspect, provided herein are methods of immunizing a subject against an antigen in a subject, comprising administering to the subject a recombinant Influenza A virus described herein, or a composition described herein. In some embodiments, the subject is a human.

[0016] In another aspect, provided herein are methods for treating cancer in a subject in need thereof, comprising administering to the subject a recombinant Influenza A virus described herein, or a composition described herein. In some embodiments, the cancer is a thoracic cancer. In some embodiments, the thoracic cancer is non-small lung cancer, or a carcinoma that has metastasized to the lungs. In some embodiments, the subject is human.

[0017] In another aspect, provided herein is a recombinant Influenza A virus described herein, or a composition described herein for use in a method for delivering IL-2 or IL- 15 to a subject, wherein the method comprises administering to the subject the recombinant Influenza A virus or the composition. In some embodiments, provided herein is a recombinant Influenza A virus described herein, or a composition described herein for use in6NAI-5003686295vla method for delivering IL-2 or IL-15 to a subject, wherein the method comprises administering to the subject the recombinant Influenza A virus or the composition. In some embodiments, the subject is human.

[0018] In another aspect, provided herein is a recombinant Influenza A virus described herein, or a composition described herein for use in a method of inducing an immune response to Influenza A virus in a subject, wherein the method comprises administering to the subject the recombinant Influenza A virus or the composition. In another aspect, provided herein is a recombinant Influenza A virus described herein, or a composition described herein for use in a method of inducing an immune response to an antigen in a subject, wherein the method comprises administering to the subject the recombinant Influenza A virus or the composition. In another aspect, provided herein is a recombinant Influenza A virus described herein, or a composition described herein for use in a method of immunizing a subject against Influenza A virus, wherein the method comprises administering to the subject the recombinant Influenza A virus or the composition. In another aspect, provided herein is a recombinant Influenza A virus described herein, or a composition described herein for use in a method of immunizing a subject against an antigen in a subject, wherein the method comprises administering to the subject the recombinant Influenza A virus or the composition. In some embodiments, the subject is human.

[0019] In another aspect, provided herein is a recombinant Influenza A virus described herein, or a composition described herein for use in a method for treating cancer in a subject in need thereof, wherein the method comprises administering to the subject the recombinant Influenza A virus or the composition. In some embodiments, the cancer is a thoracic cancer. In some embodiments, the thoracic cancer is non-small lung cancer, or a carcinoma that has metastasized to the lungs. In some embodiments, the subject is human.

[0020] In another aspect, provided herein is the use of a recombinant Influenza A virus described herein, or a composition described herein in the preparation of a medicament for delivering a biomolecule to a subject. In some embodiments, provided herein is the use of a recombinant Influenza A virus described herein, or a composition described herein in the preparation of a medicament for delivering IL-2 or IL- 15 to a subject. In some embodiments, the subject is a human.

[0021] In another aspect, provided herein is the use of a recombinant Influenza A virus described herein, or a composition described herein in the preparation of a medicament for inducing an immune response to Influenza A virus. In another aspect, provided herein is the7NAI-5003686295vluse of a recombinant Influenza A virus described herein, or a composition described herein in the preparation of a medicament for inducing an immune response to an antigen.

[0022] In another aspect, provided herein is the use of a recombinant Influenza A virus described herein, or a composition described herein in the preparation of a medicament for immunizing a subject against Influenza A virus. In another aspect, provided herein is the use of a recombinant Influenza A virus described herein, or a composition described herein in the preparation of a medicament for immunizing a subject against an antigen. In some embodiments, the subject is human.

[0023] In another aspect, provided herein is the use of a recombinant Influenza A virus described herein, or a composition described herein in the preparation of a medicament for treating cancer. In some embodiments, the cancer is a thoracic cancer. In some embodiments, the thoracic cancer is non-small lung cancer, or a carcinoma that has metastasized to the lungs. In some embodiments, the subject is human.

[0024] In another aspect, provided herein are kits comprising a container containing an NS nucleotide sequence described herein. In some embodiments, the kits further comprise one or more containers containing a nucleotide sequence of a PB1 genomic segment, a nucleotide sequence of a PB2 genomic segment, a nucleotide sequence of a PA genomic segment, a nucleotide sequence of an NP genomic segment, a nucleotide sequence of an M genomic segment, a nucleotide sequence of an HA genomic segment, and a nucleotide sequence of an NA genomic segment.

[0025] In another aspect, provided herein are kits comprising a container containing an NS genomic segment described herein. In some embodiments, the kits further comprise one or more containers containing PB2, PA, NP, M, HA and NA genomic segments.

[0026] In another aspect, provided herein are kits comprising a container containing a vector described herein. In some embodiments, the kits further comprise one or more containers containing vectors comprising nucleotide sequences encoding PB2, PA, NP, M, HA and NA genomic segments. In some embodiments, the kits further comprise one or more containers containing one or more vectors comprising a nucleotide sequence encoding an Influenza A virus PB1 protein, a nucleotide sequence encoding an Influenza A virus PB2 protein, a nucleotide sequence encoding an Influenza A virus PA protein, and a nucleotide sequence encoding an Influenza A virus NP protein.

[0027] In another aspect, provided herein are kits comprising a container containing a recombinant Influenza A virus of described herein, or a composition described herein.8NAI-5003686295vl3.1 TERMINOLOGY

[0028] Unless specifically stated or apparent from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

[0029] The terms “include(s)” or “such as,” and the like are intended to convey inclusion without limitation, unless otherwise specifically indicated.

[0030] The terms “or” and “and” can be used interchangeably and can be understood to mean “and / or.”

[0031] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0032] As used herein, and unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0033] Unless otherwise indicated, the terms “at least,” “at most,” or “about” preceding a series of elements is to be understood to refer to every element in the series.

[0034] The term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within (±): 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. The term “about” in relation to a reference numerical value can include the numerical value itself and a range of values, for example, plus or minus 10% from that numerical value. In some embodiments, the amount “about 10” includes 10 and any amounts from 9 to 11. In some cases, the numerical disclosed throughout can be “about” that numerical value even without specifically mentioning the term “about.”

[0035] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other9NAI-5003686295vlinteger or group of integers and are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Additionally, the terms “comprise(s)”, “includes”, “comprising” and “including” are intended to include examples encompassed by the term “consisting of’. Consequently, the term “consisting of’ can be used in place of the terms “comprise(s)”, “includes”, “comprising” and “including” to provide for more specific embodiments.

[0036] As used herein, the term “consists of,” or variations such as “consist of’ or “consisting of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, but that no additional integer or group of integers can be added to the specified method, structure, or composition.

[0037] As used herein, the term “consists essentially of,” or variations such as “consist essentially of’ or “consisting essentially of,” indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition.

[0038] The term “heterologous” has the meaning known to one of skill in the art. Generally, a heterologous sequence is not naturally present with another sequence.

[0039] As used herein, the term "isolated" in the context of nucleic acids or nucleotide sequences refers to a nucleic acid molecule which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized; however, "isolated" excludes members of a library of clones such as a cDNA library. In specific embodiments, an isolated nucleic acid molecule substantially free of chemical precursors or other chemicals contains less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%) of chemical precursors or other chemicals. In a specific embodiment, a nucleic acid or nucleotide sequence described herein is isolated.

[0040] In specific embodiments, a nucleic acid sequence, nucleic acid molecule, or nucleotide sequence may comprise deoxyribonucleotides, deoxyribonucleic acids, ribonucleotides, or ribonucleic acids, or polymeric forms thereof. In some embodiments, a10NAI-5003686295vlnucleic acid sequence, nucleic acid molecule, or nucleotide sequence is single-stranded. In some embodiments, a nucleic acid sequence, nucleic acid molecule, or nucleotide sequence is double-stranded. In certain embodiments, a nucleic acid sequence, nucleic acid molecule, or nucleotide sequence may comprise known analogues of natural nucleotides, for example, peptide nucleic acids (“PNAs”), that have similar binding properties as the reference nucleic acid. In some embodiments, a nucleic acid sequence, nucleic acid molecule, or nucleotide sequence comprises deoxyribonucleic acids (e.g., cDNA or DNA). In some embodiments, a nucleic acid sequence, nucleic acid molecule, or nucleotide sequence comprises ribonucleic acids (e.g., mRNA or RNA).

[0041] In some embodiments, a nucleic acid sequence, nucleic acid molecule, or nucleotide sequence comprises deoxyribonucleotides, deoxyribonucleic acids, and polymeric forms thereof, and is single- or double-stranded. In some embodiments, a nucleic acid sequence, nucleic acid molecule, or nucleotide sequence comprises ribonucleotides, ribonucleic acids, and polymeric forms thereof, and is single- or double-stranded. In some embodiments, a nucleic acid sequence, nucleic acid molecule, or nucleotide sequence comprises known analogues of natural nucleotides, for example, peptide nucleic acids (“PNAs”), that have similar binding properties as the reference nucleic acid. In some embodiments, a nucleic acid sequence, nucleic acid molecule, or nucleotide sequence is a cDNA. In some embodiments, a nucleic acid sequence, nucleic acid molecule, or nucleotide sequence is an RNA sequence (e.g., a negative-sense RNA sequence or positive-sense RNA sequence).

[0042] “Percent identity:” Techniques known to one of skill in the art can be used to determine the percent identity between two amino acid sequences or between two nucleotide sequences. Generally, to determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions X 100%). In one embodiment, the two sequences are the same length. In a certain embodiment, the percent identity is determined over the entire length of an amino acid sequence or nucleotide sequence. In some11NAI-5003686295vlembodiments, the length of sequence identity comparison may be over the full-length of the two sequences being compared (e.g., the full-length of a gene coding sequence, or a fragment thereof). In some embodiments, a fragment of a nucleotide sequence is at least 25, at least 50, at least 75, or at least 100 nucleotides. Similarly, "percent sequence identity" may be readily determined for amino acid sequences, over the full-length of a protein, or a fragment thereof. In some embodiments, a fragment of a protein comprises at least 20, at least 30, at least 40, at least 50 or more contiguous amino acids of the protein. In certain embodiments, a fragment of a protein comprises at least 75, at least 100, at least 125, at least 150 or more contiguous amino acids of the protein.

[0043] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264 2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873 5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et aL, 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul etal., 1997, Nucleic Acids Res. 25:3389 3402. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4: 11 17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. In a specific embodiment, Clustal W or Clustal Omega is used to compare amino acid sequences.12NAI-5003686295vl

[0044] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0045] As used herein, the terms "subject", "subjects", “patient”, and “patients” refer to an animal(s) (e.g., a bird(s), reptile(s), or mammal(s)), preferably a mammal including a nonprimate^) (e.g., a camel(s), donkey(s), zebra(s), cow(s), pig(s), horse(s), cat(s), dog(s), rat(s), or mouse(s)) and a primate (e.g., a monkey(s), chimpanzee(s), or human(s)). In specific embodiments, the subject or patient is human.4. BRIEF DESCRIPTION OF THE FIGURES

[0046] FIGs. 1A-1C. Schematic representation of FIG. 1A) pDZ ambisense plasmid expression system, FIG. IB) alternative splicing of Influenza A virus mRNA encoded from segment 8, FIG. 1C) alternative splicing of Influenza A virus mRNA encoded from reengineered segment 8 carrying a transgene. Icon descriptions are given in the figure. NCR: Non-coding region. Not to scale.

[0047] FIGs. 2A-2C. FIG. 2A) Schematic representation of Influenza A virus mRNA encoded from segment 8 carrying a transgene expressed as a 2A fusion upstream of the NEP protein (IL-2-2A-ANS1). Icon descriptions are given in the figure. Not to scale. FIG. 2B) Representative agarose gel image of segment 4 (HA) and segment 8 (NS) specific RT-PCR products amplified from viral RNA of IAV IL-2-2A-ANS1. FIG. 2C) Interleukin-2 (IL-2) levels in supernatants of MDCK-NS1 cells infected with indicated viruses at 72 hours postinfection at an MOI of 0.002. Representative data from two independent experiments. Data is depicted as Mean ± SD (n=3). Ng: nanogram; ml: milliliter.

[0048] FIGs. 3A-3E. FIG. 3A) Representative agarose gel image of segment 8 specific RT-PCR products amplified from viral RNAs of IAV wild type (WT), IAV Empty-ANSl and IAV IL-2-ANS1. 1 kb+ DNA ladder (NEB, USA) is given as a size control. FIG. 3B) Infectious viral titers in supernatants of MDCK-NS1 cells at indicated time points following infection with shown viruses at MOI (multiplicity of infection): 0.002. Representative data from two independent experiments. Data is depicted as Mean SD (n=3). Detection limit is 100 pfu. Two-way ANOVA is applied for statistical analysis (* / #: p<0.05, ** / ##: p<0.01, ns: not significant, *: WT vs Empty ANSI, #: WT vs IL12ANS1). hpi: hours post-infection, pfu: plaque forming unit, ml: milliliter. FIG. 3C) Representative images of a plaque assay at 48 hours post-infection with estimated input of indicated viruses at 33° C or 37° C on MDCK WT or MDCK-NS1 cells. Plaques were stained by standard immuno-staining against IAV NP protein. FIGs. 3D-3E) Interleukin 2 (IL-2) levels (FIG.3D) and bioactivity of vector sourced13NAI-5003686295vlIL-2 (FIG. 3E) in the supernatants of MDCK-NS1 cells infected with indicated viruses at 72 hours-post-infection at an MOI of 0.002. Representative data from two independent experiments. Data is depicted as Mean ± SD (n=3). Concentrations were calculated based on a standard curve of IL-2 recombinant protein supplied by the vendor, ng: nanogram, ml: milliliter.

[0049] FIGs. 4A-4B. FIG.4A) Interleukin-2 (IL-2) levels in the supernatants of consecutive passages of IAV Empty ANSI and IAV IL2ANS1 on MDCK-NS1 cells. Each passage indicates a blind passage of virus containing supernatants into fresh MDCK-NS1 cells at a dilution of 1 :2000. Data is depicted as Mean ± SD (n=3). ng: nanogram, ml: milliliter. FIG.4B) Next generation sequencing data showing acquired mutations on each segment of the indicated viruses at passage 10. Hashtag (#) indicates the technical replicates in which the mutation detected.

[0050] FIGs. 5A-5C. MDCK, 293T and A549 cells were infected with IAV Empty ANS 1 or IAV IL2ANS 1 at a multiplicity of infection of 1. Percentages of infected cells (FIG. 5A), Interleukin-2 (IL-2) levels in the supernatants (FIG. 5B) and bioactivity of vector sourced IL- 2 (FIG. 5C) at indicated time points were quantified and depicted in the figures.Representative data from two independent experiments. Data is depicted as Mean ± SD (n=3). (FIGs. 5B, 5C) Concentrations were calculated based on a standard curve of IL-2 recombinant protein supplied by the vendor. At each time point of the plots, the data for the cells infected with wild-type H1N1 IAV is shown in the left bar, the data for the cells infected with H1N1 IAV Empty ANSI is shown in the middle bar, and the data for the cells infected with H1N1 IAV IL2ANS1 is shown in the right bar. IAV: Influenza A virus. NP: Nucleoprotein, ng: nanogram, ml: milliliter.

[0051] FIGs. 6A-6B. MDCK, 293T and A549 cells were infected with H1N1 IL15ANS1 or H1N1 IL2ssIL15ANSl at a multiplicity of infection of 1. Interleukin- 15 (IL-2) levels in the supernatants (FIG. 6A) and bioactivity of vector sourced IL- 15 (FIG. 6B) at indicated time points were quantified and depicted in the figures. Representative data from two independent experiments. Data is depicted as Mean ± SD (n=3). (FIGs. 6A, 6B) Concentrations were calculated based on a standard curve of IL- 15 recombinant protein supplied by the vendor. At each time point of the plots, the data for the cells infected with H1N1 IL15ANS1 is shown in the left bar, and the data for the cells infected with H1N1 IL2ssIL15ANSl is shown in the right bar. ng: nanogram, ml: milliliter.14NAI-5003686295vl

[0052] FIGs. 7A-7D. Anti-tumor activity of IAV-ANS 1 , IAV-IL2-ANS 1 in a CT26. WT murine colon carcinoma bilateral tumor model. FIG. 7A) Schematic representation of the study. CT26.WT cells were intradermally implanted on the right (RF) and left (LF) hind legs. Tumor-bearing mice were treated intratumorally every other day with a total of four doses of 107PFU of IAV-ANS1, IAV-IL2-ANS1 or PBS for control mice (days 0, 2, 4 and 6). Tumor volume was monitored every 48 hours. EEP of 1,000 mm3. FIG. 7B) shows average of tumor volumes for each experimental group up to the first death (± SEM). FIG. 7C) Individual tumor growth curves. Each point represents tumor volume per mice at the indicated time point. FIG. 7D) Survival analysis, p values obtained through Log-Rank (Mantel-Cox) statistical analysis. CR, complete response. N= animals included in each experimental group. *p <0.05; ***p <0.001; ****p <0.0001; ns: non-significant.

[0053] FIGs. 8A-8G. Anti-tumor activity of IAV-ANS1, IAV-IL15-ANS1. A, CT26.WT murine colon carcinoma bilateral tumor model. FIG. 8A) Schematic representation of the study. CT26.WT cells were intradermally implanted on the right (RF) and left (LF) hind legs. Tumor-bearing mice were treated intratumorally every other day with a total of four doses of 107PFU of IAV-ANS1, IAV-IL15-ANS1 or PBS for control mice (days 0, 2, 4 and 6). Tumor volume was monitored every 48 hours. EEP of 1,000 mm3. FIG. 8B) shows average of tumor volumes for each experimental group up to the first death (± SEM). FIG. 8C) Individual tumor growth curves. Each point represents tumor volume per mice at the indicated time point. FIG. 8D) Survival analysis. / ? values obtained through Log-Rank (Mantel-Cox) statistical analysis. CR, complete response. N= animals included in each experimental group. *p <0.05; ***p <0.001; ****p <0.0001; ns: non-significant. FIG. 8E) Experimental lung metastasis. Schematic representation of the study. Lower panel: representative IVIS images showing the progression of CT26-Fluc cancer metastasis in the lung of experimental animals. FIG. 8F) shows average of tumor burden for each experimental group up to the first death (± SEM); N=7. FIG. 8G) Survival analysis. / ? values obtained through Log-Rank (Mantel-Cox) statistical analysis. * / ? <0.05; ***p <0.001; **** / ? <0.0001; ns: non-significant.5. DETAILED DESCRIPTION5.1 INFLUENZA A VIRUS NS SEGMENT

[0054] In one aspect, provided herein is an Influenza A virus NS genomic segment nucleotide sequence that does not produce NS1 protein. The NS genomic segment nucleotide sequence may be engineered such as described in Example 1.15NAI-5003686295vl

[0055] In some embodiments, provided herein is an NS nucleotide sequence comprising in 5’ to 3’ order: a nucleic acid sequence of a non-coding region (NCR) of segment 8 of an Influenza A virus, a nucleic acid sequence of a partial NS1 open reading frame of an Influenza A virus (e.g., a nucleic acid sequence corresponding to nucleotides 27 to 64 of segment 8 of an Influenza A virus) with the ATG changed to TTG, a nucleic acid sequence of a splicing acceptor motif, a nucleic acid sequence of a nuclear export protein (NEP) open reading frame of an Influenza A virus, and a nucleic acid sequence of a NCR of segment 8 of an Influenza A virus, wherein the partial NS1 open reading frame comprises a splicing donor motif. In some embodiments, the NS nucleotide sequence does not comprise a transgene. In some embodiments, the NS nucleotide sequence comprises a transgene. In some embodiments, the NS nucleotide sequence does not further comprise a nucleotide sequence encoding a biomolecule, such as, e.g., a protein. In some embodiments, the NS nucleotide sequence further comprises a nucleotide sequence encoding a biomolecule, such as, e.g., a protein. In specific embodiments, the NS nucleotide sequence does not encode an Influenza A virus NS1 protein. The NS nucleotide sequence may be a positive sense RNA sequence (e.g., a pre-mRNA transcript) or a DNA sequence (e.g., a cDNA). In some embodiments, the NS nucleotide sequence does not encode a fragment of the Influenza A virus NS 1 protein. Segment 8 of an Influenza A virus is sometimes referred to herein as an Influenza A virus NS segment. As the skilled person will appreciate, the genomic segments of an Influenza A virus are negative-sense and single-stranded RNA. Thus, the skilled person would understand that an Influenza A virus genomic segment would comprise the reverse complement of the nucleotide sequence of a mRNA sequence. In some embodiments, provided herein is an NS genomic segment comprising the negative-sense RNA sequence corresponding to the NS nucleotide sequence. In specific embodiments, the NS genomic segment does not encode a fragment of an Influenza A virus NS1 protein. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 60, 27 to 61, 27 to 62, 27 to 63, 27 to 64, 27 to 65, 27 to 66, or 27 to 67 of segment 8 of an Influenza A virus. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 62, 27 to 63, 27 to 64, 27 to 65, 27 to 66, 27 to 67, or 27 to 68 of segment 8 of an Influenza A virus. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame of an Influenza A virus with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 64 of segment 8 of a reference Influenza A virus (e.g., Influenza A virus A / Puerto Rico / 8 / 1934).16NAI-5003686295vlThe Influenza A virus may be one described herein or known in the art e.g., Influenza A virus A / Puerto Rico / 8 / 1934, A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14). In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934. Nucleotide positions of an Influenza A virus NS 1 open reading fame that correspond to certain nucleotide positions of an NS1 open reading frame of a reference Influenza A virus (e.g., Influenza A virus A / Puerto Rico / 8 / 1934) may be determined by one of skill in the art by aligning the NS segments. For example, the nucleotide position in the NS1 open reading frame of an Influenza A virus that corresponds to nucleotide position 27 of the partial NS1 open reading frame of Influenza A virus A / Puerto Rico / 8 / 1934 may be determined by aligning the NS segments of the two viruses. Programs, such as, e.g., Clustal W or Clustal Omega may be used to align the two sequences. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of Influenza A virus A / Califomia / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northem shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / 10BM01929 / 10, A / Jiangxi-Donghu / 346- 1 / 2013 , A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, or A / Hong Kong / 1 / 68. In specific embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotide sequence of SEQ ID NO:5. In specific embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG17NAI-5003686295vlcomprises a nucleotide sequence that is at least 90%, at least 92%, at least 95%, or at least 96% identical to the nucleotide sequence of SEQ ID NO: 5, provided that the nucleotide sequence comprises TTG as the first three nucleotides and the splicing donor motif (SEQ ID NO: 11). In specific embodiments, the nucleic acid sequence of the splicing donor motif comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the NS nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1. In specific embodiments, the nucleic acid sequence of the splicing acceptor motif comprises the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of any Influenza A virus NEP open reading frame (e.g., NEP open reading frame of an Influenza A virus described herein or known in the art). In some embodiments, the NEP open reading frame comprises the nucleotide sequence of the NEP open reading frame of Influenza A virus A / Puerto Rico / 8 / 1934. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of the NEP open reading frame of Influenza A virus A / Califomia / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, or A / Hong Kong / 1 / 68. In some embodiments, the nucleic acid sequence of the NEP open reading frame comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 8. In specific embodiments, the nucleic acid sequence of the NEP open reading frame comprises the nucleotide sequence of SEQ ID NO: 8. In specific embodiments, the NS nucleotide sequence comprises a nucleic acid sequence of a 5’ NCR and a nucleic acid sequence of a 3’ NCR. In specific embodiments, the nucleic acid sequence of the 5’ NCR comprises the nucleotide sequence of SEQ ID NO: 9. In specific embodiments, the nucleic acid sequence of the 3’ NCR comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the 3’ NCR, the partial NS1 open reading frame, and the NEP open reading frame, and the 5’ NCR are from or derived from the same Influenza A virus. In some embodiments, one or more of the 3’ NCR, the partial NS1 open reading frame, and the NEP18NAI-5003686295vlopen reading frame, and the 5’ NCR are from or derived from the different Influenza A viruses. The NS nucleotide sequence may be engineered such as described in Example 1.

[0056] In a specific embodiment, provided herein is an NS nucleotide sequence comprising in 5’ to 3’ order: a nucleic acid sequence of a 3’ non-coding region (NCR) of segment 8 of an Influenza A virus, a nucleic acid sequence of a partial NS1 open reading frame of an Influenza A virus (e.g., a nucleic acid sequence corresponding to nucleotides 27 to 64 of segment 8 of an Influenza A virus) with the ATG changed to TTG, a nucleic acid sequence of a splicing acceptor motif, a nucleic acid sequence of a nuclear export protein (NEP) open reading frame of an Influenza A virus, and a nucleic acid sequence of a 5’ NCR of segment 8 of an Influenza A virus, wherein the partial NS1 open reading frame comprises a splicing donor motif. In some embodiments, the NS nucleotide sequence does not comprise a transgene. In some embodiments, the NS nucleotide sequence does not further comprise a nucleotide sequence encoding a biomolecule, such as, e.g., a protein. In specific embodiments, the NS nucleotide sequence does not encode an Influenza A virus NS1 protein. The NS nucleotide sequence may be a positive sense RNA sequence (e.g., a pre-mRNA transcript) or a DNA sequence (e.g., a cDNA). In some embodiments, the NS nucleotide sequence does not encode a fragment of the Influenza A virus NS1 protein. Segment 8 of an Influenza A virus is sometimes referred to herein as an Influenza A virus NS segment. As the skilled person will appreciate, the genomic segments of an Influenza A virus are negativesense and single-stranded RNA. Thus, the skilled person would understand that an Influenza A virus genomic segment would comprise the reverse complement of the nucleotide sequence of a mRNA sequence. In some embodiments, provided herein is an NS genomic segment comprising the negative-sense RNA sequence corresponding to the NS nucleotide sequence. In specific embodiments, the NS genomic segment does not encode a fragment of an Influenza A virus NS1 protein. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 60, 27 to 61, 27 to 62, 27 to 63, 27 to 64, 27 to 65, 27 to 66, or 27 to 67 of segment 8 of an Influenza A virus. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 62, 27 to 63, 27 to 64, 27 to 65, 27 to 66, 27 to 67, or 27 to 68 of segment 8 of an Influenza A virus. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame of an Influenza A virus with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 64 of segment 8 of a reference Influenza A virus (e.g., Influenza A virus A / Puerto Rico / 8 / 1934). The Influenza A virus may19NAI-5003686295vlbe one described herein or known in the art e.g., Influenza A virus A / Puerto Rico / 8 / 1934, A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interi or Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14). In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934. Nucleotide positions of an Influenza A virus NS 1 open reading fame that correspond to certain nucleotide positions of an NS1 open reading frame of a reference Influenza A virus (e.g., Influenza A virus A / Puerto Rico / 8 / 1934) may be determined by one of skill in the art by aligning the NS segments. For example, the nucleotide position in the NS1 open reading frame of an Influenza A virus that corresponds to nucleotide position 27 of the partial NS1 open reading frame of Influenza A virus A / Puerto Rico / 8 / 1934 may be determined by aligning the NS segments of the two viruses. Programs, such as, e.g., Clustal W or Clustal Omega may be used to align the two sequences. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of Influenza A virus A / Califomia / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northem shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / 10BM01929 / 10, A / Jiangxi-Donghu / 346- 1 / 2013 , A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, or A / Hong Kong / 1 / 68. In specific embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotide sequence of SEQ ID NO:5. In specific embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG20NAI-5003686295vlcomprises a nucleotide sequence that is at least 90%, at least 92%, at least 95%, or at least 96% identical to the nucleotide sequence of SEQ ID NO: 5, provided that the nucleotide sequence comprises TTG as the first three nucleotides and the splicing donor motif (SEQ ID NO: 11). In specific embodiments, the nucleic acid sequence of the splicing donor motif comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the NS nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1. In specific embodiments, the nucleic acid sequence of the splicing acceptor motif comprises the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of any Influenza A virus NEP open reading frame (e.g., NEP open reading frame of an Influenza A virus described herein or known in the art). In some embodiments, the NEP open reading frame comprises the nucleotide sequence of the NEP open reading frame of Influenza A virus A / Puerto Rico / 8 / 1934. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of the NEP open reading frame of Influenza A virus A / Califomia / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, or A / Hong Kong / 1 / 68. In some embodiments, the nucleic acid sequence of the NEP open reading frame comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 8. In specific embodiments, the nucleic acid sequence of the NEP open reading frame comprises the nucleotide sequence of SEQ ID NO: 8. In specific embodiments, the nucleic acid sequence of the 5’ NCR comprises the nucleotide sequence of SEQ ID NO: 9. In specific embodiments, the nucleic acid sequence of the 3’ NCR comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the 3’ NCR, the partial NS1 open reading frame, and the NEP open reading frame, and the 5’ NCR are from or derived from the same Influenza A virus. In some embodiments, one or more of the 3’ NCR, the partial NS1 open reading frame, and the NEP open reading frame, and the 5’ NCR are from or derived from the different21NAI-5003686295vlInfluenza A viruses. The NS nucleotide sequence may be engineered such as described in Example 1.

[0057] In another aspect, provided herein is an Influenza A virus NS genomic segment nucleotide sequence comprising a transgene, wherein the Influenza A virus NS genomic segment nucleotide sequence does not produce NS1 protein. The NS genomic segment nucleotide sequence may be engineered such as described in Example 1. In some embodiments, the transgene encodes a biomolecule, such as, e.g., in Section 5.2 or Example 1.

[0058] In a specific embodiment, provided herein is an NS nucleotide sequence comprising in 5’ to 3’ order: a nucleic acid sequence of a 3’ non-coding region (NCR) of segment 8 of an Influenza A virus, a nucleic acid sequence of a partial NS1 open reading frame of an Influenza A virus (e.g., a nucleic acid sequence corresponding to nucleotides 27 to 64 of segment 8 of an Influenza A virus) with the ATG changed to TTG, a transgene, a nucleic acid sequence of a splicing acceptor motif, a nucleic acid sequence of a nuclear export protein (NEP) open reading frame of an Influenza A virus, and a nucleic acid sequence of a 5’ NCR of segment 8 of an Influenza A virus, wherein the partial NS1 open reading frame comprises a splicing donor motif. The NS nucleotide sequence may be a positive sense RNA sequence (e.g., a pre-mRNA transcript) or a DNA sequence (e.g., a cDNA). As the skilled person will appreciate, the genomic segments of an Influenza A virus are negative-sense and singlestranded RNA. Thus, the skilled person would understand that an Influenza A virus genomic segment would comprise the reverse complement of the nucleotide sequence of a mRNA sequence. In some embodiments, provided herein is a NS genomic segment comprising the negative-sense RNA sequence corresponding to the NS nucleotide sequence. In specific embodiments, the NS genomic segment does not encode an Influenza A virus NS1 protein. In specific embodiments, the NS genomic segment does not encode a fragment of an Influenza A virus NS1 protein. The transgene may encode a biomolecule, such as, e.g., a protein. See, e.g., Section 5.2, infra, regarding transgenes and examples of biomolecules that may be encoded by a transgene. In some embodiments, the transgene encodes a secretory protein (e.g, a chemokine (e.g, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, or CXCL10), an antigen (e.g., a viral antigen, a bacterial antigen, or a fungal antigen), a cytokine (e.g., IL-2 or IL- 15)), or a protein with a signal sequence (e.g., a membrane anchored protein described herein). In some embodiments, the transgene encodes a cytokine (e.g., an immunostimulatory cytokine). In some embodiments, the transgene encodes IL-2 (e.g., human IL-2). In some embodiments, the transgene encodes IL- 15 (e.g., human IL- 15). In some embodiments, the22NAI-5003686295vltransgene encodes an RNA molecule (e.g., shRNA). In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 60, 27, to 61, 27 to 62, 27 to 63, 27 to 64, 27 to 65, 27 to 66, or 27 to 67 of segment 8 of an Influenza A virus. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 62, 27 to 63, 27 to 64, 27 to 65, 27 to 66, 27 to 67, or 27 to 68 of segment 8 of an Influenza A virus. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame of an Influenza A virus with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 64 of segment 8 of a reference Influenza A virus (e.g., Influenza A virus A / Puerto Rico / 8 / 1934). The Influenza A virus may be one described herein or known in the art (e.g., Influenza A virus A / Puerto Rico / 8 / 1934, A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14). In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934. Nucleotide positions of an Influenza A virus NS 1 open reading fame that correspond to certain nucleotide positions of an NS1 open reading frame of a reference Influenza A virus (e.g., Influenza A virus A / Puerto Rico / 8 / 1934) may be determined by one of skill in the art by aligning the NS segments. For example, the nucleotide position in the NS1 open reading frame of an Influenza A virus that corresponds to nucleotide position 27 of the partial NS1 open reading frame of Influenza A virus A / Puerto Rico / 8 / 1934 may be determined by aligning the NS segments of the two viruses. Programs, such as, e.g., Clustal W or Clustal Omega may be used to align the two sequences. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of Influenza A virus A / Califomia / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northem shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed23NAI-5003686295vlgull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / 1 OBMO 1929 / 10, A / Jiangxi-Donghu / 346- 1 / 2013 , A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, or A / Hong Kong / 1 / 68. In specific embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotide sequence of SEQ ID NO: 5. In specific embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises a nucleotide sequence that is at least 90%, at least 92%, at least 95%, or at least 96% identical to the nucleotide sequence of SEQ ID NO:5, provided that the nucleotide sequence comprises TTG as the first three nucleotides and the splicing donor motif (SEQ ID NO: 11). In specific embodiments, the nucleic acid sequence of the splicing donor motif comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the NS nucleotide sequence comprises SEQ ID NO: 1. In specific embodiments, the nucleic acid sequence of the splicing acceptor motif comprises the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of any Influenza A virus NEP open reading frame (e.g., NEP open reading frame of an Influenza A virus described herein or known in the art). In some embodiments, the NEP open reading frame comprises the nucleotide sequence of the NEP open reading frame of segment 8 of Influenza A virus A / Puerto Rico / 8 / 1934. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of the NEP open reading frame of Influenza A virus A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interi or Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / 1 OBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of the NEP open reading frame of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, or A / Hong Kong / 1 / 68. In some embodiments, the nucleic acid sequence of the NEP open reading frame comprises a nucleotide sequence that is at least 80%, at least 85%, at least24NAI-5003686295vl90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 8. In specific embodiments, the nucleic acid sequence of the NEP open reading frame comprises the nucleotide sequence of SEQ ID NO: 8. In specific embodiments, the nucleic acid sequence of the 5’ NCR comprises the nucleotide sequence of SEQ ID NO: 9. In specific embodiments, the nucleic acid sequence of the 3’ NCR comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the 3’ NCR, the partial NS1 open reading frame, and the NEP open reading frame, and the 5’ NCR are from or derived from the same Influenza A virus. In some embodiments, one or more of the 3’ NCR, the partial NS1 open reading frame, and the NEP open reading frame, and the 5’ NCR are from or derived from the different Influenza A viruses. The NS nucleotide sequence may be engineered such as described in Example 1.

[0059] In a specific embodiment, provided herein is an NS nucleotide sequence comprising in 5’ to 3’ order: a nucleic acid sequence of a 3’ non-coding region (NCR) of segment 8 of an Influenza A virus, a nucleic acid sequence of a partial NS1 open reading frame of an Influenza A virus (e.g., a nucleic acid sequence corresponding to nucleotides 27 to 64 of segment 8 of an Influenza A virus) with the ATG changed to TTG, a nucleotide sequence encoding a protein, a nucleic acid sequence of a splicing acceptor motif, a nucleic acid sequence of a nuclear export protein (NEP) open reading frame of an Influenza A virus, and a nucleic acid sequence of a 5’ NCR of segment 8 of an Influenza A virus, wherein the partial NS1 open reading frame comprises a splicing donor motif. The nucleotide sequence may be a positive sense RNA sequence (e.g., a pre-mRNA transcript) or a DNA sequence (e.g., a cDNA). Segment 8 of an Influenza A virus is sometimes referred to herein as an Influenza A virus NS segment. As the skilled person will appreciate, the genomic segments of an Influenza A virus are negative-sense and single-stranded. Thus, the skilled person would understand that an Influenza A virus genomic segment would comprise the reverse complement of the nucleotide sequence of a mRNA sequence. In some embodiments, provided herein is a NS genomic segment comprising the negative-sense RNA sequence corresponding to the NS nucleotide sequence. In specific embodiments, the NS genomic segment does not encode an Influenza A virus NS1 protein. In specific embodiments, the NS genomic segment does not encode a fragment of an Influenza A virus NS1 protein. The protein may be a secretory protein (e.g, a chemokine (e.g, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, or CXCL10), an antigen (e.g., a viral antigen, a bacterial antigen, or a fungal antigen), a cytokine (e.g., IL-2 or IL- 15), or a protein with a signal sequence (e.g., a membrane anchored protein described herein). See Section 5.2, infra, for examples of25NAI-5003686295vlproteins. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 60, 27, to 61, 27 to 62, 27 to 63, 27 to 64, 27 to 65, 27 to 66, or 27 to 67 of segment 8 of an Influenza A virus. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 62, 27 to 63, 27 to 64, 27 to 65, 27 to 66, 27 to 67, or 27 to 68 of segment 8 of an Influenza A virus. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 64 of segment 8 of an Influenza A virus. The Influenza A virus may be one described herein or known in the art (e.g., Influenza A virus A / Puerto Rico / 8 / 1934, A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interi or Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14). In some embodiments, the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934. Nucleotide positions of an Influenza A virus NS1 open reading fame that correspond to certain nucleotide positions of an NS1 open reading frame of a reference Influenza A virus (e.g., Influenza A virus A / Puerto Rico / 8 / 1934) may be determined by one of skill in the art by aligning the NS segments. For example, the nucleotide position in the NS 1 open reading frame of an Influenza A virus that corresponds to nucleotide position 27 of the partial NS1 open reading frame of Influenza A virus A / Puerto Rico / 8 / 1934 may be determined by aligning the NS segments of the two viruses. Programs, such as, e.g., Clustal W or Clustal Omega may be used to align the two sequences. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of Influenza A virus A / Califomia / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior26NAI-5003686295vlAlaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, or A / Hong Kong / 1 / 68. In specific embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotide sequence of SEQ ID NO: 5. In specific embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises a nucleotide sequence that is at least 90%, at least 92%, at least 95%, or at least 96% identical to the nucleotide sequence of SEQ ID NO: 5, provided that the nucleotide sequence comprises TTG as the first three nucleotides and the splicing donor motif (SEQ ID NO: 11). In specific embodiments, the splicing donor motif comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the NS nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1. In specific embodiments, the splicing acceptor motif comprises the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of any Influenza A virus NEP open reading frame (e.g. , NEP open reading frame of an Influenza A virus described herein or known in the art). In some embodiments, the NEP open reading frame comprises the nucleotide sequence of the NEP open reading frame of segment 8 of Influenza A virus A / Puerto Rico / 8 / 1934. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of the NEP open reading frame of Influenza A virus A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interi or Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of the NEP open reading frame of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, or A / Hong Kong / 1 / 68. In some embodiments, the nucleic acid sequence of the NEP open reading frame comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 8. In specific embodiments, the nucleic acid sequence of27NAI-5003686295vlthe NEP open reading frame comprises the nucleotide sequence of SEQ ID NO: 8. In specific embodiments, the nucleic acid sequence of the 5’ NCR comprises the nucleotide sequence of SEQ ID NO: 9. In specific embodiments, the nucleic acid sequence of the 3’ NCR comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the 3’ NCR, the partial NS1 open reading frame, and the NEP open reading frame, and the 5’ NCR are from or derived from the same Influenza A virus. In some embodiments, one or more of the 3’ NCR, the partial NS1 open reading frame, and the NEP open reading frame, and the 5’ NCR are from or derived from the different Influenza A viruses. In some embodiments, the protein is a secretory protein. In some embodiments, the secretory protein is a cytokine (e.g., an immunostimulatory cytokine). In some embodiments, the cytokine is one described in Section 5.2. In specific embodiments, the cytokine is IL-2 (e.g., human IL-2). In some embodiments, the nucleotide sequence encoding the cytokine comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the cytokine comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 6 without the signal sequence. In some embodiments, the nucleotide sequence encoding the cytokine comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 20. In specific embodiments, the nucleotide sequence encoding the cytokine comprises the nucleotide sequence of SEQ ID NO: 6. In specific embodiments, the nucleotide sequence encoding the cytokine comprises the nucleotide sequence of SEQ ID NO: 6 without the signal sequence. In specific embodiments, the nucleotide sequence encoding the cytokine comprises the nucleotide sequence of SEQ ID NO: 20. In some embodiments, the cytokine encoded by the nucleotide sequence comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the cytokine encoded by the nucleotide sequence comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 10 without the signal sequence. In some embodiments, the cytokine encoded by the nucleotide sequence comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:28NAI-5003686295vl19. In specific embodiments, the cytokine encoded by the nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 10. In specific embodiments, the cytokine encoded by the nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 10 without the signal sequence. In specific embodiments, the cytokine encoded by the nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 19. In specific embodiments, the cytokine is IL-15 (e.g., human IL-15). In some embodiments, the nucleotide sequence encoding the cytokine comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 14 or 15. In some embodiments, the nucleotide sequence encoding the cytokine comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 14 without the signal sequence. In some embodiments, the nucleotide sequence encoding the cytokine comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 22. In specific embodiments, the nucleotide sequence encoding the cytokine comprises the nucleotide sequence of SEQ ID NO: 14 or 15. In specific embodiments, the nucleotide sequence encoding the cytokine comprises the nucleotide sequence of SEQ ID NO: 14 without the signal sequence. In specific embodiments, the nucleotide sequence encoding the cytokine comprises the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the cytokine encoded by the nucleotide sequence comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the cytokine encoded by the nucleotide sequence comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 16 without the signal sequence. In some embodiments, the cytokine encoded by the nucleotide sequence comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 21. In specific embodiments, the cytokine encoded by the nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 16. In specific embodiments, the cytokine encoded by the nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 16 without the signal sequence. In specific embodiments, the cytokine encoded by the nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 21. In specific embodiments, the cytokine29NAI-5003686295vlencoded by the nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 21 fused to the signal sequence of IL-2 (e.g., the amino acid sequence of SEQ ID NO: 12). In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 60, 27, to 61, 27 to 62, 27 to 63, 27 to 64, 27 to 65, 27 to 66, or 27 to 67 of segment 8 of an Influenza A virus. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 62, 27 to 63, 27 to 64, 27 to 65, 27 to 66, 27 to 67, or 27 to 68 of segment 8 of an Influenza A virus. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame of an Influenza A virus with the ATG changed to TTG comprises nucleotides corresponding to nucleotides 27 to 64 of segment 8 of a reference Influenza A virus (e.g., Influenza A virus A / Puerto Rico / 8 / 1934). The Influenza A virus may be one described herein or known in the art (e.g., Influenza A virus A / Puerto Rico / 8 / 1934, A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northem shovel er / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / 10BM01929 / 10, A / Jiangxi-Donghu / 346- 1 / 2013 , A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14). In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934. Nucleotide positions of an Influenza A virus NS1 open reading fame that correspond to certain nucleotide positions of an NS1 open reading frame of a reference Influenza A virus (e.g., Influenza A virus A / Puerto Rico / 8 / 1934) may be determined by one of skill in the art by aligning the NS segments. For example, the nucleotide position in the NS1 open reading frame of an Influenza A virus that corresponds to nucleotide position 27 of the partial NS1 open reading frame of Influenza A virus A / Puerto Rico / 8 / 1934 may be determined by aligning the NS segments of the two viruses. Programs, such as, e.g., Clustal W or Clustal Omega may be used to align the two sequences. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of Influenza A virus A / Califomia / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99,30NAI-5003686295vlA / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interi or Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, or A / Hong Kong / 1 / 68. In specific embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotide sequence of SEQ ID NO: 5. In specific embodiments, the nucleic acid sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises a nucleotide sequence that is at least 90%, at least 92%, at least 95%, or at least 96% identical to the nucleotide sequence of SEQ ID NO: 5, provided that the nucleotide sequence comprises TTG as the first three nucleotides and the splicing donor motif (SEQ ID NO: 11). In specific embodiments, the nucleic acid sequence of the splicing donor motif comprises the nucleotide sequence of SEQ ID NO: 11. In specific embodiments, the nucleic acid sequence of the splicing acceptor motif comprises the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of any Influenza A virus NEP open reading frame (e.g., NEP open reading frame of an Influenza A virus described herein or known in the art). In some embodiments, the NEP open reading frame comprises the nucleotide sequence of the NEP open reading frame of Influenza A virus A / Puerto Rico / 8 / 1934. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of the NEP open reading frame of Influenza A virus A / Califomia / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of the NEP open reading frame of segment 8 of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, or A / Hong Kong / 1 / 68. In some embodiments, the nucleic acid sequence of the NEP open reading frame comprises a nucleotide sequence that is at least 80%, at least31NAI-5003686295vl85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 8. In specific embodiments, the nucleic acid sequence of the NEP open reading frame comprises the nucleotide sequence of SEQ ID NO: 8. In specific embodiments, the nucleic acid sequence of the 5’ NCR comprises the nucleotide sequence of SEQ ID NO: 9. In specific embodiments, the nucleic acid sequence of the 3’ NCR comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the 3’ NCR, the partial NS1 open reading frame, and the NEP open reading frame, and the 5’ NCR are from or derived from the same Influenza A virus. In some embodiments, one or more of the 3’ NCR, the partial NS1 open reading frame, and the NEP open reading frame, and the 5’ NCR are from or derived from the different Influenza A viruses.

[0060] In specific embodiments, provided herein is an NS nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 3. In specific embodiments, provided herein is an NS nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 23. In specific embodiments, provided herein is an NS nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 24.

[0061] In some embodiments, provided herein is an NS genomic segment comprising in 3’ to 5’ order of the following: a 3’ NCR of segment 8 of an Influenza A virus, a partial NS1 open reading frame of an Influenza A virus (e.g., nucleotides 27 to 64 of segment 8 of an Influenza A virus) with the AUG changed to UUG, a splicing acceptor motif, NEP open reading frame of an Influenza A virus, and 5’ NCR of segment 8 of an Influenza A virus, wherein the partial NS1 open reading frame comprises a splicing donor motif. In specific embodiments, the NS genomic segment does not encode an Influenza A virus NS1 protein. In some embodiments, provided herein is an NS genomic segment comprising in 3’ to 5’ order of the following: a 3’ NCR of segment 8 of an Influenza A virus, a partial NS1 open reading frame of an Influenza A virus (e.g., nucleotides 27 to 64 of segment 8 of an Influenza A virus) with the AUG changed to UUG, a transgene, a splicing acceptor motif, NEP open reading frame of an Influenza A virus, and 5’ NCR of segment 8 of an Influenza A virus, wherein the partial NS1 open reading frame comprises a splicing donor motif. In some embodiments, the transgene encodes a biomolecule, such as described herein (e.g., in Section 5.2). In specific embodiments, the NS genomic segment does not encode an Influenza A virus NS1 protein. In some embodiments, provided herein is an NS genomic segment comprising in 3’ to 5’ order of the following: a 3’ NCR of segment 8 of an Influenza A virus, a partial NS1 open reading frame of an Influenza A virus (e.g., nucleotides 27 to 64 of segment 8 of an Influenza A virus) with the AUG changed to UUG, a negative-sense RNA32NAI-5003686295vlsequence coding a protein, such as, e.g., a secretory protein (e.g., a chemokine (e.g., CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, or CXCL10), an antigen (e.g., a viral antigen, a bacterial antigen, or a fungal antigen), or a cytokine (e.g., IL-2 or IL- 15)) or a protein with a signal sequence (e.g., a membrane anchored protein described herein), a splicing acceptor motif, NEP open reading frame of an Influenza A virus, and 5’ NCR of segment 8 of an Influenza A virus, wherein the partial NS1 open reading frame comprises a splicing donor motif. In specific embodiments, the NS genomic segment does not encode an Influenza A virus NS 1 protein. In specific embodiments, the NS genomic segment does not encode a fragment of an Influenza A virus NS1 protein. In some embodiments, the partial NS1 open reading frame with the AUG changed to UUG comprises nucleotides 27 to 64 of segment 8 of an Influenza A virus. The Influenza A virus may one described herein or known to one of skill in the art. In some embodiments, the partial NS1 open reading frame with the AUG changed to UUG comprises nucleotides corresponding to nucleotides 27 to 60, 27, to 61, 27 to 62, 27 to 63, 27 to 64, 27 to 65, 27 to 66, or 27 to 67 of segment 8 of an Influenza A virus. In some embodiments, the partial NS1 open reading frame with the AUG changed to UUG comprises nucleotides corresponding to nucleotides 27 to 62, 27 to 63, 27 to 64, 27 to 65, 27 to 66, 27 to 67, or 27 to 68 of segment 8 of an Influenza A virus. In some embodiments, the partial NS1 open reading frame of an Influenza A virus with the AUG changed to UUG comprises nucleotides corresponding to nucleotides 27 to 64 of segment 8 of a reference Influenza A virus (e.g., Influenza A virus A / Puerto Rico / 8 / 1934). The Influenza A virus may be one described herein or known in the art (e.g., Influenza A virus A / Puerto Rico / 8 / 1934, A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interi or Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14). In some embodiments, the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934. Nucleotide positions of an Influenza A virus NS1 open reading fame that correspond to certain nucleotide positions of an NS1 open reading frame of a reference Influenza A virus (e.g., Influenza A virus A / Puerto Rico / 8 / 1934) may be determined by one of skill in the art by aligning the NS segments. For example, the33NAI-5003686295vlnucleotide position in the NS 1 open reading frame of an Influenza A virus that corresponds to nucleotide position 27 of the partial NS1 open reading frame of Influenza A virus A / Puerto Rico / 8 / 1934 may be determined by aligning the NS segments of the two viruses. Programs, such as, e.g., Clustal W or Clustal Omega may be used to align the two sequences. In some embodiments, the partial NS1 open reading frame with the AUG changed to UUG comprises nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934. In some embodiments, the partial NS1 open reading frame with the AUG changed to UUG comprises nucleotides 27 to 64 of segment 8 of Influenza A virus A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northem shovel er / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / 10BM01929 / 10, A / Jiangxi-Donghu / 346- 1 / 2013 , A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the partial NS1 open reading frame with the AUG changed to UUG comprises nucleotides 27 to 64 of segment 8 of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, or A / Hong Kong / 1 / 68. In some embodiments, the partial NS1 open reading frame with the AUG changed to UUG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934. In specific embodiments, the partial NS1 open reading frame with the AUG changed to UUG comprises the negative-sense RNA complement of the nucleotide sequence of SEQ ID NO: 5. In specific embodiments, the partial NS1 open reading frame with the AUG changed to UUG comprises a nucleotide sequence that is at least 90%, at least 92%, at least 95%, or at least 96% identical to the negative- sense RNA complement of the nucleotide sequence of SEQ ID NO: 5, provided that the nucleotide sequence comprises UUG as the first three nucleotides and the splicing donor motif (negative-sense RNA complement of SEQ ID NO: 11). In specific embodiments, the splicing donor motif comprises the negative-sense RNA complement of the nucleotide sequence of SEQ ID NO: 11. In specific embodiments, the splicing acceptor motif comprises the negative-sense RNA complement of the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the NEP open reading frame comprises the nucleotide sequence of any Influenza A virus NEP open reading frame (e.g., NEP open reading frame of an Influenza A virus described herein or known in the art). In some embodiments, the NEP open reading frame comprises the NEP open reading frame of Influenza A virus A / Puerto Rico / 8 / 1934. In some embodiments, the34NAI-5003686295vlNEP open reading frame comprises the NEP open reading frame of Influenza A virus A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interi or Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the NEP open reading frame comprises the NEP open reading frame of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, or A / Hong Kong / 1 / 68. In some embodiments, the NEP open reading frame comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the negative-sense RNA complement of the nucleotide sequence of SEQ ID NO: 8. In specific embodiments, the NEP open reading frame comprises the negative-sense RNA complement of the nucleotide sequence of SEQ ID NO: 8. In specific embodiments, the 5’ NCR comprises the negative-sense RNA complement of the nucleotide sequence of SEQ ID NO: 9. In specific embodiments, the 3’ NCR comprises the negativesense RNA complement of the nucleotide sequence of SEQ ID NO: 4. In some embodiments, provided herein is an NS nucleotide sequence encoding the NS genomic segment.

[0062] In specific embodiments, provided herein is an NS genomic segment comprising the negative-sense RNA complement of the nucleotide sequence of SEQ ID NO: 3. In specific embodiments, provided herein is an NS genomic segment comprising the negative-sense RNA complement of the nucleotide sequence of SEQ ID NO: 23. In specific embodiments, provided herein is NS genomic segment comprising the negative-sense RNA complement of the nucleotide sequence of SEQ ID NO: 24.

[0063] In some embodiments, an NS genomic segment described herein is isolated. In some embodiments, an NS nucleotide sequence described herein is isolated.

[0064] In a specific embodiment, provided herein is an NS genomic segment described in Section 8. In a specific embodiment, provided herein is an NS genomic segment nucleotide sequence described in Section 8. In some embodiments, provided herein is an NS nucleotide sequence depicted in FIG. IB or 1C. In a specific embodiment, provided herein is an NS nucleotide sequence with the elements depicted in FIG. IB or 1C. In a specific embodiment, provided herein is an NS nucleotide sequence which results in the transcription of the messenger RNA depicted in FIG. IB. In a specific embodiment, provided herein is an NS35NAI-5003686295vlnucleotide sequence which results in the transcription of the messenger RNA depicted in FIG. 1C. In a specific embodiment, provided herein is an Empty ANSI described in Section 8, infra. In a specific embodiment, provided herein is an ANS1-IL2NEP described in Section 8, infra. In a specific embodiment, provided herein is an ANS1-IL15NEP described in Section 8, infra. In a specific embodiment, provided herein is an ANSl-IL2ssIL15NEP described in Section 8, infra.

[0065] In some embodiments, provided herein is a vector comprising an NS genomic segment nucleotide sequence described herein. In some embodiments, provided herein is a vector comprising an NS nucleotide sequence described herein. In some embodiments, the vector is a plasmid, phage, transposon, cosmid, bacmid, mini -plasmid (e.g, plasmid devoid of bacterial elements), virus, or virion. In some embodiments, the vector is a plasmid. In some embodiments, the vector is an ambisense plasmid (e.g, a pDZ plasmid). In some embodiments, the vector is an pDZ plasmid (see, e.g., Quinlivan et al., 2005, J. of Virology 79: 8431-8439 for information relating to the pDZ plasmid). In some embodiments, the vector is a pHW2000 plasmid (see, e.g., Hoffmann et al., 2000, Proc Natl Acad Sci U S A. 97(11): 6108- 13 for information relating to the pHW2000 plasmid). In some embodiments, the vector is a pAD3000 plasmid (see, e.g., Hoffmann et al., 2000, Proc Natl Acad Sci U S A. 97(11): 6108- 13 for information relating to the pAD3000 plasmid). In some embodiments, the vector is a pAD4000 plasmid (see, e.g., Wang et al., 2007, J. of Virology 4: 102 for information relating to the pAD4000 plasmid).

[0066] Techniques known to one of skill in the art or described herein (e.g., in Section 8) may be used to produce an NS nucleotide sequence, a vector comprising an NS nucleotide sequence, an NS genomic segment nucleotide sequence, or a vector comprising an NS genomic segment nucleotide sequence. In specific embodiments, the methods described herein (e.g., in Section 8) are used to produce EmptyANSl. In specific embodiments, the methods described herein (e.g., in Section 8) are used to produce ANS1-IL2NEP. In specific embodiments, the methods described herein (e.g., in Section 8) are used to produce NS1- IL15NEP. In specific embodiments, the methods described herein (e.g., in Section 8) are used to produce ANS l-IL2ssIL 15 NEP.

[0067] In a specific embodiment, provided herein is a plasmid described in Section 8.5.2 TRANSGENES

[0068] In some embodiments, a transgene is incorporated into or present in an NS genomic segment described herein (e.g., in Section 5.1 or 8) or an NS nucleotide sequence described herein (e.g., in Section 5.1 or 8). In some embodiments, a transgene comprises a nucleotide36NAI-5003686295vlsequence encoding a biomolecule, such as a protein, polypeptide, peptide, RNA (e.g., siRNA, miRNA, pre-miRNA, IncRNA, snoRNA, small hairpin RNA (shRNA), trans-splicing RNA, or antisense RNA), an antisense oligonucleotide (AON), antisense ribo-oligonucleotide, an antigen, or a dominant negative mutant. In some embodiments, the biomolecule is a protein. In some embodiments, the biomolecule is a secretory protein. In some embodiments, the biomolecule is a protein with a signal sequence. In some embodiments, the biomolecule is a membrane anchored protein. In some embodiments, the biomolecule is a polypeptide. In some embodiments, the biomolecule is a peptide. In some embodiments, the biomolecule is a cytokine (e.g., IL-2 or IL- 15). In some embodiments, the biomolecule is an immunostimulatory cytokine. In some embodiments, the biomolecule is a chemokine. The chemokine may be CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, or CXCL10. In some embodiments, the biomolecule is an antigen (e.g., a viral antigen, a bacterial antigen, or a fungal antigen). In some embodiments, a transgene comprises a nucleotide sequence encoding a protein. In some embodiments, a transgene comprises a regulatory control sequence or element operably linked to a nucleotide sequence encoding a biomolecule. In some embodiments, a transgene comprises two or more regulatory control sequences or elements operably linked to a nucleotide sequence encoding a biomolecule. A regulatory control sequence or element may permit the transcription, translation and / or expression of a biomolecule in a cell transfected or transduced with a vector or Influenza A virus described herein. As used herein, “operably linked” includes both expression control sequences that are contiguous with the nucleotide sequence of interest and expression control sequences that act in trans or at a distance (e.g., an enhancer) to control the nucleotide sequence of interest. Exemplary of regulatory control elements that can be used, include but are not limited to, transcription initiation, termination, promoter and / or enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.

[0069] In some embodiments, a nucleotide sequence encoding a protein, such as a secretory protein or a protein with a signal sequence (e.g., a membrane anchored protein) is incorporated into or present in an NS genomic segment described herein or an NS nucleotide sequence described herein. See, e.g., Section 5.1 and Section 8 for a description of a nucleotide encoding secretory protein or a protein with a signal sequence (e.g., a membrane anchored protein) being incorporated into or present in an NS genomic segment or an NS37NAI-5003686295vlnucleotide sequence. In a specific embodiment, a nucleotide sequence encoding a protein with a signal sequence is incorporated into or present in an NS genomic segment as described herein or an NS nucleotide sequence described herein. In a specific embodiment, a nucleotide sequence encoding a membrane anchored protein is incorporated into or present in an NS genomic segment described herein or an NS nucleotide sequence described herein. In specific embodiments, the membrane anchored protein is one that uses the same ER / Golgi export pathway as a secretory protein. In specific embodiments, the membrane anchored protein is one that locates and attaches to cellular membrane covalently or non-covalently. The membrane anchored protein may be an integral membrane protein or a transmembrane protein. In a specific embodiment, a nucleotide sequence encoding a secretory protein is incorporated into or present in an NS genomic segment described herein or an NS nucleotide sequence described herein. In some embodiments, the secretory protein is a chemokine. The chemokine may be CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, or CXCL10. In some embodiments, a nucleotide sequence encoding an antigen is incorporated into or present in an NS genomic segment described herein or an NS nucleotide sequence described herein. In some embodiments, the secretory protein is a cytokine, such as, e.g., IL-2 or IL- 15, is incorporated into or present in an NS genomic segment as described herein or an NS nucleotide sequence described herein.

[0070] IL-2

[0071] In a specific embodiment, a nucleotide sequence encoding IL-2 is incorporated into or present in an NS genomic segment described herein or an NS nucleotide sequence described herein. See, e.g., Section 5.1 and Section 8 for a description of a cytokine being incorporated into or in present an NS genomic segment described herein, or an NS nucleotide sequence described herein. In a specific embodiment, the nucleotide sequence encodes human IL-2. One of skill in the art would be able to use such sequence information to produce a nucleotide sequence for incorporation into an NS genomic segment described herein or an NS nucleotide sequence described herein. In a specific embodiment, a nucleotide sequence encoding a human IL-2 comprising the amino acid sequence set forth in GenBank Accession No. NP_000577.2, NM_000586, or SEQ ID NO: 10 or 19, may be incorporated into or present in an NS genomic segment as described herein or an NS nucleotide sequence described herein. Given the degeneracy of the nucleic acid code, there are a number of different nucleic acid sequences that may encode the same IL-2 protein. In a specific embodiment, a nucleotide sequence comprises the reverse complement RNA sequence of the sequence of set forth in SEQ ID NO: 6, or the sequence set forth in SEQ ID NO: 6. In a38NAI-5003686295vlspecific embodiment, a nucleotide sequence comprises the corresponding negative sense RNA sequence of the sequence of set forth in SEQ ID NO: 6 without the signal sequence, or the sequence set forth in SEQ ID NO: 6 without the signal sequence. In some embodiments, a nucleotide sequence comprises the corresponding negative sense RNA sequence of the sequence of set forth in SEQ ID NO: 20, or the sequence set forth in SEQ ID NO: 20. In some embodiments, in place of the signal sequence of SEQ ID NO: 6 a different signal sequence (e.g., a signal sequence heterologous to IL-2) may be present. In some embodiments, the human IL-2 encoded by a nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 10 without the signal sequence. In some embodiments, the human IL-2 encoded by a nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 19 and a signal sequence heterologous to IL-2, and the nucleotide sequence is incorporated or present in an NS genomic segment described herein or an NS nucleotide sequence described herein. In some embodiments, a nucleotide sequence comprises SEQ ID NO: 20 and a heterologous signal sequence. In a specific embodiment, a nucleotide sequence comprising the nucleotide sequence encoding IL-2 (e.g., human IL-2) is codon optimized. See, e.g., Section 5.2.1, infra, for a discussion regarding codon optimization. In some embodiments, the human IL-2 encoded by a nucleotide sequence comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 10. In some embodiments, the human IL-2 encoded by a nucleotide sequence comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 10 without the signal sequence. In some embodiments, the human IL-2 encoded by a nucleotide sequence comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 19. In a specific embodiment, the nucleotide sequence encoding IL-2 comprises the sequence set forth in SEQ ID NO: 6 or 20. In a specific embodiment, human IL-2 encoded by a nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 10 or 19, and the nucleotide sequence is incorporated into or present in an NS genomic segment described herein or an NS nucleotide sequence described herein.

[0072] “Interleukin-2” and “IL-2” refer to any IL-2 known to those of skill in the art. In preferred embodiments, the IL-2 is bioactive. In preferred embodiments, the IL-2 exhibits one or more functions of IL-2. Examples of functions of IL-2 include regulation of signals to T cells, B cells, and NK cells, promotion of the development of T regulatory cells, and the maintenance of self-tolerance. In certain embodiments, the IL-2 may be human, dog, cat,39NAI-5003686295vlhorse, pig, or cow IL-2. In a specific embodiment, the IL-2 is human IL-2. GenBank™ accession numbers NG_016779.1 (GI number 291219938) and NM_000586 provide exemplary human IL-2 nucleic acid sequences. GenBank™ accession numbers NP_000577.2 (GI number 28178861) and NM_000586 provide exemplary human IL-2 amino acid sequences. As used herein, the terms “interleukin-2” and “IL-2” encompass interleukin-2 polypeptides that are modified by post-translational processing such as signal peptide cleavage, disulfide bond formation, glycosylation (e.g, N-linked glycosylation), protease cleavage and lipid modification (e.g., S-palmitoylation). The IL-2 may be the mature or immature form. The IL-2 may be a naturally occurring isoform of human IL-2 or another mammalian IL-2. In some embodiments, IL-2 consists of a single polypeptide chain that includes a signal sequence (otherwise sometimes referred to herein as a signal peptide). In other embodiments, IL-2 consists of a single polypeptide chain that does not include a signal sequence. The signal sequence can be the naturally occurring signal peptide sequence or a variant thereof. In some embodiments, the signal peptide is an IL-2 signal peptide. In some embodiments, the signal peptide is heterologous to IL-2.

[0073] In a specific embodiment, a nucleotide sequence encoding an IL-2 derivative is incorporated into or present in an NS genomic segment described herein or an NS nucleotide sequence described herein. See, e.g., Section 5.1 and Section 8 for a description of a cytokine being incorporated into or present in an NS genomic segment described herein or an NS nucleotide sequence described herein. In a specific embodiment, the nucleotide sequence encodes a human IL-2 derivative. One of skill in the art would be able to use such sequence information to produce a nucleotide sequence for incorporation into an NS genomic segment described herein or an NS nucleotide sequence described herein. In some embodiments, an IL-2 derivative has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% amino acid sequence identity to an IL-2 described herein or known to those of skill in the art. In some embodiments, an IL-2 derivative comprises an amino acid sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 10. In some embodiments, an IL-2 derivative comprises an amino acid sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 10 without the signal sequence. In some embodiments, an IL-2 derivative comprises an amino acid sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 19. In some embodiments, an IL-2 derivative comprises deleted forms of a known IL-2 (e.g, human IL-2), wherein up to about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are deleted from the known IL-2 (e.g.,40NAI-5003686295vlhuman IL-2). Also provided herein are IL-2 derivatives comprising deleted forms of a known IL-2, wherein about 1-3, 3-5, 5-7, 7-10, 10-15, or 15-20 amino acid residues are deleted from the known IL-2 (e.g., human IL-2). The amino acid residue deletions may be at the N- terminus and / or the C-terminus. Further provided herein are IL-2 derivatives comprising altered forms of a known IL-2 (e.g., human IL-2), wherein up to about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues of the known IL-2 are substituted (e.g., conservatively substituted) with other amino acids. In a specific embodiment, the known IL-2 is human IL-2, such as, e.g., provided in GenBank™ accession number NP 000577.2 (GI number 28178861) or NM 000586, or SEQ ID NO: 10. In some embodiments, an IL-2 derivative comprises up to about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservatively substituted amino acids. Examples of conservative amino acid substitutions include, e.g., replacement of an amino acid of one class with another amino acid of the same class. In some embodiments, a conservative substitution does not alter the structure or function, or both, of a polypeptide. Classes of amino acids may include hydrophobic (Met, Ala, Vai, Leu, He), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gin, His, Lys, Arg), conformation disruptors (Gly, Pro) and aromatic (Trp, Tyr, Phe).

[0074] In some embodiments, an IL-2 derivative is at least 80%, 85%, 90%, 95%, 98%, or 99% or is 80% to 85%, 80% to 90%, 80% to 95%, 90% to 95%, 85% to 99%, or 95% to 99% identical (e.g., sequence identity) to a naturally occurring IL-2 (e.g., human IL-2). In some embodiments, an IL-2 derivative is a polypeptide encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% or is 80% to 85%, 80% to 90%, 80% to 95%, 90% to 95%, 85% to 99%, or 95% to 99% identical (e.g., sequence identity) to a nucleic acid sequence encoding a naturally occurring IL-2. In some embodiments, the naturally occurring IL-2 is human IL-2, such as, e.g., provided in GenBank™ accession number NP 000577.2 (GI number 28178861), GenBank™ accession number NM 000586, or GenBank™ accession number NG_016779.1 (GI number 291219938), or SEQ ID NOTO. In some embodiments, an IL-2 derivative contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, or 2 to 5, 2 to 10, 5 to 10, 5 to 15, 5 to 20, 10 to 15, or 15 to 20 amino acid mutations (i.e., additions, deletions, substitutions or any combination thereof) relative to a naturally occurring IL-2 (e.g., human IL-2). In some embodiments, an IL-2 derivative is a polypeptide encoded by nucleic acid sequence that can hybridize under high, moderate or typical stringency hybridization conditions to a nucleic acid sequence encoding a naturally occurring IL-2 (e.g., human IL-2). In some embodiments, an IL-2 derivative is a polypeptide encoded by nucleic acid sequence that can hybridize under high, moderate or typical41NAI-5003686295vlstringency hybridization conditions to across an entire nucleic acid sequence encoding a naturally occurring IL-2 (e.g., human IL-2). Hybridization conditions are known to one of skill in the art (see, e.g., U.S. Patent Application No. 2005 / 0048549 at, e.g., paragraphs 72 and 73). In some embodiments, an IL-2 derivative is a polypeptide encoded by a nucleic acid sequence that can hybridize under high, moderate or typical stringency hybridization conditions to a nucleic acid sequence encoding a fragment of a naturally occurring IL-2 (e.g., human IL-2) of at least 10 contiguous amino acids, at least 12 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, at least 125 contiguous amino acids, at least 150 contiguous amino acids, or 10 to 20, 20 to 50, 25 to 75, 25 to 100, 25 to 150, 50 to 75, 50 to 100, 75 to 100, 50 to 150, 75 to 150, 100 to 150, or 100 to 200 contiguous amino acids. In some embodiments, an IL-2 derivative is a fragment of a naturally occurring IL-2 (e.g, human IL-2). IL-2 derivatives also include polypeptides that comprise the amino acid sequence of a naturally occurring mature form of IL-2 and a heterologous signal peptide amino acid sequence. In addition, IL-2 derivatives include polypeptides that have been modified by, e.g., glycosylation, acetylation, pegylation, phosphorylation, amidation, derivitization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein moiety, etc. Further, IL-2 derivatives include polypeptides comprising one or more non-classical amino acids. In a preferred embodiment, the IL-2 derivative is bioactive. In preferred embodiments, the IL-2 derivative retains one, two, or more, or all of the functions of the naturally occurring IL-2 (e.g., human IL-2) from which it was derived. Examples of functions of IL-2 include regulation of signals to T cells, B cells, and NK cells, promotion of the development of T regulatory cells, and the maintenance of self-tolerance. Tests for determining whether or not an IL-2 derivative retains one or more functions of the naturally occurring IL-2 (e.g., human IL-2) from which it was derived are known to one of skill in the art.

[0075] In specific embodiments, a nucleotide sequence encoding IL-2 or a derivative thereof is codon optimized. See, e.g., Section 5.2.1 regarding codon optimization.

[0076] IL-15

[0077] In a specific embodiment, a nucleotide sequence encoding IL-15 is incorporated into or present in an NS genomic segment described herein or an NS nucleotide sequence described herein. See, e.g., Section 5.1 and Section 8 for a description of a cytokine being incorporated into or in present an NS genomic segment described herein, or an NS nucleotide42NAI-5003686295vlsequence described herein. In a specific embodiment, the nucleotide sequence encodes human IL-15. One of skill in the art would be able to use such sequence information to produce a nucleotide sequence for incorporation into an NS genomic segment described herein or a nucleotide sequence thereof. In a specific embodiment, a nucleotide sequence encoding a human IL- 15 comprising the amino acid sequence set forth in GenBank Accession No. NM_172174 or NM_000585.5, or SEQ ID NO: 16 or 21, may be incorporated into or present in an NS genomic segment as described herein or an NS nucleotide sequence described herein. Given the degeneracy of the nucleic acid code, there are a number of different nucleic acid sequences that may encode the same IL- 15 protein. In a specific embodiment, a nucleotide sequence comprises the reverse complement RNA sequence of the sequence of set forth in SEQ ID NO: 14, or the sequence set forth in SEQ ID NO: 14. In a specific embodiment, a nucleotide sequence comprises the corresponding negative sense RNA sequence of the sequence of set forth in SEQ ID NO: 14 without the signal sequence, or the sequence set forth in SEQ ID NO: 14 without the signal sequence. In a specific embodiment, a nucleotide sequence comprises the reverse complement RNA sequence of the sequence of set forth in SEQ ID NO: 15, or the sequence set forth in SEQ ID NO: 15. In some embodiments, a nucleotide sequence comprises the corresponding negative sense RNA sequence of the sequence of set forth in SEQ ID NO: 22, or the sequence set forth in SEQ ID NO: 22. In some embodiments, in place of the signal sequence of SEQ ID NO: 14 a different signal sequence (e.g, a signal sequence heterologous to IL- 15) may be present. In some embodiments, the human IL- 15 encoded by a nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 16 without the signal sequence. In some embodiments, the human IL-15 encoded by a nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 21 and the amino acid sequence of the IL- 15 is fused to a signal sequence heterologous to IL- 15. In some embodiments, a nucleotide sequence comprises SEQ ID NO: 22 and a nucleic acid sequence of a heterologous signal sequence (e.g, nucleotide the signal sequence of IL-2, such as, e.g., SEQ ID NO: 13). In a specific embodiment, a nucleotide sequence comprising the nucleotide sequence encoding IL- 15 (e.g., human IL- 15) is codon optimized. See, e.g., Section 5.2.1, infra, for a discussion regarding codon optimization. In some embodiments, the human IL- 15 encoded by a nucleotide sequence comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 16. In some embodiments, the human IL-15 encoded by a nucleotide sequence comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 16 without the signal sequence. In43NAI-5003686295vlsome embodiments, the human IL-15 encoded by a nucleotide sequence comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 21. In a specific embodiment, the nucleotide sequence encoding IL-15 comprises the sequence set forth in SEQ ID NO: 14 or 15. In a specific embodiment, human IL-15 encoded by a nucleotide sequence comprises the amino acid sequence of SEQ ID NO: 16 or 21, and the nucleotide sequence is incorporated into or present in an NS genomic segment described herein or an NS nucleotide sequence described herein.

[0078] “Interleukin- 15” and “IL- 15” refer to any IL- 15 known to those of skill in the art. In preferred embodiments, the IL- 15 is bioactive. In preferred embodiments, the IL- 15 exhibits one or more functions of IL-15. Examples of functions of IL- 15 include development, differentiation, and survival of NK cell, differentiation of dendritic cells, promotion of neutrophil cytoskeletal rearrangement, and stimulation of T cell proliferation. In certain embodiments, the IL- 15 may be human, dog, cat, horse, pig, or cow IL-15. In a specific embodiment, the IL-15 is human IL-15. GenBank™ accession number NM 000585.5 provides an exemplary human IL-15 nucleic acid sequence. GenBank™ accession number NM 000585.5 provides an exemplary human IL-15 amino acid sequence. As used herein, the terms “interleukin- 15” and “IL- 15” encompass interleukin- 15 polypeptides that are modified by post-translational processing such as signal peptide cleavage, disulfide bond formation, glycosylation (e.g., N-linked glycosylation), protease cleavage and lipid modification (e.g., S- palmitoylation). The IL- 15 may be the mature or immature form. The IL- 15 may be a naturally occurring isoform of human IL-15 or another mammalian IL-15. In some embodiments, IL-15 includes of a single polypeptide chain that includes a signal sequence (otherwise sometimes referred to herein as a signal peptide). In other embodiments, IL- 15 includes of a single polypeptide chain that does not include a signal sequence. The signal sequence can be the naturally occurring signal peptide sequence or a variant thereof. In some embodiments, the signal peptide is an IL-15 signal peptide. In some embodiments, the signal peptide is heterologous to IL-15.

[0079] In a specific embodiment, a nucleotide sequence encoding an IL- 15 derivative is incorporated into or present in an NS genomic segment described herein or an NS nucleotide sequence described herein. See, e.g., Section 5.1 and Section 8 for a description of a cytokine being incorporated into or present in an NS genomic segment described herein or an NS nucleotide sequence described herein. In a specific embodiment, the nucleotide sequence encodes a human IL- 15 derivative. One of skill in the art would be able to use such sequence44NAI-5003686295vlinformation to produce a nucleotide sequence for incorporation into an NS genomic segment described herein or an NS nucleotide sequence described herein. In some embodiments, an IL-15 derivative has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% amino acid sequence identity to an IL- 15 described herein or known to those of skill in the art. In some embodiments, an IL-15 derivative comprises an amino acid sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 16. In some embodiments, an IL-15 derivative comprises an amino acid sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 16 without the signal sequence. In some embodiments, an IL-15 derivative comprises an amino acid sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 21. In some embodiments, an IL-15 derivative comprises deleted forms of a known IL-15 (e.g., human IL-15), wherein up to about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are deleted from the known IL-15 (e.g., human IL-15). Also provided herein are IL-15 derivatives comprising deleted forms of a known IL-15, wherein about 1-3, 3-5, 5-7, 7-10, 10-15, or 15-20 amino acid residues are deleted from the known IL-15 (e.g., human IL-15). The amino acid residue deletions may be at the N-terminus and / or the C-terminus. Further provided herein are IL- 15 derivatives comprising altered forms of a known IL- 15 (e.g., human IL- 15), wherein up to about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues of the known IL-15 are substituted (e.g., conservatively substituted) with other amino acids. In a specific embodiment, the known IL-15 is human IL-15, such as, e.g., provided in GenBank™ accession number NM_172174 or NM_000585.5, or SEQ ID NO: 16 or 21. In some embodiments, an IL-15 derivative comprises up to about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservatively substituted amino acids. Examples of conservative amino acid substitutions include, e.g., replacement of an amino acid of one class with another amino acid of the same class. In some embodiments, a conservative substitution does not alter the structure or function, or both, of a polypeptide. Classes of amino acids may include hydrophobic (Met, Ala, Vai, Leu, He), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gin, His, Lys, Arg), conformation disruptors (Gly, Pro) and aromatic (Trp, Tyr, Phe).

[0080] In some embodiments, an IL- 15 derivative is at least 80%, 85%, 90%, 95%, 98%, or 99% or is 80% to 85%, 80% to 90%, 80% to 95%, 90% to 95%, 85% to 99%, or 95% to 99% identical (e.g., sequence identity) to a naturally occurring IL-15 (e.g., human IL-15). In some embodiments, an IL- 15 derivative is a polypeptide encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% or is 80% to 85%, 80% to 90%, 80% to 95%,45NAI-5003686295vl90% to 95%, 85% to 99%, or 95% to 99% identical (e.g., sequence identity) to a nucleic acid sequence encoding a naturally occurring IL-15. In some embodiments, the naturally occurring IL-15 is human IL-15, such as, e.g., provided in GenBank™ accession number NM_172174 or NM_000585.5, or SEQ ID NO: 16 or 21. In some embodiments, an IL-15 derivative contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, or 2 to 5, 2 to 10, 5 to 10, 5 to 15, 5 to 20, 10 to 15, or 15 to 20 amino acid mutations (i.e., additions, deletions, substitutions or any combination thereof) relative to a naturally occurring IL- 15 (e.g., human IL- 15). In some embodiments, an IL- 15 derivative is a polypeptide encoded by nucleic acid sequence that can hybridize under high, moderate or typical stringency hybridization conditions to a nucleic acid sequence encoding a naturally occurring IL- 15 (e.g., human IL- 15). In some embodiments, an IL- 15 derivative is a polypeptide encoded by nucleic acid sequence that can hybridize under high, moderate or typical stringency hybridization conditions to across an entire nucleic acid sequence encoding a naturally occurring IL- 15 (e.g., human IL- 15). Hybridization conditions are known to one of skill in the art (see, e.g., U.S. Patent Application No. 2005 / 0048549 at, e.g., paragraphs 72 and 73). In some embodiments, an IL- 15 derivative is a polypeptide encoded by a nucleic acid sequence that can hybridize under high, moderate or typical stringency hybridization conditions to a nucleic acid sequence encoding a fragment of a naturally occurring IL- 15 (e.g., human IL- 15) of at least 10 contiguous amino acids, at least 12 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, at least 125 contiguous amino acids, at least 150 contiguous amino acids, or 10 to 20, 20 to 50, 25 to 75, 25 to 100, 25 to 150, 50 to 75, 50 to 100, 75 to 100, 50 to 150, 75 to 150, 100 to 150, or 100 to 200 contiguous amino acids. In some embodiments, an IL-15 derivative is a fragment of a naturally occurring IL-15 (e.g., human IL-15). IL-15 derivatives also include polypeptides that comprise the amino acid sequence of a naturally occurring mature form of IL- 15 and a heterologous signal peptide amino acid sequence. In addition, IL- 15 derivatives include polypeptides that have been modified by, e.g., glycosylation, acetylation, pegylation, phosphorylation, amidation, derivitization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein moiety, etc. Further, IL-15 derivatives include polypeptides comprising one or more non-classical amino acids. In a preferred embodiment, the IL- 15 derivative is bioactive. In preferred embodiments, the IL- 15 derivative retains one, two, or more, or all of the functions of the naturally occurring IL- 1546NAI-5003686295vl(e.g., human IL- 15) from which it was derived. Examples of functions of IL- 15 include development, differentiation, and survival of NK cell, differentiation of dendritic cells, promotion of neutrophil cytoskeletal rearrangement, and stimulation of T cell proliferation. Tests for determining whether or not an IL- 15 derivative retains one or more functions of the naturally occurring IL-15 (e.g., human IL-15) from which it was derived are known to one of skill in the art.

[0081] In specific embodiments, a nucleotide sequence encoding IL- 15 or a derivative thereof is codon optimized. See, e.g., Section 5.2.1 regarding codon optimization.5.2.1 CODON OPTIMIZATION

[0082] Any codon optimization technique known to one of skill in the art may be used to codon optimize a nucleic acid sequence encoding a protein, such as a secretory protein (e.g., a cytokine (e.g., IL-2 or IL- 15), a chemokine, an antigen (e.g., a viral antigen, a bacterial antigen, or a fungal antigen)) or a protein with a signal sequence (e.g., a membrane anchored protein). Methods of codon optimization are known in the art, e.g, the OptimumGene™ (GenScript®) protocol and Genewiz® protocol, which are incorporated by reference herein in its entirety. See also U.S. Patent No. 8,326,547 for methods for codon optimization, which is incorporated herein by reference in its entirety.

[0083] As an exemplary method for codon optimization, each codon in the open frame of the nucleic acid sequence encoding a protein, polypeptide, or peptide is replaced by the codon most frequently used in mammalian proteins (e.g., human proteins). This may be done using a web-based program (www.encorbio.com / protocols / Codon.htm) that uses the Codon Usage Database, maintained by the Department of Plant Gene Research in Kazusa, Japan.5.3 RECOMBINANT INFLUENZA A VIRUSES

[0084] In one aspect, provided herein are recombinant Influenza A viruses comprising an NS genomic segment described herein (e.g., in Section 5.1, or 8). In some embodiments, provided herein is a recombinant Influenza A virus comprising: (1) an NS genomic segment described herein (e.g., in Section 5.1, or 8), (2) an HA genomic segment, (3) an NA genomic segment, (4) a PB1 genomic segment, (5) a PB2 genomic segment, (6) a PA agenomic segment, (7) an M genomic segment, and (8) an NP genomic segment. In some embodiments, the NS genomic segment comprises the negative sense RNA sequence corresponding to a nucleotide sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at leaset 60%, at least 65%, at least 70%, or at least 75% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS genomic segment comprises the negative sense RNA sequence corresponding to a nucleotide sequence that is at least 80% or47NAI-5003686295vlat least 85% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS genomic segment comprises the negative sense RNA sequence corresponding to a nucleotide sequence that is at least 90% or at least 95% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleotide sequence encoding the NS genomic segment comprises the negative sense RNA sequence corresponding to a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS genomic segment comprises the negative sense RNA sequence corresponding to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS genomic segment comprises the negative sense RNA sequence corresponding to a nucleotide sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at leaset 60%, at least 65%, at least 70%, or at least 75% identical to the nucleotide sequence of SEQ ID NO:23 or 24. In some embodiments, the NS genomic segment comprises the negative sense RNA sequence corresponding to a nucleotide sequence that is at least 80% or at least 85% identical to the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the NS genomic segment comprises the negative sense RNA sequence corresponding to a nucleotide sequence that is at least 90% or at least 95% identical to the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the nucleotide sequence encoding the NS genomic segment comprises the negative sense RNA sequence corresponding to a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the NS genomic segment comprises the negative sense RNA sequence corresponding to the nucleotide sequence of SEQ ID NO: 23 or 24.

[0085] In some embodiments, the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from any Influenza A virus (e.g., any Influenza A virus described herein or known to one of skill in the art). In some embodiments, one or more of the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from the same Influenza A virus or different Influenza A viruses. In some embodiments, the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from the same Influenza A virus. In some embodiments, two or more of the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from different Influenza A viruses. In some embodiments, one or more of the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from Influenza A viruses of the same subtype. In some embodiments, the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from Influenza A viruses of the same subtype. In some embodiments, one or more of the PB1, PB2, PA, NP, M, HA and NA genomic segments are48NAI-5003686295vlfrom or derived from different Influenza A virus subtypes. In some embodiments, one or more of the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from the different strains of Influenza A virus. The strains of Influenza A virus may be from the same subtype or different subtypes. In some embodiments, one or more of the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from the same strain of Influenza A virus. In some embodiments, the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from the same strain of Influenza A virus. In some embodiments, one or more of the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from Influenza A virus A / Puerto / 8 / 1934. In some embodiments, the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from Influenza A virus A / Puerto / 8 / 1934. In some embodiments, one or more of the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from Influenza A virus A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northem shovel er / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / 10BM01929 / 10, A / Jiangxi-Donghu / 346- 1 / 2013 , A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from Influenza A virus A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interi or Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In some embodiments, one or more of the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from a cold-adapted Influenza A virus. In some embodiments, the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from a cold- adapted Influenza A virus. In some embodiments, one or more of the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from an attenuated Influenza A virus. In some embodiments, the PB1, PB2, PA, NP, M, HA and NA genomic segments are from or derived from an attenuated Influenza A virus.49NAI-5003686295vl

[0086] In some embodiments, the HA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strain(s) from which the PB1, PB2, PA, NP, and M genomic segments are from or derived from. In some embodiments, the HA genomic segment is from or derived from a seasonal influenza virus. In some embodiments, the HA genomic segment is from or derived from an Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18. In some embodiments, the HA genomic segment is from or derived from a naturally occurring Influenza A virus. In some embodiments, the HA genomic segment is from an exotic Influenza A virus (e.g., an Influenza A virus not prevalent in humans). In some embodiments, the HA genomic segment is artificially created or a consensus HA genomic segment. In some embodiments, the use of an exotic Influenza A virus HA genomic segment or an artificially created HA genomic allows for the evasion of pre-existing immunity to HA in a subject. In some embodiments, the HA genomic segment is from or derived from the same Influenza A virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, or M genomic segment is from or derived from. In some embodiments, the HA genomic segment is from or derived from the same Influenza A virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, and M genomic segments are from or derived from. In some embodiments, the NA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strain(s) from which the PB1, PB2, PA, NP, M, and NS genomic segments are from or derived from. In some embodiments, the NA genomic segment is from or derived from a seasonal Influenza A virus. In some embodiments, the NA genomic segment is from or derived from a naturally occurring Influenza A virus. In some embodiments, the NA genomic segment is from or derived from an Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, or Nl l subtype. In some embodiments, the NA genomic segment is artificially created or a consensus NA genomic segment. In some embodiments, the NA genomic segment is from or derived from the same Influenza A virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, or M genomic segment is from or derived from. In some embodiments, the NA genomic segment is from or derived from the same influenza virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, and M genomic segments are from or derived from. In some embodiments, the HA genomic segment and NA genomic segment are from or derived from the same Influenza A virus strain. In some embodiments, the HA genomic segment and NA genomic segment are from or derived from different Influenza A virus strains. In some embodiments, the PB1, PB2, PA, NP, M, HA, and NA genomic segments are from or derived50NAI-5003686295vlfrom the same Influenza A virus strain. In some embodiments, the PB1, PB2, PA, NP, M, HA, and NA genomic segments are from or derived from a first Influenza A virus and the NS genomic segment described herein is from or derived from a second Influenza A virus, wherein the first and second Influenza A viruses are different. In some embodiments, the first and second Influenza A viruses are from the same subtype. In some embodiments, the first and second Influenza A viruses are different strains. In some embodiments, the HA and NA genomic segments are from or derived from a first Influenza A virus and the PB1, PB2, PA, NP, M, and NS genomic segments are from or derived from a second Influenza A virus, wherein the first and second Influenza A viruses are different. In some embodiments, the first Influenza A viruses is a seasonal Influenza A virus. In some embodiments, the first and second Influenza A viruses are different subtypes. In some embodiments, the first and second Influenza A viruses are different strains. In some embodiments, the first and second Influenza A viruses are from different subtypes. In some embodiments, the PB1, PB2, PA, NP, and M genomic segments are from or derived from a first Influenza A virus, an NS genomic segment described herein is from or derived from a second Influenza A virus, and the HA and NA genomic segments are from or derived from a third Influenza A virus, wherein the first, second, and third Influenza A viruses are different. In some embodiments, the first, second, and third Influenza A viruses are from the same subtype. In some embodiments, the first, second, and third Influenza A viruses are from different strains. In some embodiments, the first, second, and third Influenza A viruses are from different subtypes. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1, PB2, PA, NP, M, HA, and NA genomic segments comprise one or more mutations that attenuate the Influenza A virus. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1, PB2, PA, NP, M, HA, and NA genomic segments comprise a heterologous nucleotide sequence. The heterologous nucleotide sequence may be any nucleotide sequence segment not found in the Influenza A virus genomic segment of interest or any Influenza A virus genomic segments. In a specific embodiment, the heterologous nucleotide sequence encodes an antigen. The antigen may be any antigen not found in nature in an Influenza A virus.

[0087] Non-limiting examples of Influenza A viruses include subtype H1N1, subtype H1N2, subtype H1N3, subtype H10N4, subtype H10N5, subtype H10N7, subtype H10N8, subtype H10N9, subtype Hl INI, subtype Hl 1N13, subtype Hl 1N2, subtype Hl 1N4, subtype Hl 1N6, subtype Hl 1N8, subtype Hl 1N9, subtype H12N1, subtype H12N4, subtype H12N5, subtype H12N8, subtype H13N2, subtype H13N3, subtype H13N6, subtype H13N7, subtype Hl 4N5, subtype H14N6, subtype H15N8, subtype H15N9, subtype H16N3, subtype51NAI-5003686295vlH1N1, subtype H1N2, subtype H1N3, subtype H1N6, subtype H1N9, subtype H2N1, subtype H2N2, subtype H2N3, subtype H2N5, subtype H2N7, subtype H2N8, subtype H2N9, subtype H3N1, subtype H3N2, subtype H3N3, subtype H3N4, subtype H3N5, subtype H3N6, subtype H3N8, subtype H3N9, subtype H4N1, subtype H4N2, subtype H4N3, subtype H4N4, subtype H4N5, subtype H4N6, subtype H4N8, subtype H4N9, subtype H5N1, subtype H5N2, subtype H5N3, subtype H5N4, subtype H5N6, subtype H5N7, subtype H5N8, subtype H5N9, subtype H6N1, subtype H6N2, subtype H6N3, subtype H6N4, subtype H6N5, subtype H6N6, subtype H6N7, subtype H6N8, subtype H6N9, subtype H7N1, subtype H7N2, subtype H7N3, subtype H7N4, subtype H7N5, subtype H7N7, subtype H7N8, subtype H7N9, subtype H8N4, subtype H8N5, subtype H9N1, subtype H9N2, subtype H9N3, subtype H9N5, subtype H9N6, subtype H9N7, subtype H9N8, and subtype H9N9.

[0088] Specific examples of the Influenza A virus strains include A / Califomia / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, and A / HK / 4801 / 14.

[0089] Other specific examples of strains of Influenza A virus include, but are not limited to: A / sw / Iowa / 15 / 30 (H1N1); A / WSN / 33 (H1N1); A / eq / Prague / 1 / 56 (H7N7); A / PR / 8 / 34; A / mallard / Potsdam / 178-4 / 83 (H2N2); A / herring gull / DE / 712 / 88 (H16N3); A / sw / Hong Kong / 168 / 1993 (H1N1); A / mallard / Alberta / 211 / 98 (H1N1); A / shorebird / Delaware / 168 / 06 (H16N3); A / sw / Netherlands / 25 / 80 (H1N1); A / sw / Germany / 2 / 81 (H1N1);A / sw / Hannover / 1 / 81 (H1N1); A / sw / Potsdam / 1 / 81 (H1N1); A / sw / Potsdam / 15 / 81 (H1N1); A / sw / Potsdam / 268 / 81 (H1N1); A / sw / Finistere / 2899 / 82 (H1N1); A / sw / Potsdam / 35 / 82 (H3N2); A / sw / Cote d'Armor / 3633 / 84 (H3N2); A / sw / Gent / 1 / 84 (H3N2);A / sw / Netherlands / 12 / 85 (H1N1); A / sw / Karrenzien / 2 / 87 (H3N2); A / sw / Schwerin / 103 / 89 (H1N1); A / turkey / Germany / 3 / 91 (H1N1); A / sw / Germany / 8533 / 91 (H1N1);A / sw / Belgium / 220 / 92 (H3N2); A / sw / Gent / V230 / 92 (H1N1); A / sw / Leipzig / 145 / 92 (H3N2); A / sw / Re220 / 92hp (H3N2); A / sw / Bakum / 909 / 93 (H3N2); A / sw / Schleswig-Holstein / 1 / 93 (H1N1); A / sw / Scotland / 419440 / 94 (H1N2); A / sw / Bakum / 5 / 95 (H1N1); A / sw / Best / 5C / 96 (H1N1); A / sw / England / 17394 / 96 (H1N2); A / sw / Jena / 5 / 96 (H3N2); A / sw / Oedenrode / 7C / 96 (H3N2); A / sw / Lohne / 1 / 97 (H3N2); A / sw / Cote d'Armor / 790 / 97 (H1N2);52NAI-5003686295vlA / sw / Bakum / 1362 / 98 (H3N2); A / sw / Italy / 1521 / 98 (H1N2); A / sw / Italy / 1553-2 / 98 (H3N2);A / sw / Italy / 1566 / 98 (H1N1); A / sw / Italy / 1589 / 98 (H1N1); A / sw / Bakum / 8602 / 99 (H3N2);A / sw / Cotes d'Armor / 604 / 99 (H1N2); A / sw / Cote d'Armor / 1482 / 99 (H1N1);A / sw / Gent / 7625 / 99 (H1N2); A / Hong Kong / 1774 / 99 (H3N2); A / sw / Hong Kong / 5190 / 99 (H3N2); A / sw / Hong Kong / 5200 / 99 (H3N2); A / sw / Hong Kong / 5212 / 99 (H3N2); A / sw / Ille et Villaine / 1455 / 99 (H1N1); A / sw / Italy / 1654- 1 / 99 (H1N2); A / sw / Italy / 2034 / 99 (H1N1);A / sw / Italy / 2064 / 99 (H1N2); A / sw / Berlin / 1578 / 00 (H3N2); A / sw / Bakum / 1832 / 00 (H1N2);A / sw / Bakum / 1833 / 00 (H1N2); A / sw / Cote d'Armor / 800 / 00 (H1N2); A / sw / Hong Kong / 7982 / 00 (H3N2); A / sw / Italy / 1081 / 00 (H1N2); A / sw / Belzig / 2 / 01 (H1N1);A / sw / Belzig / 54 / 01 (H3N2); A / sw / Hong Kong / 9296 / 01 (H3N2); A / sw / Hong Kong / 9745 / 01 (H3N2); A / sw / Spain / 33601 / 01 (H3N2); A / sw / Hong Kong / 1144 / 02 (H3N2); A / sw / Hong Kong / 1197 / 02 (H3N2); A / sw / Spain / 39139 / 02 (H3N2); A / sw / Spain / 42386 / 02 (H3N2);A / Switzerland / 8808 / 2002 (H1N1); A / sw / Bakum / 1769 / 03 (H3N2);A / sw / Bissendorf / IDTl 864 / 03 (H3N2); A / sw / Ehren / IDT2570 / 03 (H1N2);A / sw / Gescher / IDT2702 / 03 (H1N2); A / sw / Haseliinne / 2617 / 03hp (H1N1);A / sw / L6ningen / IDT2530 / 03 (H1N2); A / sw / IVD / IDT2674 / 03 (H1N2);A / sw / Nordkirchen / IDT 1993 / 03 (H3N2); A / sw / Nordwalde / IDT2197 / 03 (H1N2);A / sw / Norden / IDT2308 / 03 (H1N2); A / sw / Spain / 50047 / 03 (H1N1); A / sw / Spain / 51915 / 03 (H1N1); A / sw / Vechta / 2623 / 03 (H1N1); A / sw / Visbek / IDT2869 / 03 (H1N2);A / sw / Waltersdorf / IDT2527 / 03 (H1N2); A / sw / Damme / IDT2890 / 04 (H3N2);A / sw / Geldem / IDT2888 / 04 (H1N1); A / sw / Granstedt / IDT3475 / 04 (H1N2);A / sw / Greven / IDT2889 / 04 (H1N1); A / sw / Gudensberg / IDT2930 / 04 (H1N2);A / sw / Gudensberg / IDT2931 / 04 (H1N2); A / sw / Lohne / IDT3357 / 04 (H3N2);A / sw / Nortrup / IDT3685 / 04 (H1N2); A / sw / Seesen / IDT3055 / 04 (H3N2);A / sw / Spain / 53207 / 04 (H1N1); A / sw / Spain / 54008 / 04 (H3N2); A / sw / Stolzenau / IDT3296 / 04 (H1N2); A / sw / Wedel / IDT2965 / 04 (H1N1); A / sw / Bad Griesbach / IDT4191 / 05 (H3N2);A / sw / Cloppenburg / IDT4777 / 05 (H1N2); A / sw / D6tlingen / IDT3780 / 05 (H1N2);A / sw / D6tlingen / IDT4735 / 05 (H1N2); A / sw / Egglham / IDT5250 / 05 (H3N2);A / sw / Harkenblek / IDT4097 / 05 (H3N2); A / sw / Hertzen / IDT4317 / 05 (H3N2);A / sw / Krogel / IDT4192 / 05 (H1N1); A / sw / Laer / IDT3893 / 05 (H1N1); A / sw / Laer / IDT4126 / 05 (H3N2); A / sw / Merzen / IDT4114 / 05 (H3N2); A / sw / Muesleringen-S. / IDT4263 / 05 (H3N2); A / sw / Osterhofen / IDT4004 / 05 (H3N2); A / sw / Sprenge / IDT3805 / 05 (H1N2);A / sw / Stadtlohn / IDT3853 / 05 (H1N2); A / sw / Voglarn / IDT4096 / 05 (H1N1);A / sw / Wohlerst / IDT4093 / 05 (H1N1); A / sw / Bad Griesbach / IDT5604 / 06 (H1N1);53NAI-5003686295vlA / sw / Herzlake / IDT5335 / 06 (H3N2); A / sw / Herzlake / IDT5336 / 06 (H3N2);A / sw / Herzlake / IDT5337 / 06 (H3N2); and A / wild boar / Germany / Rl 69 / 2006 (H3N2).

[0090] Other specific examples of strains of Influenza A virus include, but are not limited to: A / Toronto / 3141 / 2009 (H1N1); A / Regensburg / D6 / 2009 (H1N1); A / Bayern / 62 / 2009 (H1N1); A / Bayem / 62 / 2009 (H1N1); A / Bradenburg / 19 / 2009 (H1N1); A / Bradenburg / 20 / 2009 (H1N1); A / Distrito Federal / 2611 / 2009 (H1N1); A / Mato Grosso / 2329 / 2009 (H1N1); A / Sao Paul o / l 454 / 2009 (H1N1); A / Sao Paulo / 2233 / 2009 (H1N1); A / Stockholm / 37 / 2009 (H1N1); A / Stockholm / 41 / 2009 (H1N1); A / Stockholm / 45 / 2009 (H1N1); A / swine / Alberta / OTH-33- 1 / 2009 (H1N1); A / swine / Alberta / OTH-33-14 / 2009 (H1N1); A / swine / Alberta / OTH-33- 2 / 2009 (H1N1); A / swine / Alberta / OTH-33-21 / 2009 (H1N1); A / swine / Alberta / OTH-33- 22 / 2009 (H1N1); A / swine / Alberta / OTH-33 -23 / 2009 (H1N1); A / swine / Alberta / OTH-33- 24 / 2009 (H1N1); A / swine / Alberta / OTH-33 -25 / 2009 (H1N1); A / swine / Alberta / OTH-33- 3 / 2009 (H1N1); A / swine / Alberta / OTH-33 -7 / 2009 (H1N1); A / Beijing / 502 / 2009 (H1N1);A / Firenze / 10 / 2009 (H1N1); A / Hong Kong / 2369 / 2009 (H1N1); A / Italy / 85 / 2009 (H1N1); A / Santo Domingo / 572N / 2009 (H1N1); A / Catalonia / 385 / 2009 (H1N1);A / Catalonia / 386 / 2009 (H1N1); A / Catalonia / 387 / 2009 (H1N1); A / Catalonia / 390 / 2009 (H1N1); A / Catalonia / 394 / 2009 (H1N1); A / Catalonia / 397 / 2009 (H1N1);A / Catalonia / 398 / 2009 (H1N1); A / Catalonia / 399 / 2009 (H1N1); A / Sao Paulo / 2303 / 2009 (H1N1); A / Akita / 1 / 2009 (H1N1); A / Castro / JXP / 2009 (H1N1); A / Fukushima / 1 / 2009 (H1N1); A / Israel / 276 / 2009 (H1N1); A / Israel / 277 / 2009 (H1N1); A / Israel / 70 / 2009 (H1N1); A / Iwate / 1 / 2009 (H1N1); A / Iwate / 2 / 2009 (H1N1); A / Kagoshima / 1 / 2009 (H1N1);A / Osaka / 180 / 2009 (H1N1); A / Puerto Montt / Bio87 / 2009 (H1N1); A / Sao Paulo / 2303 / 2009 (H1N1); A / Sapporo / 1 / 2009 (H1N1); A / Stockholm / 30 / 2009 (H1N1); A / Stockholm / 31 / 2009 (H1N1); A / Stockholm / 32 / 2009 (H1N1); A / Stockholm / 33 / 2009 (H1N1);A / Stockholm / 34 / 2009 (H1N1); A / Stockholm / 35 / 2009 (H1N1); A / Stockholm / 36 / 2009 (H1N1); A / Stockholm / 38 / 2009 (H1N1); A / Stockholm / 39 / 2009 (H1N1);A / Stockholm / 40 / 2009 (H1N1;) A / Stockholm / 42 / 2009 (H1N1); A / Stockholm / 43 / 2009 (H1N1); A / Stockholm / 44 / 2009 (H1N1); A / Utsunomiya / 2 / 2009 (H1N1);A / WRAIR / 0573N / 2009 (H1N1); and A / Zhejiang / DTID-ZJU01 / 2009 (H1N1).

[0091] In specific embodiments, the Influenza A virus from which one or more (e.g., two, three, four, five, six, seven, or all) genomic segments is from or derived from is A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interi or Alaska / 7MP0167 / 07, A / black54NAI-5003686295vlheaded gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14. In specific embodiments, the Influenza A virus from which one or more (e.g., two, three, four, five, six, seven, or all) genomic segments is from or derived from is A / California / 04 / 09, A / Vietnam / 1203 / 04, A / Hong Kong / 1 / 68, or A / PR / 8 / 34.

[0092] In a specific embodiment, provided herein is a recombinant Influenza A virus described in Section 8 or 9. In a preferred embodiment, provided herein is a recombinant Influenza A virus described in Section 8 that secretes bioactive IL-2 (e.g., bioactive human IL-2). In a preferred embodiment, provided herein is a recombinant Influenza A virus described in Section 8 that secretes bioactive IL-15 (e.g., bioactive human IL-15).

[0093] In specific embodiments, a recombinant Influenza A virus described herein (e.g., in Section 5.1, 8, or 9) reduces contaminant peptides and / or improves protein secretion (e.g., cytokine secretion). In specific embodiments, a recombinant Influenza A virus described herein reduces contaminant peptides and improves IL-2 secretion. In specific embodiments, a recombinant Influenza A virus described herein reduces contaminant peptides and improves IL- 15 secretion. In specific embodiments, the reduction and improvement are relative to the contaminant peptides and / or secretion by a recombinant Influenza A virus with an NS genomic segment depicted in FIG. 2A.

[0094] In specific embodiments, a recombinant Influenza A virus comprising an NS genomic segment described herein secretes a protein, a polypeptide, or a peptide, which is detectable in the supernatant of cells infected with the recombinant Influenza A virus. In some embodiments, the protein, polypeptide, or peptide is detectable by an ELISA. In some embodiments, the protein, polypeptide, or peptide is detectable by a Western blot assay or another immunoassay.

[0095] In specific embodiments, a recombinant Influenza A virus comprising an NS genomic segment described herein secretes a cytokine (e.g., IL-2 or IL- 15), which is detectable in the supernatant of cells infected with the recombinant Influenza A virus. In some embodiments, the cytokine is detectable by an ELISA. In some embodiments, the cytokine is detectable by a Western blot assay or another immunoassay.

[0096] In specific embodiments, a recombinant Influenza A virus comprising an NS genomic segment described herein secretes a chemokine, which is detectable in the supernatant of cells infected with the recombinant Influenza A virus. In some embodiments,55NAI-5003686295vlthe chemokine is detectable by an ELISA. In some embodiments, the chemokine is detectable by a Western blot assay or another immunoassay.

[0097] In specific embodiments, a recombinant Influenza A virus comprising an NS genomic segment described herein secretes an antigen, which is detectable in the supernatant of cells infected with the recombinant Influenza A virus. In some embodiments, the antigen is detectable by an ELISA. In some embodiments, the antigen is detectable by a Western blot assay or another immunoassay.

[0098] In some embodiments, a recombinant Influenza A virus comprising an NS genomic segment described herein produces a protein with a signal sequence, which is detectable in cells infected with the recombinant Influenza A virus. In specific embodiments, a recombinant Influenza A virus comprising an NS genomic segment described herein produces a membrane anchored protein, which is detectable in the cells infected with the recombinant Influenza A virus. In some embodiments, the antigen is detectable by a Western blot assay or another immunoassay.

[0099] In some embodiments, the supernatant of cells infected with a recombinant Influenza A virus comprising an NS genomic segment described herein (e.g., in Section 5.1, Section 8, or FIG. 1C) contains 1 ng / ml to 10 ng / ml of protein encoded by the NS genomic segment, as detected by an immunoassay (e.g., an ELISA). In some embodiments, the cells infected are 293T cells. In some embodiments, the cells are MDCK-NS1 cells.

[0100] In some embodiments, the supernatant of cells infected with a recombinant Influenza A virus comprising an NS genomic segment described herein (e.g., in Section 5.1, Section 8, or FIG. 1C) contains 1 ng / ml to 10 ng / ml of cytokine (e.g., IL-2 or IL- 15) encoded by the NS genomic segment, as detected by an immunoassay (e.g., an ELISA). In some embodiments, the cells infected are 293T cells. In some embodiments, the cells are MDCK- NS1 cells.

[0101] In some embodiments, the supernatant of cells infected with a recombinant Influenza A virus comprising an NS genomic segment described herein (e.g., in Section 5.1, Section 8, or FIG. 1C) contains 1 ng / ml to 10 ng / ml of chemokine encoded by the NS genomic segment, as detected by an immunoassay (e.g., an ELISA). In some embodiments, the cells infected are 293T cells. In some embodiments, the cells are MDCK-NS1 cells.

[0102] In some embodiments, the supernatant of cells infected with a recombinant Influenza A virus comprising an NS genomic segment described herein (e.g, in Section 5.1, Section 8, or FIG. 1C) contains 1 ng / ml to 10 ng / ml of antigen encoded by the NS genomic56NAI-5003686295vlsegment, as detected by an immunoassay (e.g., an ELISA). In some embodiments, the cells infected are 293T cells. In some embodiments, the cells are MDCK-NS1 cells.

[0103] In some embodiments, the supernatant of cells infected with a first recombinant Influenza A virus comprising an NS genomic segment described herein (e.g., in Section 5.1, or FIG. 1C) contains a higher concentration of a secretory protein (e.g., a cytokine) encoded by the NS genomic segment than the supernatant of the same type of cells infected with a second recombinant Influenza A virus cultured under the same conditions as the cells infected with the first recombinant Influenza A virus, wherein the second recombinant Influenza A virus comprises the same genomic segments as the first recombinant Influenza A virus other than the NS1 genomic segment, and wherein the NS1 genomic segment of the second recombinant Influenza A virus comprises a nucleotide sequence encoding the same secretory protein (e.g., cytokine) as the NS1 genomic segment of the first recombinant Influenza A virus, a nucleotide sequence encoding a linker, a nucleotide sequence encoding a 2A peptide, and a nucleotide sequence of an NEP open reading frame. In some embodiments, the supernatant of cells infected with a first recombinant Influenza A virus comprising an NS genomic segment described herein (e.g., in Section 5.1, or FIG. 1C) contains a higher concentration of a secretory protein (e.g., a cytokine) encoded by the NS genomic segment than the supernatant of the same type of cells infected with a second recombinant Influenza A virus cultured under the same conditions as the cells infected with the first recombinant Influenza A virus, wherein the second recombinant Influenza A virus comprises the same genomic segments as the first recombinant Influenza A virus other than the NS1 genomic segment, and wherein the NS 1 genomic segment of the second recombinant Influenza A virus comprises a nucleotide sequence encoding the same secretory protein (e.g., cytokine), a nucleotide sequence encoding a linker, a nucleotide sequence encoding a 2A peptide, and a nucleotide sequence of an NEP open reading frame, such as described in FIG. 2A. In some embodiments, the higher concentration is statistically significant. In some embodiments, the higher concentration is a concentration of 1 ng / ml to 10 ng / ml higher. In some embodiments, the higher concentration is a concentration of 1 ng / ml to 8 ng / ml higher. In some embodiments, the higher concentration is a concentration of 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, or 5 ng / ml higher. In some embodiments, the higher concentration is a concentration of 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, or 10 ng / ml higher. In some embodiments, the secretory protein (e.g., cytokine) is detected by an immunoassay (e.g., an ELISA).

[0104] In some embodiments, the supernatant of cells infected with a first recombinant Influenza A virus comprising an NS genomic segment described in FIG. 1C contains a57NAI-5003686295vlhigher concentration of a secretory protein (e.g., cytokine) encoded by the NS genomic segment than the supernatant of the same type of cells infected with a second recombinant Influenza A virus cultured under the same conditions as the cells infected with the first recombinant Influenza A virus, wherein the second recombinant Influenza A virus comprises the same genomic segments as the first recombinant Influenza A virus other than the NS1 genomic segment, and wherein the NS1 genomic segment of the second recombinant Influenza A virus is an NS genomic segment depicted in FIG. 2A, which encodes the same secretory protein (e.g., cytokine). In some embodiments, the higher concentration is statistically significant. In some embodiments, the higher concentration is a concentration of 1 ng / ml to 10 ng / ml higher. In some embodiments, the higher concentration is a concentration of 1 ng / ml to 8 ng / ml higher. In some embodiments, the higher concentration is a concentration of 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, or 5 ng / ml higher. In some embodiments, the higher concentration is a concentration of 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, or 10 ng / ml higher. In some embodiments, the secreted protein (e.g., cytokine) is detected by an immunoassay (e.g., an ELISA).

[0105] In specific embodiments, a recombinant Influenza A virus described herein lacks the NS1 gene and is attenuated in vitro and in vivo, but is replication competent in the interferon deficient systems. In specific embodiments, this activity makes the recombinant Influenza A virus useful as a live-attenuated vaccine and for anti-cancer therapy.5.4 METHODS FOR PRODUCING RECOMBINANT INFLUENZA A VIRUSES

[0106] Techniques known to one skilled in the art may be used to produce a recombinant influenza virus described herein. For example, reverse genetics techniques may be used to generate such an influenza virus. Briefly, reverse genetics techniques generally involve the preparation of synthetic recombinant viral RNAs that contain the non-coding regions of the negative- strand, viral RNA which are essential for the recognition by viral polymerases and for packaging signals necessary to generate a mature virion. The recombinant RNAs are synthesized from a recombinant DNA template and reconstituted in vitro with purified viral polymerase complex to form recombinant ribonucleoproteins (RNPs) which can be used to trans feet cells. A more efficient transfection is achieved if the viral polymerase proteins are present during transcription of the synthetic RNAs either in vitro or in vivo. The synthetic recombinant RNPs can be rescued into infectious virus particles. The foregoing techniques are described in U.S. Patent No. 5,166,057 issued November 24, 1992; in U.S. Patent No. 5,854,037 issued December 29, 1998; in European Patent Publication EP 0702085A1, published February 20, 1996; in U.S. Patent Application Serial No. 09 / 152,845; in58NAI-5003686295vlInternational Patent Publications PCT WO 97 / 12032 published April 3, 1997; WO 96 / 34625 published November 7, 1996; in European Patent Publication EP A780475; WO 99 / 02657 published January 21, 1999; WO 98 / 53078 published November 26, 1998; WO 98 / 02530 published January 22, 1998; WO 99 / 15672 published April 1, 1999; WO 98 / 13501 published April 2, 1998; WO 97 / 06270 published February 20, 1997; and EPO 780 475 Al published June 25, 1997, each of which is incorporated by reference herein in its entirety.

[0107] Alternatively, helper-free plasmid technology may be used to produce a recombinant influenza virus containing a modified influenza virus genomic segment. Briefly, full length cDNAs of viral segments are amplified using PCR with primers that include unique restriction sites, which allow the insertion of the PCR product into the plasmid vector (Flandorfer et al, 2003, J. Virol. 77:9116-9123; Nakaya et al, 2001, J. Virol. 75: 11868-11873; both of which are incorporated herein by reference in their entireties). The plasmid vector is designed so that an exact negative (vRNA sense) transcript is expressed. For example, the plasmid vector may be designed to position the PCR product between a truncated human RNA polymerase I promoter and a hepatitis delta virus ribozyme sequence such that an exact negative (vRNA sense) transcript is produced from the polymerase I promoter. Separate plasmid vectors comprising each viral segment as well as expression vectors comprising necessary viral proteins may be transfected into cells leading to production of recombinant viral particles. In another example, plasmid vectors from which both the viral genomic RNA and mRNA encoding the necessary viral proteins are expressed may be used. For a detailed description of helper-free plasmid technology, see, e.g., International Publication No. WO 01 / 04333; U.S. Patent Nos. 6,951,754, 7,384,774, 6,649,372, and 7,312,064; Fodor et al.,1999, J. Virol. 73:9679-9682; Quinlivan et al., 2005, J. Virol. 79:8431-8439; Hoffmann et al.,2000, Proc. Natl. Acad. Sci. USA 97:6108-6113; and Neumann et al., 1999, Proc. Natl. Acad. Sci. USA 96:9345-9350, which are incorporated herein by reference in their entireties.

[0108] In specific embodiments, genomic segments or the complements thereof are transfected into a cell that expresses the proteins necessary for production of viral particles. In some embodiments, ambisense plasmids, such as pDZ, each containing one of the eight influenza A viral genes are co-transfected in cells to generate Influenza A virus, such as described in, e.g., Martinez-Sobrido and Garcia-Sastre, 2010, J Vis Exp. 42: 2057.

[0109] Techniques known in the art can be used to isolate / purify the recombinant influenza virus that results. For example, the Influenza A virus can be plaque purified using techniques known to one of skill in the art.59NAI-5003686295vl

[0110] In some embodiments, provided herein is a method for producing a recombinant influenza virus, comprising: culturing cells comprising Influenza A virus HA, NA, PB1, PB2, PA, M, and NP genomic segments, an Influenza A virus NS genomic segment described herein, and nucleotide sequences encoding Influenza A virus PB 1 protein, Influenza A virus PB2 protein, Influenza A virus PA protein, and Influenza A virus NP protein. In some embodiments, provided herein is a method for producing a recombinant influenza virus, comprising: culturing cells transfected with one or more vectors (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 vectors) comprising Influenza A virus HA, NA, PB1, PB2, PA, M, and NP genomic segments, an Influenza A virus NS genomic segment described herein, a nucleotide sequence encoding Influenza A virus PB1 protein, a nucleotide sequence encoding Influenza A virus PB2 protein, a nucleotide sequence encoding Influenza A virus PA protein, and a nucleotide sequence encoding Influenza A virus NP protein. In some embodiments, provided herein is a method for producing a recombinant influenza virus, comprising: culturing cells transfected with one or more vectors (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 vectors) comprising nucleotide sequences of Influenza A virus HA, NA, PB1, PB2, PA, M, and NP genomic segments, a nucleotide sequence of an Influenza A virus NS genomic segment described herein, a nucleotide sequence encoding an Influenza A virus PB1 protein, a nucleotide sequence sequence encoding an Influenza A virus PB2 protein, a nucleotide sequence sequence encoding Influenza A virus PA protein, and a nucleotide sequence encoding an Influenza A virus NP protein. In some embodiments, the nucleotide sequence of the NS genomic segment comprises the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleotide sequence of the NS genomic segment comprises the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:23 or 24. In some embodiments, the HA genomic segment is from or derived from an Influenza A virus strain, which is different60NAI-5003686295vlthan the Influenza A virus strain(s) from which the PB1, PB2, PA, NP, and M genomic segments are from or derived from. In some embodiments, the HA genomic segment is from or derived from a seasonal influenza virus. In some embodiments, the HA genomic segment is from or derived from a naturally occurring Influenza A virus. In some embodiments, the HA genomic segment is from or derived from an Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl 1, H12, H13, H14, H15, H16, H17, or H18. In some embodiments, the HA genomic segment is from an exotic Influenza A virus (e.g., an Influenza A virus not prevalent in humans). In some embodiments, the HA genomic segment is artificially created or a consensus HA genomic segment. In some embodiments, the use of an exotic Influenza A virus HA genomic segment or an artificially created HA genomic allows for the evasion of pre-existing immunity to HA in a subject. In some embodiments, the HA genomic segment is from or derived from the same Influenza A virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, or M genomic segment is from or derived from. In some embodiments, the HA genomic segment is from or derived from the same Influenza A virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, and M genomic segments are from or derived from. In some embodiments, the NA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strain(s) from which the PB1, PB2, PA, NP, M, and NS genomic segments are from or derived from. In some embodiments, the NA genomic segment is from or derived from a seasonal Influenza A virus. In some embodiments, the NA genomic segment is from or derived from a naturally occurring Influenza A virus. In some embodiments, the NA genomic segment is from or derived from an Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, or N11 subtype. In some embodiments, the NA genomic segment is artificially created or a consensus NA genomic segment. In some embodiments, the NA genomic segment is from or derived from the same Influenza A virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, or M genomic segment is from or derived from. In some embodiments, the NA genomic segment is from or derived from the same influenza virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, and M genomic segments are from or derived from. In some embodiments, the HA genomic segment and NA genomic segment are from or derived from the same Influenza A virus strain. In some embodiments, the HA genomic segment and NA genomic segment are from or derived from different Influenza A virus strains. In some embodiments, the PB1, PB2, PA, NP, M, HA, and NA genomic segments are from or derived from the same Influenza A virus strain. In some embodiments, the PB1, PB2, PA, NP, M, NS, HA, and NA genomic segments are from or61NAI-5003686295vlderived from the same Influenza A virus strain. In some embodiments, the PB1, PB2, PA, NP, M, HA, and NA genomic segments are from or derived from a first Influenza A virus and the NS genomic segment described herein is from or derived from a second Influenza A virus, wherein the first and second Influenza A viruses are different. In some embodiments, the first and second Influenza A viruses are the same subtype. In some embodiments, the first and second Influenza A viruses are different strains. In some embodiments, the first and second Influenza A viruses are different subtypes. In some embodiments, the HA and NA genomic segments are from or derived from a first Influenza A virus and the PB1, PB2, PA, NP, M, and NS genomic segments are from or derived from a second Influenza A virus, wherein the first and second Influenza A viruses are different. In some embodiments, the first Influenza A viruses is a seasonal Influenza A virus. In some embodiments, the first and second Influenza A viruses are different subtypes. In some embodiments, the first and second Influenza A viruses are different strains. In some embodiments, the PB1, PB2, PA, NP, and M genomic segments are from or derived from a first Influenza A virus, an NS genomic segment described herein is from or derived from a second Influenza A virus, and the HA and NA genomic segments are from or derived from a third Influenza A virus, wherein the first, second, and third Influenza A viruses are different. In some embodiments, the first, second, and third Influenza A viruses are the same subtype. In some embodiments, the first, second, and third Influenza A viruses are different strains. In some embodiments, the first, second, and third Influenza A viruses are different subtypes. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1, PB2, PA, NP, M, HA, and NA genomic segments comprise one or more mutations that attenuate the Influenza A virus. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1, PB2, PA, NP, M, HA, and NA genomic segments comprise a heterologous nucleotide sequence. The heterologous nucleotide sequence may encode an antigen. In some embodiments, the cells are cells known in the art or described herein. In some embodiments, the cells are 293T cells, MDCK cells, or Vero cells. In some embodiments, the cells are interferon (IFN)-deficient. In some embodiments, the method further comprises harvesting the supernatant from the cells, infecting MDCK cells (e.g., MDCK-NS1 cells) or embryonated eggs, and harvesting the supernatant from the cells or allantoic fluid from the eggs. In some embodiments, the embryonated eggs are embryonated chicken eggs or other embryonated eggs described in the art. In some embodiments, the embryonated eggs are IFN-deficient. In some embodiments, the cells are MDCK cells (e.g., MDCK-NS1 cells), 293T cells, or Vero cells. In some embodiments, the embryonated chicken eggs are about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days old.62NAI-5003686295vlIn some embodiments, the method further comprises plaque purifying the recombinant Influenza A virus.

[0111] In some embodiments, provided herein is a method for producing a recombinant Influenza A virus, comprising: culturing cells transfected with a PB1 bidirectional plasmid comprising a nucleotide sequence of an Influenza A virus PB1 genomic segment, a PB2 bidirectional plasmid comprising a nucleotide sequence of an Influenza A virus PB2 genomic segment, a PA bidirectional plasmid comprising a nucleotide sequence of an Influenza A virus PA genomic segment, an NP bidirectional plasmid comprising a nucleotide sequence of an Influenza A virus NP genomic segment, an M plasmid comprising a nucleotide sequence of an Influenza A virus M genomic segment, an NS plasmid comprising a nucleotide sequence of an Influenza A virus NS genomic segment described herein, an HA plasmid comprising a nucleotide sequence of an Influenza A virus HA genomic segment, and an NA plasmid comprising a nucleotide sequence of an Influenza A virus NA genomic segment, wherein each bidirectional plasmid comprises a human RNA polymerase I promoter, a mouse terminator sequence, and a polymerase II transcription cassette. In some embodiments, the nucleotide sequence of the NS genomic segment comprises the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 75%, at least 80%, at least 80%, at least 90% or at least 95% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleotide sequence of the NS genomic segment comprises the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 75%, at least 80%, at least 80%, at least 90% or at least 95% identical to the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the HA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strain(s) from which the PB1, PB2, PA, NP, and M genomic segments are from or derived from. In some embodiments, the HA genomic segment is from or derived from a seasonal influenza virus. In some embodiments, the HA genomic segment is from or derived from a naturally occurring Influenza A virus. In some embodiments, the HA genomic segment is from or derived from an Hl, H2, H3, H4, H5, H6,63NAI-5003686295vlH7, H8, H9, H10, Hll, H12, H13, H14, H15, H16, H17, or H18. In some embodiments, the HA genomic segment is from an exotic Influenza A virus (e.g., an Influenza A virus not prevalent in humans). In some embodiments, the HA genomic segment is artificially created or a consensus HA genomic segment. In some embodiments, the use of an exotic Influenza A virus HA genomic segment or an artificially created HA genomic allows for the evasion of pre-existing immunity to HA in a subject. In some embodiments, the HA genomic segment is from or derived from the same Influenza A virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, or M genomic segment is from or derived from. In some embodiments, the HA genomic segment is from or derived from the same Influenza A virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, and M genomic segments are from or derived from. In some embodiments, the NA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strain(s) from which the PB1, PB2, PA, NP, M, and NS genomic segments are from or derived from. In some embodiments, the NA genomic segment is from or derived from a seasonal Influenza A virus. In some embodiments, the NA genomic segment is from or derived from a naturally occurring Influenza A virus. In some embodiments, the NA genomic segment is from or derived from an Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, or Ni l. In some embodiments, the NA genomic segment is artificially created or a consensus NA genomic segment. In some embodiments, the NA genomic segment is from or derived from the same Influenza A virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, or M genomic segment is from or derived from. In some embodiments, the NA genomic segment is from or derived from the same influenza virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, and M genomic segments are from or derived from. In some embodiments, the HA genomic segment and NA genomic segment are from or derived from the same Influenza A virus strain. In some embodiments, the HA genomic segment and NA genomic segment are from or derived from different Influenza A virus strains. In some embodiments, the PB1, PB2, PA, NP, M, HA, and NA genomic segments are from or derived from the same Influenza A virus strain. In some embodiments, the PB1, PB2, PA, NP, M, NS, HA, and NA genomic segments are from or derived from the same Influenza A virus strain. In some embodiments, the PB1, PB2, PA, NP, M, HA, and NA genomic segments are from or derived from a first Influenza A virus and the NS genomic segment described herein is from or derived from a second Influenza A virus, wherein the first and second Influenza A viruses are different. In some embodiments, the first and second Influenza A viruses are the same subtype. In some embodiments, the first and second64NAI-5003686295vlInfluenza A viruses are different strains. In some embodiments, the first and second Influenza A viruses are different subtypes. In some embodiments, the HA and NA genomic segments are from or derived from a first Influenza A virus and the PB1, PB2, PA, NP, M, and NS genomic segments are from or derived from a second Influenza A virus, wherein the first and second Influenza A viruses are different. In some embodiments, the first Influenza A viruses is a seasonal Influenza A virus. In some embodiments, the first and second Influenza A viruses are different subtypes. In some embodiments, the first and second Influenza A viruses are different strains. In some embodiments, the PB1, PB2, PA, NP, and M genomic segments are from or derived from a first Influenza A virus, an NS genomic segment described herein is from or derived from a second Influenza A virus, and the HA and NA genomic segments are from or derived from a third Influenza A virus, wherein the first, second, and third Influenza A viruses are different. In some embodiments, the first, second, and third Influenza A viruses are the same subtype. In some embodiments, the first, second, and third Influenza A viruses are different strains. In some embodiments, the first, second, and third Influenza A viruses are different subtypes. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1, PB2, PA, NP, M, HA, and NA genomic segments comprise one or more mutations that attenuate the Influenza A virus. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1, PB2, PA, NP, M, HA, and NA genomic segments comprise a heterologous nucleotide sequence. The heterologous nucleotide sequence may encode an antigen. In some embodiments, the cells are cells known in the art or described herein. In some embodiments, the cells are 293T cells, MDCK cells, or Vero cells. In some embodiments, the cells are interferon (IFN)-deficient.. In some embodiments, the method further comprises harvesting the supernatant from the cells, infecting MDCK cells (e.g., MDCK-NS1 cells) or embryonated eggs (e.g., embryonated eggs described in the art), and harvesting the supernatant from the cells or allantoic fluid from the eggs. In some embodiments, the embryonated eggs are IFN-deficient. In some embodiments, the embryonated eggs are embryonated chicken eggs or other bird eggs. In some embodiments, the cells are MDCK cells (e.g., MDCK-NS1 cells), 293T cells, or Vero cells. In some embodiments, the embryonated chicken eggs are about 6 days old. In some embodiments, the embryonated chicken eggs are about 7 days old. In some embodiments, the embryonated chicken eggs are about 8 days old. In some embodiments, the embryonated chicken eggs are about 9 days old. In some embodiments, the embryonated chicken eggs are about 10 days old. In some embodiments, the method further comprises plaque purifying the recombinant Influenza A virus.65NAI-5003686295vl

[0112] In some embodiments, each bidirectional plasmid is the ambisense plasmid pDZ.

[0113] In some embodiments, provided herein is a method for producing a recombinant Influenza A virus, comprising: culturing cells transfected with eight vectors comprising nucleotide sequences of the genomic segments of Influenza A virus, and four vectors encoding Influenza A virus PB1 protein, PB2 protein, PA protein, and NP protein, wherein each of the four vector encodes a different Influenza A virus protein, wherein each of the eight vectors comprises a nucleotide sequence of a different genomic segment, and wherein the eight vectors include: (1) a PB1 vector comprising a PB1 genomic segment nucleotide sequence, (2) a PB2 vector comprising a PB2 genomic segment nucleotide sequence, (3) a PA vector comprising a PA genomic segment nucleotide sequence, (4) an NP vector comprising an NP genomic segment nucleotide sequence, (5) an M vector comprising an M genomic segment nucleotide sequence, (6) an NS vector comprising an NS genomic segment nucleotide sequence described herein (e.g., in Section 5.1 or 8), (7) an HA vector comprising an HA genomic segment nucleotide sequence, and (8) an NA vector comprising NA genomic segment nucleotide sequence. In some embodiments, the NS genomic segment nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS genomic segment nucleotide sequence comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS genomic segment nucleotide sequence comprises a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS genomic segment nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the NS genomic segment nucleotide sequence comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the NS genomic segment nucleotide sequence comprises a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the HA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strain(s) from which the PB1, PB2, PA, NP, and M genomic segments are from or derived from. In some embodiments, the HA genomic segment is from or derived from a seasonal influenza virus. In some embodiments, the HA genomic segment is from or derived from a naturally occurring Influenza A virus. In some embodiments, the HA genomic segment is from or derived from an Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16,66NAI-5003686295vlor Hl 7. In some embodiments, the HA genomic segment is from an exotic Influenza A virus (e.g., an Influenza A virus not prevalent in humans). In some embodiments, the HA genomic segment is artificially created or a consensus HA genomic segment. In some embodiments, the use of an exotic Influenza A virus HA genomic segment or an artificially created HA genomic allows for the evasion of pre-existing immunity to HA in a subject. In some embodiments, the HA genomic segment is from or derived from the same Influenza A virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, or M genomic segment is from or derived from. In some embodiments, the HA genomic segment is from or derived from the same Influenza A virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, and M genomic segments are from or derived from. In some embodiments, the NA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strain(s) from which the PB1, PB2, PA, NP, M, and NS genomic segments are from or derived from. In some embodiments, the NA genomic segment is from or derived from a seasonal Influenza A virus. In some embodiments, the NA genomic segment is from or derived from a naturally occurring Influenza A virus. In some embodiments, the NA genomic segment is from or derived from an Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, or Nl 1. In some embodiments, the NA genomic segment is artificially created or a consensus NA genomic segment. In some embodiments, the NA genomic segment is from or derived from the same Influenza A virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, or M genomic segment is from or derived from. In some embodiments, the NA genomic segment is from or derived from the same influenza virus strain as the Influenza A virus strain from which the PB1, PB2, PA, NP, and M genomic segments are from or derived from. In some embodiments, the HA genomic segment and NA genomic segment are from or derived from the same Influenza A virus strain. In some embodiments, the HA genomic segment and NA genomic segment are from or derived from different Influenza A virus strains. In some embodiments, the PB1, PB2, PA, NP, M, HA, and NA genomic segments are from or derived from the same Influenza A virus strain. In some embodiments, the PB1, PB2, PA, NP, M, NS, HA, and NA genomic segments are from or derived from the same Influenza A virus strain. In some embodiments, the PB1, PB2, PA, NP, M, HA, and NA genomic segments are from or derived from a first Influenza A virus and the NS genomic segment described herein is from or derived from a second Influenza A virus, wherein the first and second Influenza A viruses are different. In some embodiments, the first and second Influenza A viruses are the same subtype. In some embodiments, the first and second Influenza A viruses are different strains. In some67NAI-5003686295vlembodiments, the HA and NA genomic segments are from or derived from a first Influenza A virus and the PB1, PB2, PA, NP, M, and NS genomic segments are from or derived from a second Influenza A virus, wherein the first and second Influenza A viruses are different. In some embodiments, the first Influenza A viruses is a seasonal Influenza A virus. In some embodiments, the first and second Influenza A viruses are different subtypes. In some embodiments, the first and second Influenza A viruses are different strains. In some embodiments, the first and second Influenza A viruses are different subtypes. In some embodiments, the PB1, PB2, PA, NP, and M genomic segments are from or derived from a first Influenza A virus, an NS genomic segment described herein is from or derived from a second Influenza A virus, and the HA and NA genomic segments are from or derived from a third Influenza A virus, wherein the first, second, and third Influenza A viruses are different. In some embodiments, the first, second, and third Influenza A viruses are the same subtype. In some embodiments, the first, second, and third Influenza A viruses are from different strains. In some embodiments, the first, second, and third Influenza A viruses are different subtypes. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1, PB2, PA, NP, M, HA, and NA genomic segments comprise one or more mutations that attenuate the Influenza A virus. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1, PB2, PA, NP, M, HA, and NA genomic segments comprise a heterologous nucleotide sequence. The heterologous nucleotide sequence may encode an antigen. In some embodiments, the cells are cells known in the art or described herein. In some embodiments, the cells are 293T cells, MDCK cells, or Vero cells. In some embodiments, the cells are interferon (IFN)-deficient. In some embodiments, the method further comprises harvesting the supernatant from the cells, infecting MDCK cells (e.g., MDCK-NS1 cells) or embryonated eggs (e.g., embryonated eggs described in the art), and harvesting the supernatant from the cells or allantoic fluid from the eggs In some embodiments, the embryonated eggs are embryonated chicken eggs or other bird embryonated eggs. In some embodiments, the embryonated eggs are IFN-deficient. In some embodiments, the cells are MDCK cells (e.g., MDCK-NS1 cells), 293T cells, or Vero cells. In some embodiments, the embryonated chicken eggs are about 6 days old. In some embodiments, the embryonated chicken eggs are about 7 days old. In some embodiments, the embryonated chicken eggs are about 8 days old. In some embodiments, the embryonated chicken eggs are about 9 days. In some embodiments, the embryonated chicken eggs are about 10 days old. In some embodiments, the method further comprises plaque purifying the recombinant Influenza A virus.68NAI-5003686295vl

[0114] In a specific embodiment, a method described herein (e.g., in Examples) is used to produce a recombinant Influenza A virus.5.5 METHODS FOR PROPAGATING INFLUENZA A VIRUSES

[0115] Recombinant Influenza A viruses described herein (e.g., Sections 5.3, 8, or 9) can be propagated in any substrate that allows the virus to grow to titers that permit the uses of the viruses described herein.

[0116] Recombinant Influenza A viruses described herein (e.g., Sections 5.3, 8, or 9) may be grown in cells (e.g., avian cells, chicken cells, etc.) that are susceptible to infection by the viruses, embryonated eggs (e.g., chicken eggs or quail eggs) or animals (e.g., birds). Such methods are well known to those skilled in the art. In a specific embodiment, a recombinant Influenza A virus described herein may be propagated in a cell line, e.g., cancer cell line such as HeLa cells, MCF7 cells, THP-1 cells, U87 cells, DU145 cells, Lncap cells, and T47D cells. In certain embodiments, the cells or cell lines (e.g., cancer cells or cancer cell lines) are obtained, derived, or obtained and derived from a human(s). In a specific embodiment, a recombinant Influenza A virus described herein is propagated in interferon deficient systems or interferon (IFN) deficient substrates, such as, e.g., IFN deficient cells (e.g., IFN deficient cell lines) or IFN deficient embryonated eggs. In some embodiments, a recombinant Influenza A virus described herein is propagated in chicken cells or embryonated chicken eggs. Representative chicken cells include, but are not limited to, chicken embryo fibroblasts and chicken embryo kidney cells. In a specific embodiment, a recombinant Influenza A virus described herein is propagated in Vero cells. In another specific embodiment a recombinant Influenza A virus described herein is propagated in chicken eggs or quail eggs. In certain embodiments a recombinant Influenza A virus described herein is first propagated in embryonated eggs and then propagated in cells (e.g., a cell line). In another specific embodiment, a recombinant Influenza A virus described herein is propagated as described in Section 8, infra.

[0117] As used herein, the phrases “IFN deficient systems” or “IFN-deficient substrates” refer to systems, e.g., cells, cell lines and animals, such as mice, chickens, turkeys, rabbits, rats, horses etc., which do not produce one, two or more types of IFN (e.g., IFN-gamma, IFN- alpha, and IFN-beta), or do not produce any type of IFN, or produce low levels of one, two or more types of IFN, or produce low levels of any IFN (i.e., a reduction in any IFN expression of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or more when compared to IFN-competent systems under the same conditions), do not respond or respond less efficiently to one, two or more types of IFN, or do not respond to any type of69NAI-5003686295vlIFN, have a delayed response to one, two or more types of IFN, are deficient in the activity of antiviral genes induced by one, two or more types of IFN, or induced by any type of IFN, or any combination thereof.

[0118] A recombinant Influenza A virus described herein may be propagated in embryonated eggs (e.g., chicken embryonated eggs), e.g., from 6 to 10 days old, 6 to 9 days old, 6 to 8 days old, 6 to 7 days old, 7 to 8 days old, 7 to 9 days old, 8 to 9 days old or 8 to 10 days old. In a specific embodiment, 6 days old embryonated chicken eggs are used to propagate a recombinant influenza virus described herein. Young or immature embryonated eggs (e.g., chicken embryonated eggs) can be used to propagate the recombinant influenza viruses described herein. Immature embryonated eggs encompass eggs which are less than ten day old eggs, e.g., eggs 6 to 8 days old or 8 to 10 day old that are IFN-deficient. Immature embryonated eggs also encompass eggs which artificially mimic immature eggs up to, but less than 10 days old, as a result of alterations to the growth conditions, e.g., changes in incubation temperatures; treatment with drugs; or any other alteration which results in an egg with a retarded development, such that the IFN system is not fully developed as compared with 10 to 12 day old eggs, or eggs older than 12 days old. A recombinant Influenza A virus described herein can be propagated in different locations of the embryonated egg, e.g., the allantoic cavity (such as, e.g., the allantoic cavity of chicken embryonated eggs). For a detailed discussion on the growth and propagation viruses, see, e.g., U.S. Patent No.6,852,522 and U.S. Patent No. 7,494,808, both of which are hereby incorporated by reference in their entireties.

[0119] In a specific embodiment, a recombinant Influenza A virus described herein is propagated as described in the Examples below (e.g, Section 8 or 9). In some embodiments, a recombinant Influenza A virus described herein is propagated in MDCK cells (e.g, MDCK- NS1 protein cells), A549 cells, or 293T cells.

[0120] For virus isolation, recombinant Influenza A viruses described herein can be removed from embryonated eggs or cell culture and separated from cellular components, typically by well-known clarification procedures, e.g., such as centrifugation, depth filtration, and microfiltration, and may be further purified as desired using procedures well known to those skilled in the art, e.g., tangential flow filtration (TFF), density gradient centrifugation, differential extraction, or chromatography.

[0121] In a specific embodiment, virus isolation from allantoic fluid of an infected egg (e.g., a chicken egg) begins with harvesting allantoic fluid, which is clarified using a filtration system to remove cells and other large debris.70NAI-5003686295vl

[0122] In a specific embodiment, provided herein is a cell (e.g., a cell line) or embryonated egg (e.g., a chicken embryonated egg) comprising a recombinant A influenza virus described herein. In another specific embodiment, provided herein is a method for propagating a recombinant Influenza A virus described herein, the method comprising culturing a cell (e.g., a cell line) or embryonated egg (e.g., a chicken embryonated egg) infected with the recombinant Influenza A virus. In some embodiments, the method may further comprise isolating or purifying the recombinant A influenza virus from the cell or embryonated egg. In a specific embodiment, provided herein is a method for propagating a recombinant Influenza A virus described herein, the method comprising (a) culturing a cell (e.g., a cell line) or embryonated egg infected with a recombinant Influenza A virus described herein; and (b) isolating the recombinant Influenza A virus from the cell or embryonated egg. The cell or embryonated egg may be one described herein or known to one of skill in the art. In some embodiments, the cell or embryonated egg is IFN deficient. In some embodiments, the cell(s) is one described herein. In specific embodiments, the cell(s) is in vitro or ex vivo. In specific embodiments, the cell(s) is isolated. In some embodiments, the cell(s) are not in vivo. In specific embodiments, the embryonated egg(s) is ex vivo. In some embodiments, the embryonated egg(s) is one known to one of skill in the art or described herein.

[0123] In a specific embodiment, provided herein is a method for producing a composition (e.g., an immunogenic composition) comprising a recombinant Influenza A virus described herein, the method comprising (a) propagating a recombinant Influenza A virus described herein a cell(s) (e.g., a cell line) or embryonated egg; and (b) isolating the recombinant Influenza A virus from the cell(s) or embryonated egg. The method may further comprise adding the recombinant Influenza A virus to a container along with a pharmaceutically acceptable carrier.

[0124] In an aspect, provided herein are cells (e.g., a cell line) or embryonated eggs (e.g., a chicken embryonated egg) comprising a PB1 genomic segment of an Influenza A virus, a PB2 genomic segment of an Influenza A virus, a PA genomic segment of an Influenza A virus, an NP genomic segment of an Influenza A virus, a M genomic segment of an Influenza A virus, and an NS genomic segment described herein. In some embodiments, the cells (e.g., cell line) or embryonated (e.g., a chicken embryonated egg) further comprises an HA genomic segment and an NA genomic segment. In some embodiments, the NS genomic segment nucleotide sequence comprises the negative-sense RNA complement of the nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS genomic segment nucleotide sequence comprises the negative-sense RNA complement of a nucleotide71NAI-5003686295vlsequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS genomic segment nucleotide sequence comprises the negative-sense RNA complement of the nucleotide sequence of SEQ ID NO:23 or 24. In some embodiments, the NS genomic segment nucleotide sequence comprises the negative-sense RNA complement of a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:23 or 24. In some embodiments, the HA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strains from which the PB1, PB2, PA, NP, M and NS genomic segments are from or derived from. In some embodiments, the HA genomic segment is from or derived from a seasonal Influenza A virus. In some embodiments, the HA genomic segment is from or derived from a naturally occurring influenza virus. In some embodiments, the HA genomic segment is from an exotic Influenza A virus (e.g., an Influenza A virus not prevalent in humans). In some embodiments, the HA genomic segment is artificially created or a consensus HA genomic segment. In some embodiments, the use of an exotic Influenza A virus HA genomic segment or an artificially created HA genomic allows for the evasion of pre-existing immunity to HA in a subject. In some embodiments, the NA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strains from which the PB1, PB2, PA, NP, M and NS genomic segments are from or derived from. In some embodiments, the NA genomic segment is from or derived from a seasonal Influenza A virus. In some embodiments, the NA genomic segment is from or derived from a naturally occurring influenza virus. In some embodiments, the NA genomic segment is artificially created or a consensus NA genomic segment. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the genomic segments comprise one or more mutations that attenuate the Influenza A virus. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1, PB2, PA, NP, M, HA, and NA genomic segments comprise a heterologous nucleotide sequence. The heterologous nucleotide sequence may encode an antigen. In some embodiments, the cell (e.g., a cell line) or embryonated egg (e.g., a chicken embryonated egg) further comprises a nucleotide sequence encoding a PB1 protein, a nucleotide sequence encoding a PB2 protein, a nucleotide sequence encoding a PA protein, and a nucleotide sequence encoding a NP protein.

[0125] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) genomic segments described herein are incorporated in one or more vectors (e.g., plasmids, such as, e.g., a plasmid described herein). In some embodiments, the one or more vectors is a bidirectional72NAI-5003686295vlplasmid, wherein the bidirectional plasmid comprises a human RNA polymerase I promoter, a mouse terminator sequence, and a polymerase II transcription cassette. In some embodiments, the one or more vectors is an ambisense plasmid. In some embodiments, the one or more vectors is an pDZ plasmid (see, e.g., Quinlivan et al., 2005, J. of Virology 79: 8431-8439 for information relating to the pDZ plasmid). In some embodiments, the one or more vectors is a pHW2000 plasmid (see, e.g., Hoffmann et al., 2000, Proc Natl Acad Sci U S A. 97(11) :6108-13 for information relating to the pHW2000 plasmid). In some embodiments, the one or more vectors is a pAD3000 plasmid (see, e.g., Hoffmann et al., 2000, Proc Natl Acad Sci U S A. 97(11) :6108-13 for information relating to the pAD3000 plasmid). In another embodiment, the one or more vectors is a pAD4000 plasmid (see, e.g., Wang et al., 2007, J. of Virology 4: 102 for information relating to the pAD4000 plasmid).

[0126] In some embodiments, provided herein are cells (e.g., a cell line) or embryonated eggs (e.g., a chicken embryonated egg) comprising an NS genomic segment described herein or an NS nucleotide sequence. In some embodiments, provided herein are cells (e.g., a cell line) or embryonated eggs (e.g., a chicken embryonated egg) comprising a vector comprising an NS nucleotide sequence described herein.

[0127] In specific embodiments, the cells are ones known to one of skill in the art or described herein. In specific embodiments, the cells are isolated. In specific embodiments, the cell(s) is in vitro or ex vivo. In specific embodiments, the cell(s) are not in vivo. In specific embodiments, the embryonated egg(s) is ex vivo. In some embodiments, the embryonated egg(s) is one known to one of skill in the art or described herein.5.6 COMPOSITIONS COMPRISING RECOMBINANT INFLUENZA A VIRUSES

[0128] In one aspect, provided herein are compositions comprising an NS genomic segment described herein (e.g., in Section 5.1, or 8). In specific embodiments, provided herein is a composition comprising an NS genomic segment described herein (e.g., in Section 5.1, or 8). In some embodiments, the NS genomic segment comprises the corresponding negative sense RNA sequence of the nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS genomic segment comprises a nucleotide sequence that is at least 75%, at least 80%, at leat 85%, at least 90% or at least 95% identical to the corresponding negative sense RNA sequence of the nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS genomic segment comprises a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the corresponding negative sense RNA sequence of the nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS genomic segment73NAI-5003686295vlcomprises the corresponding negative sense RNA sequence of the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the NS genomic segment comprises a nucleotide sequence that is at least 75%, at least 80%, at leat 85%, at least 90% or at least 95% identical to the corresponding negative sense RNA sequence of the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the NS genomic segment comprises a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the corresponding negative sense RNA sequence of the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the composition further comprises one or more, or all of the following: a PB1 genomic segment, a PB2 genomic segment, a PA genomic segment, an NP genomic segment, and a M genomic segment. In some embodiments, the composition further comprises a PB1 genomic segment, a PB2 genomic segment, a PA genomic segment, an NP genomic segment, and a M genomic segment. In some embodiments, the composition further comprises one or more, or all of the following: a PB1 genomic segment, a PB2 genomic segment, a PA genomic segment, an NP genomic segment, an HA genomic segment, an NA genomic segment, and a M genomic segment. In some embodiments, the composition further comprises a PB1 genomic segment, a PB2 genomic segment, a PA genomic segment, an NP genomic segment, an HA genomic segment, an NA genomic segment, and a M genomic segment.

[0129] In specific embodiments, provided herein is a composition comprising an NS nucleotide sequence described herein (e.g., in Section 5.1, or 8). In some embodiments, the NS nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS nucleotide sequence comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS nucleotide sequence comprises a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the NS nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the NS nucleotide sequence comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the NS nucleotide sequence comprises a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the composition further comprises one or more, or all of the following: a nucleotide sequence of a PB1 genomic segment, a nucleotide sequence of a PB2 genomic segment, a nucleotide sequence of a PA genomic segment, a74NAI-5003686295vlnucleotide sequence of an NP genomic segment, and a nucleotide sequence of a M genomic segment. In some embodiments, the composition further comprises a nucleotide sequence of a PB1 genomic segment, a nucleotide sequence of a PB2 genomic segment, a nucleotide sequence of a PA genomic segment, a nucleotide sequence of an NP genomic segment, and a nucleotide sequence of a M genomic segment. In some embodiments, the composition further comprises one or more, or all of the following: a nucleotide sequence of a PB1 genomic segment, a nucleotide sequence of a PB2 genomic segment, a nucleotide sequence of a PA genomic segment, a nucleotide sequence of an NP genomic segment, a nucleotide sequence of an HA genomic segment, a nucleotide sequence of an NA genomic segment, and a M genomic segment. In some embodiments, the composition further comprises a nucleotide sequence of a PB 1 genomic segment, a nucleotide sequence of a PB2 genomic segment, a nucleotide sequence of a PA genomic segment, a nucleotide sequence of an NP genomic segment, a nucleotide sequence of an HA genomic segment, a nucleotide sequence of an NA genomic segment, and a nucleotide sequence of a M genomic segment. In some embodiments, the HA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strains from which the PB1, PB2, PA, NP, M and NS genomic segments are from or derived from. In some embodiments, the HA genomic segment is from or derived from a seasonal Influenza A virus. In some embodiments, the HA genomic segment is from or derived from a naturally occurring influenza virus. In some embodiments, the HA genomic segment is from or derived from an Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, or H17. In some embodiments, the HA genomic segment is from an exotic Influenza A virus (e.g., an Influenza A virus not prevalent in humans). In some embodiments, the HA genomic segment is artificially created or a consensus HA genomic segment. In some embodiments, the use of an exotic Influenza A virus HA genomic segment or an artificially created HA genomic allows for the evasion of pre-existing immunity to HA in a subject. In some embodiments, the NA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strains from which the PB1, PB2, PA, NP, M and NS genomic segments are from or derived from. In some embodiments, the NA genomic segment is from or derived from a seasonal Influenza A virus. In some embodiments, the NA genomic segment is from or derived from a naturally occurring influenza virus. In some embodiments, the NA genomic segment is from or derived from an Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10,or N11. In some embodiments, the NA genomic segment is artificially created or a consensus NA genomic segment. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1,75NAI-5003686295vlPB2, PA, NP, M, HA, and NA genomic segments comprise one or more mutations that attenuate the Influenza A virus. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1, PB2, PA, NP, M, HA, and NA genomic segments comprise a heterologous nucleotide sequence. The heterologous nucleotide sequence may encode an antigen.

[0130] In specific embodiments, provided herein is a composition comprising a nucleotide sequence of an NS genomic segment described herein (e.g., in Section 5.1, or 8). In some embodiments, the nucleotide sequence of the NS genomic segment comprises the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleotide sequence of the NS genomic segment comprises the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the composition further comprises a nucleotide sequence of a PB 1 genomic segment, a nucleotide sequence of a PB2 genomic segment, a nucleotide sequence of a PA genomic segment, a nucleotide sequence of an NP genomic segment, and a nucleotide sequence of a M genomic segment. In some embodiments, the composition further comprises a nucleotide sequence of a PB1 genomic segment, a nucleotide sequence of a PB2 genomic segment, a nucleotide sequence of a PA genomic segment, a nucleotide sequence of an NP genomic segment, a nucleotide sequence of an HA genomic segment, a nucleotide sequence of an NA genomic segment, and a nucleotide sequence of a M genomic segment. In some embodiments, the HA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strains from which the PB1, PB2, PA, NP, M and NS genomic segments are from or derived from. In some embodiments, the HA genomic segment is from or derived from a seasonal Influenza A virus. In some embodiments, the HA genomic segment is from or derived from a naturally occurring influenza virus. In some embodiments, the HA genomic segment is from or derived from an Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15,76NAI-5003686295vlHl 6, or Hl 7. In some embodiments, the HA genomic segment is from an exotic Influenza A virus (e.g., an Influenza A virus not prevalent in humans). In some embodiments, the HA genomic segment is artificially created or a consensus HA genomic segment. In some embodiments, the use of an exotic Influenza A virus HA genomic segment or an artificially created HA genomic allows for the evasion of pre-existing immunity to HA in a subject. In some embodiments, the NA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strains from which the PB1, PB2, PA, NP, M and NS genomic segments are from or derived from. In some embodiments, the NA genomic segment is from or derived from a seasonal Influenza A virus. In some embodiments, the NA genomic segment is from or derived from a naturally occurring influenza virus. In some embodiments, the NA genomic segment is from or derived from an Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10,or Nl l. In some embodiments, the NA genomic segment is artificially created or a consensus NA genomic segment. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1, PB2, PA, NP, M, HA, and NA genomic segments comprise one or more mutations that attenuate the Influenza A virus. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1, PB2, PA, NP, M, HA, and NA genomic segments comprise a heterologous nucleotide sequence. The heterologous nucleotide sequence may encode an antigen.

[0131] In some embodiments, provided herein is a composition comprising a vector (e.g., a plasmid or viral vector) comprising a nucleotide sequence of an NS genomic segment described herein (e.g., in Section 5.1, or 8). In some embodiments, provided herein is a composition comprising a vector (e.g., a plasmid or viral vector) comprising an NS nucleotide sequence described herein (e.g., in Section 5.1, or 8). In some embodiments, the nucleotide sequence of the NS genomic segment comprises the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleotide sequence of the NS genomic segment comprises the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the nucleotide sequence of the NS genomic segment comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the nucleotide sequence of the NS77NAI-5003686295vlgenomic segment comprises a nucleotide sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 23 or 24. In some embodiments, the composition further comprises one or more vectors (e.g., 1, 2, 3, 4, or 5 vectors) comprising a nucleotide sequence of a PB1 genomic segment, a nucleotide sequence of a PB2 genomic segment, a nucleotide sequence of a PA genomic segment, a nucleotide sequence of an NP genomic segment, and a nucleotide sequence of a M genomic segment. In some embodiments, the composition further comprises one or more vectors (e.g., 1, 2, 3, 4, 5, 6, or 7 vectors) comprising a nucleotide sequence of a PB1 genomic segment, a nucleotide sequence of a PB2 genomic segment, a nucleotide sequence of a PA genomic segment, a nucleotide sequence of an NP genomic segment, a nucleotide sequence of an HA genomic segment, a nucleotide sequence of an NA genomic segment, and a nucleotide sequence of a M genomic segment. In some embodiments, the vectors are plasmids. In some embodiments, the vectors are ambisense plasmids. In some embodiments, the HA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strains from which the PB1, PB2, PA, NP, M and NS genomic segments are from or derived from. In some embodiments, the HA genomic segment is from or derived from a seasonal Influenza A virus. In some embodiments, the HA genomic segment is from or derived from a naturally occurring Influenza A virus. In some embodiments, the HA genomic segment is from or derived from an Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl 1, H12, H13, H14, Hl 5, Hl 6, or Hl 7. In some embodiments, the HA genomic segment is from an exotic Influenza A virus (e.g., an Influenza A virus not prevalent in humans). In some embodiments, the HA genomic segment is artificially created or a consensus HA genomic segment. In some embodiments, the use of an exotic Influenza A virus HA genomic segment or an artificially created HA genomic allows for the evasion of pre-existing immunity to HA in a subject. In some embodiments, the NA genomic segment is from or derived from an Influenza A virus strain, which is different than the Influenza A virus strains from which the PB1, PB2, PA, NP, M and NS genomic segments are from or derived from. In some embodiments, the NA genomic segment is from or derived from a seasonal Influenza A virus. In some embodiments, the NA genomic segment is from or derived from a naturally occurring Influenza A virus. In some embodiments, the NA genomic segment is from or derived from an Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, or N11. In some embodiments, the NA genomic segment is artificially created or a consensus NA genomic segment. In some embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1, PB2, PA, NP, M, HA, and NA genomic segments comprise one or more mutations that attenuate the Influenza A virus. In78NAI-5003686295vlsome embodiments, one or more (e.g., 1, 2, 3, or more) of the PB1, PB2, PA, NP, M, HA, and NA genomic segments comprise a heterologous nucleotide sequence. The heterologous nucleotide sequence may encode an antigen.

[0132] In another aspect, provided herein are compositions comprising a recombinant Influenza A virus described herein (e.g., in Section 5.3, 8, or 9). In a specific embodiment, the compositions are pharmaceutical compositions, such as immunogenic compositions (e.g., vaccine compositions). In one embodiment, a composition comprises a recombinant Influenza A virus described herein (e.g., Section 5.3, or 8) and a pharmaceutically acceptable carrier. In a specific embodiment, provided herein are immunogenic compositions comprising a recombinant Influenza A virus described herein (e.g., in Section 5.3, 8, or 9). The compositions may be used in methods of inducing an immune response to Influenza A virus. The compositions may be used in methods of inducing an immune response to an antigen. The compositions may be used in methods for immunizing against Influenza A virus. The compositions may be used in methods for immunizing against an antigen associated with a disease or condition. The compositions may be used to express a protein (e.g., a cytokine (e.g., IL-2 or IL- 15) that has an immunologic effect.

[0133] In a specific embodiment, provided herein are pharmaceutical compositions comprising a recombinant Influenza A virus described herein (e.g., in Section 5.3, 8, or 9) and a pharmaceutically acceptable carrier. In some embodiments, the recombinant Influenza A virus does not include a transgene. In specific embodiments, the recombinant Influenza A virus comprises an NS genomic segment, wherein the NS genomic segment comprises a transgene encoding a biomolecule (see, e.g., Section 5.2 for examples of biomolecules). In specific embodiments, the recombinant Influenza A virus comprises an NS genomic segment, wherein the NS genomic segment comprises a transgene encoding a cytokine (e.g., an immunostimulatory cytokine). In specific embodiments, the recombinant Influenza A virus comprises an NS genomic segment, wherein the NS genomic segment comprises a transgene encoding IL-2 (e.g., human IL-2) or IL-15 (e.g., human IL-15). In some embodiments, the pharmaceutical composition further comprises one or more additional prophylactic or therapeutic agents (e.g., an anti-cancer agent). In a specific embodiment, a pharmaceutical composition comprises an effective amount of a recombinant Influenza A virus described herein (e.g., Section 5.3, or 8), and optionally one or more additional prophylactic or therapeutic agents (e.g., an anti-cancer agent), in a pharmaceutically acceptable carrier. In some embodiments, the recombinant Influenza A virus described herein (e.g., Section 5.3, or 8) is the only active ingredient included in the composition (e.g., immunogenic composition).79NAI-5003686295vlIn some embodiments, the recombinant Influenza A virus is a live virus (e.g., a live attenuated virus).

[0134] In one embodiment, an immunogenic composition comprises a recombinant Influenza A virus described herein (e.g., in Section 5.3, 8, or 9) and a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition further comprises one or more additional prophylactic or therapeutic agents. In a specific embodiment, an immunogenic composition comprises an effective amount of a recombinant Influenza A virus described herein (e.g., in Section 5.3, 8, or 9), and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. In some embodiments, the recombinant Influenza A virus described herein (e.g., in Section 5.3, 8, or 9) is the only active ingredient included in the immunogenic composition. In some embodiments, the recombinant Influenza A virus is a live virus (e.g., a live attenuated virus). In specific embodiments, the immunogenic composition is a vaccine.

[0135] In some embodiments, administration of an immunogenic composition described herein to a subject (e.g., a human) induces an immune response in the subject. In some embodiments, administration of an immunogenic composition described herein to a subject (e.g., a human) induces an antibody(ies) (e.g., neutralizing antibody(ies)) response. In some embodiments, administration of an immunogenic composition described herein to a subject (e.g., a human) induces an immune response against influenza virus (e.g., Influenza A virus) in the subject. In some embodiments, administration of an immunogenic composition described herein to a subject (e.g., a human) induces antibody (e.g., neutralizing antibody) against Influenza A virus. In some embodiments, administration of an immunogenic composition described herein to a subject (e.g., a human) induces neutralizing antibody against Influenza A virus. In certain embodiments, administration of an immunogenic composition described herein to a subject (e.g., a human) induces an immune response that provides some level of protection (e.g., partial or complete protection) against developing Influenza A virus disease. In some embodiments, administration of an immunogenic composition described herein to a subject (e.g., a human) reduces the number of symptoms of influenza virus disease (e.g., Influenza A virus disease), reduces the severity of one or more symptoms of influenza virus disease (e.g., Influenza A virus disease), or both. In some embodiments, administration of an immunogenic composition described herein to a subject (e.g., a human) induces an immune response against an antigen expressed by a recombinant Influenza A virus described herein, which antigen is associated with a disease or condition. In some embodiments, administration of an immunogenic composition described herein to a80NAI-5003686295vlsubject (e.g., a human) induces an antibody(ies) (e.g., a neutralizing antibody(ies) against an antigen expressed by a recombinant Influenza A virus described herein, which antigen is associated with a disease or condition. In some embodiments, administration of an immunogenic composition described herein to a subject e.g., a human) induces an immune response that provides some level of protection against developing a disease or condition associated with an antigen expressed by a recombinant Influenza A virus described herein. In some embodiments, administration of an immunogenic composition described herein to a subject (e.g., a human) reduces the number of symptoms of a disease or condition associated with an antigen expressed by a recombinant Influenza A virus described herein, reduces the severity of one or more symptoms of a disease or condition associated with an antigen expressed by a recombinant Influenza A virus described herein, or both.

[0136] In some embodiments, administration of a pharmaceutical composition described herein is immunostimulatory. In some embodiments, administration of a pharmaceutical composition described herein induces the innate immune response and / or adaptive immune response. In some embodiments, administration of a pharmaceutical composition described herein induces an anti-tumor response. In some embodiments, administration of a pharmaceutical composition described herein induces an innate immune response and / or adaptive immune response to a tumor.

[0137] In a specific embodiment, a recombinant Influenza A virus described herein that is included in a composition (e.g., an immunogenic composition) described herein is a live virus. In particular, embodiment, the recombinant Influenza A virus included in an immunogenic composition described herein is an attenuated live virus.

[0138] The compositions (e.g., immunogenic compositions) provided herein can be in any form that allows for the composition to be administered to a subject. In a specific embodiment, the pharmaceutical compositions are suitable for veterinary administration, human administration, or both.

[0139] As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried81NAI-5003686295vlskim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin. The formulation should suit the mode of administration.

[0140] In a specific embodiment, the compositions (e.g., immunogenic compositions) are formulated to be suitable for the intended route of administration to a subject. For example, a composition (e.g., an immunogenic composition) may be formulated to be suitable for parenteral, intratumoral, intraarterial, intrapleural, inhalation, intranasal, intraperitoneal, oral, intradermal, colorectal, intraperitoneal, and intracranial administration. In some embodiments, a composition (e.g., an immunogenic composition) is formulated for intravenous, intraarterial, oral, intraperitoneal, intranasal, intratracheal, intrapleural, intracranial, subcutaneous, intramuscular, topical, or pulmonary administration. In a specific embodiment, a composition (e.g., an immunogenic composition) is formulated for intranasal administration. In some embodiments, a composition (e.g, an immunogenic composition) is formulated for a nasal spray. In some embodiments, a composition (e.g, an immunogenic composition) is formulated for intramuscular administration. In some embodiments, a composition (e.g., an immunogenic composition) is formulated for intratumoral administration. In some embodiments, a composition (e.g., an immunogenic composition) is formulated for pulmonary administration.

[0141] In a specific embodiment, a composition (e.g., an immunogenic composition) comprising a recombinant Influenza A virus described herein (see, e.g., Sections 5.3, 8, and 9) is formulated to be suitable for intranasal administration to the subject (e.g., human subject).

[0142] In some embodiments, a composition (e.g., an immunogenic composition) described herein comprises an effective amount of a recombinant Influenza A virus described herein. In specific embodiments, an effective amount of a recombinant Influenza A virus described herein is an amount of recombinant Influenza A virus to generate an immune response in a subject or a population of subjects. In specific embodiments, an effective amount of a recombinant Influenza A virus described herein is 104to 1012PFU or EID50. In some embodiments, an effective amount is 1 to 15 micrograms of an Influenza A virus HA protein expressed by a recombinant Influenza A virus described herein.

[0143] In some embodiments, a composition (e.g., an immunogenic composition) described herein comprises 104to 1012EID50 of a recombinant Influenza A virus described herein. In some embodiments, a composition (e.g., an immunogenic composition) described herein82NAI-5003686295vlcomprises 1 to 15 micrograms of an Influenza A virus HA protein expressed by a recombinant Influenza A virus described herein.

[0144] In some embodiments, provided herein is a composition comprising supernatant from cells infected with a recombinant Influenza A virus described herein. In some embodiments, Influenza A virus in the supernatant has been inactivated using techniques known to one of skill in the art.5.7 USES OF RECOMBINANT INFLUENZA A VIRUSES

[0145] In another aspect, a recombinant Influenza A virus described herein (e.g., in Section 5.1, 8, or 9) or a composition thereof (e.g., a composition described in Section 5.6) may be used to induce an immune response in a subject (e.g., a human subject). In some embodiments, provided herein is a method for inducing an immune response in a subject (e.g., a human subject), comprising administering a recombinant Influenza A virus described herein to the subject. In some embodiments, provided herein is a method for inducing an immune response in a subject (e.g., a human subject), comprising administering an effective amount of a recombinant Influenza A virus described herein to the subject. The immune response induced in the subject may be against Influenza A virus, an antigen expressed by the Influenza A virus, or both. In some embodiments, the innate immune response and / or adaptive immune response is induced. In some embodiments, the recombinant Influenza A virus or composition thereof is the only active agent administered to the subject. In other embodiments, the recombinant Influenza A virus or composition thereof is administered to the subject in combination with another prophylactic or therapeutic agent. In some embodiments, a recombinant Influenza A virus or composition thereof is administered to a subject in combination with a supportive therapy or a pain relief therapy (e.g., ibuprofen and / or acetaminophen). The recombinant Influenza A virus or composition thereof may be administered before, concurrently, or after the administration of another prophylactic or therapeutic agent, a supportive therapy, or a pain relief therapy to the subject. In some embodiments, a recombinant Influenza A virus or composition thereof and another prophylactic or therapeutic agent, a supportive therapy, or a pain relief therapy are administered within 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, 18 hours, or 24 hours of each other.

[0146] In another aspect, a recombinant Influenza A virus described herein (e.g., in Section 5.1, 8, or 9) or a composition thereof (e.g., a composition described in Section 5.6) may be used to immunize a subject (e.g., a human subject). In some embodiments, provided herein is83NAI-5003686295vla method for immunizing a subject (e.g., a human subject), comprising administering a recombinant Influenza A virus described herein to the subject. In some embodiments, provided herein is a method for immunizing a subject (e.g., a human subject), comprising administering an effective amount of a recombinant Influenza A virus described herein to the subject. The subject may be immunized against Influenza A virus, an antigen expressed by the Influenza A virus, or both. In some embodiments, the recombinant Influenza A virus or composition thereof is the only active agent administered to the subject. In other embodiments, the recombinant Influenza A virus or composition thereof is administered to the subject in combination with another prophylactic or therapeutic agent. In some embodiments, a recombinant Influenza A virus or composition thereof is administered to a subject in combination with a supportive therapy or a pain relief therapy (e.g., ibuprofen and / or acetaminophen). The recombinant Influenza A virus or composition thereof may be administered before, concurrently, or after the administration of another prophylactic or therapeutic agent, a supportive therapy, or a pain relief therapy to the subject. In some embodiments, a recombinant Influenza A virus or composition thereof and another prophylactic or therapeutic agent, a supportive therapy, or a pain relief therapy are administered within 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, 18 hours, or 24 hours of each other.

[0147] In another aspect, a recombinant Influenza A virus described herein or a composition thereof (e.g., a composition described in Section 5.6) may be used in the prevention of influenza virus disease. In some embodiments, provided herein are methods for preventing influenza virus disease, comprising administering to a subject in need thereof (e.g., a human subject in need thereof) a recombinant Influenza A virus described herein or a composition thereof. In some embodiments, provided herein is a method for preventing influenza virus disease, comprising administering to a subject in need thereof (e.g., a human subject in need thereof) an effective amount of a recombinant Influenza A virus described herein or a composition thereof. In some embodiments, the recombinant Influenza A virus or composition thereof is the only active agent administered to the subject. In other embodiments, the recombinant Influenza A virus or composition thereof is administered to the subject in combination with another prophylactic or therapeutic agent. In some embodiments, a recombinant Influenza A virus or composition thereof is administered to a subject in combination with a supportive therapy, a pain relief therapy (e.g., ibuprofen and / or acetaminophen), or other therapy that does not have a therapeutic effect on influenza virus84NAI-5003686295vldisease. The recombinant Influenza A virus or composition thereof may be administered before, concurrently, or after the administration of another prophylactic or therapeutic agent, a supportive therapy, a pain relief therapy, or other therapy to the subject. In some embodiments, a recombinant Influenza A virus or composition thereof and another prophylactic or therapeutic agent, a supportive therapy, a pain relief therapy, or other therapy are administered within 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, 18 hours, or 24 hours of each other.

[0148] In some embodiments, a recombinant A influenza virus described herein, or a composition described herein, or a combination therapy described herein is administered to a patient to prevent the onset of one, two or more symptoms of influenza virus disease. In some embodiments, the administration of a recombinant Influenza A virus described herein, or an immunogenic composition described herein, or a combination therapy described herein to a subject prevents the onset or development of one, two or more symptoms of influenza virus disease, or reduces the severity of one, two or more symptoms of influenza virus disease. In some embodiments, the administration of a recombinant Influenza A virus described herein, or an immunogenic composition described herein, or a combination therapy described herein to a subject prevents the onset or development of one, two or more symptoms of influenza virus disease and reduces the severity of one, two or more symptoms of influenza virus disease. Symptoms of influenza virus disease include a congested or runny nose, cough, fever, sore throat, fatigue, headache, and muscle or body aches.

[0149] In some embodiments, a recombinant Influenza A virus described herein encoding an antigen or a composition thereof is administered to a patient to prevent the onset of one, two or more symptoms of a disease or condition associated with the antigen. In some embodiments, the administration of a recombinant Influenza A virus described herein encoding an antigen or a composition thereof to a subject prevents the onset or development of one, two or more symptoms of a disease or condition associated with the antigen, or reduces the severity of one, two or more symptoms of a disease or condition associated with the antigen. In some embodiments, the administration of a recombinant Influenza A virus described herein encoding an antigen or a composition thereof to a subject prevents the onset or development of one, two or more symptoms of a disease or condition associated with the antigen and reduces the severity of one, two or more symptoms of the disease or condition.

[0150] In some embodiments, the administration of a recombinant Influenza A virus described herein, a composition described herein, or a combination therapy described herein85NAI-5003686295vlinduces antibodies to influenza virus protein (e.g., influenza virus HA, influenza virus NA, etc.). In some embodiments, the administration of a recombinant Influenza A virus described herein, a composition described herein, or a combination therapy described herein to a subject induces neutralizing antibody to influenza virus HA and / or influenza virus NA.

[0151] In some embodiments, the administration of a recombinant Influenza A virus described herein encoding an antigen, a composition described herein comprising such a recombinant Influenza A virus, or a combination therapy described herein including such a recombinant Influenza A virus induces antibody to the antigen. In some embodiments, the administration of a recombinant Influenza A virus described herein encoding an antigen, described herein comprising such a recombinant Influenza A virus, or a combination therapy described herein including such a recombinant Influenza A virus induces neutralizing antibody to the antigen.

[0152] In another aspect, provided herein are methods for delivering a biomolecule (e.g., a biomolecule described herein) to a cell(s), comprising contacting the cell(s) with a recombinant Influenza A virus described herein. In some embodiments, the cell(s) is contacted with the recombinant Influenza A virus described herein in cell culture. In some embodiments, the cell(s) is contacted with the recombinant Influenza A virus described herein in vitro or ex vivo. In some embodiments, the cell(s) is contacted with the recombinant Influenza A virus described herein in vivo e.g., in a non-human animal or a human). In some embodiments, the cell(s) is not contacted in vivo with the recombinant Influenza A virus described herein. The recombinant Influenza A virus may be in a composition described herein. In some embodiments, about 104to about 1012PFU or EID50 of a recombinant Influenza A virus described herein is contacted with the cell(s). In some embodiments, the biomolecule comprises a protein e.g., a secretory protein). In some embodiments, the biomolecule comprises a cytokine e.g., an immunostimulatory cytokine). In preferred embodiments, the biomolecule is IL-2 e.g., human IL-2). In preferred embodiments, the biomolecule is IL- 15 e.g., human IL- 15).

[0153] In another aspect, a recombinant Influenza A virus described herein e.g., in Section 5.1, 8, or 9) or a composition thereof e.g., a composition described in Section 5.6) may be used to deliver a biomolecule in a subject e.g., a human subject). In some embodiments, provided herein is a method for delivering a biomolecule e.g., a biomolecule described herein) a subject e.g., a human subject), comprising administering a recombinant Influenza A virus described herein to the subject. In some embodiments, provided herein is a method for delivering a biomolecule in a subject e.g., a human subject), comprising administering an86NAI-5003686295vleffective amount of a recombinant Influenza A virus described herein to the subject. In some embodiments, the biomolecule comprises a protein (e.g., a secretory protein). In some embodiments, the biomolecule comprises a cytokine (e.g., an immunostimulatory cytokine). In some embodiments, the biomolecule comprises an immunomodulatory cytokine. In some embodiments, the immunomodulatory cytokine enhances to adjuvanticity of the recombinant Influenza A virus. In preferred embodiments, the biomolecule is IL-2 (e.g., human IL-2). In preferred embodiments, the biomolecule is IL- 15 (e.g., human IL- 15).

[0154] In another aspect, a recombinant Influenza A virus described herein (e.g., in Section 5.1, 8, or 9) or a composition thereof (e.g., a composition described in Section 5.6) may be used to treat cancer in a subject (e.g., a human subject). In some embodiments, provided herein is a method for treating cancer in a subject (e.g., a human subject), comprising administering a recombinant Influenza A virus described herein to the subject. In some embodiments, provided herein is a method for treating cancer in a subject (e.g., a human subject), comprising administering an effective amount of a recombinant Influenza A virus described herein to the subject. In some embodiments, the recombinant Influenza A virus comprises an NS genomic segment described herein that does not include a transgene. In some embodiments, the recombinant Influenza A virus comprises an NS genomic segment described herein, wherein the NS genomic segment comprises a transgene. In some embodiments, the transgene encodes a biomolecule (e.g., a biomolecule described herein). In some embodiments, the biomolecule comprises a protein (e.g., a secretory protein). In some embodiments, the biomolecule comprises a cytokine (e.g., an immunostimulatory cytokine). In specific embodiments, the biomolecule is IL-2 (e.g., human IL-2). In specific embodiments, the biomolecule is IL-15 (e.g., human IL-15). In some embodiments, the recombinant Influenza A virus or composition thereof is the only active agent administered to the subject. In other embodiments, the recombinant Influenza A virus or composition thereof is administered to the subject in combination with another prophylactic or therapeutic agent (e.g., an anti-cancer agent). In some embodiments, a recombinant Influenza A virus or composition thereof is administered to a subject in combination with a supportive therapy or a pain relief therapy (e.g., ibuprofen and / or acetaminophen). The recombinant Influenza A virus or composition thereof may be administered before, concurrently, or after the administration of another prophylactic or therapeutic agent (e.g., an anti-cancer agent), a supportive therapy, or a pain relief therapy to the subject. In some embodiments, a recombinant Influenza A virus or composition thereof and another prophylactic or therapeutic agent, a supportive therapy, or a pain relief therapy are administered within 1 minute, 287NAI-5003686295vlminutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, 18 hours, or 24 hours of each other. In some embodiments, the treatment of cancer reduces one or more symptoms of cancer and / or reduces the frequency of one or more symptoms of cancer. In some embodiments, the treatment of cancer reduces tumor size and / or tumor growth. In some embodiments, the treatment of cancer results in an effect described in the Examples below (e.g., in Section 9). In some embodiments, the cancer is a thoracic cancer, such as, e.g., nonsmall lung cancer or another lung carcinoma. In some embodiments, the cancer is a carcinoma that has metastasized the lungs, such as, e.g., metastatic colon carcinoma. In some embodiments, the cancer is one described in the Examples below (e.g., in Section 9).

[0155] A recombinant Influenza A virus, or a composition described herein comprising a recombinant Influenza A virus may be administered locally or systemically to a subject. For example, a recombinant Influenza A virus, or a composition described herein comprising a recombinant Influenza A virus may be administered parenterally (e.g., intraperitoneally, intravenously, intra-arterially, intradermally, intramuscularly, or subcutaneously), intratumorally, intrapleurally, intranasally, intracavitary, intracranially, orally, rectally, by inhalation, or topically to a subject. In a specific embodiment, a recombinant Influenza A virus or composition described herein comprising a recombinant Influenza A virus is administered intranasally to a subject. In a specific embodiment, a recombinant Influenza A virus or composition described herein comprising a recombinant Influenza A virus is administered intranasally to a subject. In a specific embodiment, a recombinant Influenza A virus or composition described herein comprising a recombinant Influenza A virus is administered intratumorally to a subject. In a specific embodiment, a recombinant Influenza A virus or composition described herein comprising a recombinant Influenza A virus is administered by pulmonary administration to a subject.

[0156] A recombinant Influenza A virus described herein, or a composition described herein comprising a recombinant Influenza A virus may be administered to a subject in combination with one or more other therapies. The recombinant Influenza A virus, or composition described herein comprising a recombinant Influenza A virus and one or more other therapies may be administered by the same or different routes of administration to the subject. Additional therapies that can be used in a combination with a recombinant Influenza A virus described herein, or a composition described herein comprising a recombinant Influenza A virus include, but are not limited to, acetaminophen, an anti-cancer agent (e.g., a88NAI-5003686295vlchemotherapeutic), ibuprofen, throat lozenges, cough suppressants, inhalers, antibiotics and oxygen.

[0157] A recombinant influenza virus, or a composition described herein comprising a recombinant Influenza A virus and one or more additional therapies may be administered concurrently or sequentially to the subject. In some embodiments, the recombinant influenza virus and one or more additional therapies are administered to a subject in the same composition. In some embodiments, the recombinant influenza virus and one or more additional therapies are administered to a subject in different compositions. The recombinant influenza virus and one or more other therapies may be administered by the same or different routes of administration to the subject. Any route known to one of skill in the art or described herein may be used to administer the recombinant influenza virus and one or more other therapies.

[0158] In some embodiments, a recombinant Influenza A virus is administered to a subject (e.g., human) at a dose of about 104to about 1012PFU or EID50. In some embodiments, 1 to 15 micrograms of an Influenza A virus HA protein is administered to a subject (e.g., human). In some embodiments, a recombinant Influenza A virus or a composition described herein is administered to a subject (e.g., human) once, twice, three, four or more times with intervals as often as needed. In some embodiments, dosages similar to those currently being used in clinical trials for recombinant influenza viruses are administered to a subject (e.g., human). In some embodiments, dosages similar to those dosages approved by the U.S. FDA or another regulatory authority for influenza virus vaccines.

[0159] In certain embodiments, a recombinant Influenza A virus or a composition (e.g., an immunogenic composition) described herein is administered to a subject (e.g, human) as a single dose followed by a second dose a certain number of weeks or months later. In some embodiments, a dose of a first recombinant Influenza A virus described herein or a composition described herein (e.g, immunogenic composition) is administered to a subject (e.g., human) followed by the administration of a dose of a second recombinant Influenza A virus described herein or a composition described herein (e.g., immunogenic composition) a certain number of weeks or months later. The first recombinant Influenza A virus and second recombinant Influenza A virus may be the same or different. In some embodiments, a subject is administered one or more boosters of a recombinant Influenza A virus described herein or a composition described herein (e.g., immunogenic composition). The recombinant Influenza A virus used for each booster may be the same or different. The two, three, four, or more recombinant Influenza A viruses described herein, or compositions (e.g., immunogenic89NAI-5003686295vlcompositions) described herein administered to the subject (e.g., human) may administered by the same or different routes.

[0160] In another aspect, a recombinant Influenza A virus described herein, or an immunogenic composition described herein is administered to a non-human subject (e.g., a mouse, rat, guinea, camel, etc.) and the antibodies generated in response to the polypeptide are isolated. Hybridomas may be made, and monoclonal antibodies produced using techniques known to one of skill in the art. The antibodies may also be optimized. In some embodiments, the antibodies produced are humanized or chimerized. In some embodiments, the non-human subject produces human antibodies. The antibodies produced using a recombinant Influenza A virus described herein, or an immunogenic composition described herein may be optimized, using techniques known to one of skill in the art. In some embodiments, antibodies generated using a recombinant Influenza A virus described herein, or an immunogenic composition described herein may be used to prevent, treat or prevent and treat influenza virus disease, or a disease or condition associated with an antigen encoded by the recombinant Influenza A virus.

[0161] In some embodiments, a recombinant Influenza A virus described herein is used in an immunoassay (e.g., an ELISA assay) known to one of skill in the art or described herein to detect antibody specific for an influenza virus protein (e.g., HA or NA). In some embodiments, provided herein is a method for detecting the presence of antibody specific to an Influenza A virus protein (e.g., HA or NA), comprising contacting a specimen with the recombinant Influenza A virus described herein in an immunoassay (e.g., an ELISA). In some embodiments, the specimen is a biological specimen. In some embodiments, the biological specimen comprises blood, plasma or sera from a subject (e.g., a human subject). In some embodiments, the specimen comprises an antibody or antisera.5.8 KITS

[0162] In one aspect, provided herein are kits comprising a container containing a nucleotide sequence of an NS genomic segment described herein (e.g., in Section 5.1, 8, or 9) or an NS genomic segment described herein (e.g., in Section 5.1, 8, or 9). In some embodiments, a kit comprises one or more containers (e.g., 1, 2, 3, 4, 5, or 6 containers) containing a nucleotide sequence of a PB 1 genomic segment, a nucleotide sequence of a PB2 genomic segment, a nucleotide sequence of a PA genomic segment, a nucleotide sequence of an NP genomic segment, a nucleotide sequence of a M genomic segment, and a nucleotide sequence of an NS genomic segment described herein (e.g., in Section 5.1, 8, or 9). In some embodiments, a kit comprises one or more containers (e.g., 1, 2, 3, 4, 5, or 6 containers)90NAI-5003686295vlcontaining a nucleotide sequence comprising a PB1 genomic segment, a nucleotide sequence comprising a PB2 genomic segment, a nucleotide sequence comprising a PA genomic segment, a nucleotide sequence comprising an NP genomic segment, a nucleotide sequence comprising a M genomic segment, and a nucleotide sequence comprising an NS genomic segment described herein (e.g., in Section 5.1, or 8). In some embodiments, the kit further comprises one or more containers (e.g., 1 or 2 containers) containing a nucleotide sequence of an HA genomic segment and a nucleotide sequence of an NA genomic segment. In some embodiments, the kit further comprises one or more containers e.g., 1 or 2 containers) containing a nucleotide sequence comprising an HA genomic segment and a nucleotide sequence comprising an NA genomic segment. In some embodiments, each container contains a different nucleotide sequence or genomic segment. In some embodiments, a container contains 1, 2, 3, 4, 5, 6 or more different nucleotide sequences or genomic segments.

[0163] In some embodiments, provided herein are kits comprising a container containing an NS nucleotide sequence described herein e.g., in Section 5.1, or 8). In some embodiments, a kit comprises one or more containers e.g., 1, 2, 3, 4, 5, or 6 containers) containing a nucleotide sequence of a PB 1 genomic segment, a nucleotide sequence of a PB2 genomic segment, a nucleotide sequence of a PA genomic segment, a nucleotide sequence of an NP genomic segment, a nucleotide sequence of a M genomic segment, and an NS nucleotide sequence described herein e.g., in Section 5.1, or 8). In some embodiments, a kit comprises one or more containers e.g., 1, 2, 3, 4, 5, or 6 containers) containing a nucleotide sequence comprising a PB1 genomic segment, a nucleotide sequence comprising a PB2 genomic segment, a nucleotide sequence comprising a PA genomic segment, a nucleotide sequence comprising an NP genomic segment, a nucleotide sequence comprising a M genomic segment, and NS nucleotide sequence described herein e.g., in Section 5.1, or 8). In some embodiments, the kit further comprises one or more containers e.g., 1 or 2 containers) containing a nucleotide sequence of an HA genomic segment and a nucleotide sequence of an NA genomic segment. In some embodiments, the kit further comprises one or more containers e.g., 1 or 2 containers) containing a nucleotide sequence comprising an HA genomic segment and a nucleotide sequence comprising an NA genomic segment. In some embodiments, each container contains a different nucleotide sequence or genomic segment. In some embodiments, a container contains 1, 2, 3, 4, 5, 6 or more different nucleotide sequences.91NAI-5003686295vl

[0164] In some embodiments, provided herein are kits comprising a container containing an NS construct such as in FIG. IB. In some embodiments, provides are kits comprising a container containing an NS construct such as in FIG. 1C.

[0165] In some embodiments, a kit comprises a container containing a vector comprising an NS nucleotide sequence described herein. In some embodiments, a kit comprises a container containing a vector comprising an NS genomic segment nucleotide sequence described herein. In some embodiments, a kit comprises a container containing a vector comprising NS genomic segment described in the Example below.

[0166] In some embodiments, a kit comprises one or more containers (e.g., 1, 2, 3, 4, 5, or 6 containers) containing: (1) a PB1 vector comprising a PB1 genomic segment nucleotide sequence, (2) a PB2 vector comprising a PB2 genomic segment nucleotide sequence, (3) a PA vector comprising a PA genomic segment nucleotide sequence, (4) an NP vector comprising an NP genomic segment nucleotide sequence, (5) an M vector comprising a M genomic segment nucleotide sequence, and (6) an NS vector comprising an NS genomic segment nucleotide sequence described herein (e.g., in Section 5.1, or 8). In some embodiments, the kit further comprises one or more containers (e.g., 1 or 2 containers) containing an HA vector comprising an HA genomic segment nucleotide sequence and an NA vector comprising an NA genomic segment nucleotide sequence. In some embodiments, the kit further comprises one or more containers (e.g., 1, 2, 3, or 4) containing one or more vectors (e.g., 1, 2, 3, or 4) comprising a nucleotide sequence encoding a PB1 protein, a nucleotide sequence encoding a PB2 protein, a nucleotide sequence encoding a PA protein, and a nucleotide sequence encoding a NP protein. In some embodiments, one vector comprises the nucleotide sequence encoding the Influenza A virus PB1 protein, the nucleotide sequence encoding the Influenza A virus PB2 protein, the nucleotide sequence encoding the Influenza A virus PA protein, and the nucleotide sequence encoding the Influenza A virus NP protein. In some embodiments, one vector comprises the nucleotide sequence encoding the Influenza A virus PB1 protein, a second vector the nucleotide sequence encoding the Influenza A virus PB2 protein, a third vector comprises the nucleotide sequence encoding the Influenza A virus PA protein, and a fourth vector comprises the nucleotide sequence encoding the Influenza A virus NP protein. In some embodiments, each container contains a different vector. In some embodiments, a container contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more different vectors. In some embodiments, the vectors comprising the genomic segment nucleotide sequences are in the same container. In some embodiments, PB1 vector, PB2 vector, PA vector, NP vector, M vector, and NS vector are in the same container.92NAI-5003686295vlIn some embodiments, the HA vector and NA vector are in the same container. In other embodiments, the HA vector and NA vector are in different containers.

[0167] In some embodiments, a kit comprises one or more containers (e.g., 1, 2, 3, 4, 5, or 6 containers) containing: (1) a PB1 vector comprising a PB1 genomic segment nucleotide sequence, (2) a PB2 vector comprising a PB2 genomic segment nucleotide sequence, (3) a PA vector comprising a PA genomic segment nucleotide sequence, (4) an NP vector comprising an NP genomic segment nucleotide sequence, (5) an M vector comprising a M genomic segment nucleotide sequence, and (6) an NS vector comprising an NS genomic segment nucleotide sequence described herein (e.g, in Section 5.1, or 8). In some embodiments, the kit further comprises one or more containers (e.g, 1 or 2 containers) containing an HA vector comprising an HA genomic segment nucleotide sequence and an NA vector comprising an NA genomic segment nucleotide sequence. In some embodiments, each container contains a different vector. In some embodiments, a container contains 1, 2, 3, 4, 5, 6, 7, 8, or more different vectors. In some embodiments, the vectors comprising the genomic segment nucleotide sequences are in the same container. In some embodiments, PB1 vector, PB2 vector, PA vector, NP vector, M vector, and NS vector are in the same container. In some embodiments, the HA vector and NA vector are in the same container. In other embodiments, the HA vector and NA vector are in different containers. In some embodiments, one or more vectors is an ambisense plasmid. In some embodiments, one or more vectors is an pDZ plasmid (see, e.g., Quinlivan et al., 2005, J. of Virology 79: 8431— 8439 for information relating to the pDZ plasmid). In some embodiments, one or more vectors is a pHW2000 plasmid (see, e.g., Hoffmann et al., 2000, Proc Natl Acad Sci U S A. 97(11): 6108- 13 for information relating to the pHW2000 plasmid). In some embodiments, one or more vectors is a pAD3000 plasmid (see, e.g., Hoffmann et al., 2000, Proc Natl Acad Sci U S A. 97(11) :6108-13 for information relating to the pAD3000 plasmid). In some embodiments, one or more vectors is a pAD4000 plasmid (see, e.g., Wang et al., 2007, J. of Virology 4: 102 for information relating to the pAD4000 plasmid).

[0168] In some embodiments, a kit described herein further comprises instructions for using the genomic segments, nucleotides, or vectors to produce a recombinant Influenza A virus.

[0169] In another aspect, provided herein are kits comprising a container containing a recombinant Influenza A virus described herein (e.g., Section 5.1, 8, or 9). In some embodiments, the kit further comprises instructions for using the virus.93NAI-5003686295vl

[0170] In another aspect, provided herein is a kit comprising a container containing a composition described herein. In a specific embodiment, the composition is pharmaceutical composition. In some embodiments, the composition is an immunogenic composition (e.g., vaccine composition). See, e.g., Section 5.6 for compositions, including immunogenic compositions.

[0171] In a specific embodiment, a kit comprises a container containing an NS genomic segment or NS genomic segment described in an Example below (e.g., in Section 8). In another specific embodiment, a kit comprises a container containing a recombinant Influenza A virus described in an Example.5.9 BIOLOGICAL ASSAYS

[0172] In a specific embodiment, one, two or more of the assays described in Section 8, may be used to characterize a recombinant influenza virus described herein, or genomic segment described herein. Techniques known to one of skill in the art may be used to detect a genomic segment or the gene products of a genomic segment of interest. For example, RT- PCR can be used with primers that are specific to a genomic segment to detect and quantify the genomic segment. Western blot, ELISA, radioimmunoassay, immunoprecipitation, immunocytochemistry, or immunocytochemistry in conjunction with FACS can be used to quantify the gene products of a genomic segment of interest.

[0173] Viral assays include those that measure viral replication (as determined, e.g., by plaque formation) or the production of viral proteins (as determined, e.g., by western blot analysis) or viral RNAs (as determined, e.g., by RT-PCR or northern blot analysis) in cultured cells in vitro using methods which are well known in the art.

[0174] Growth of a recombinant Influenza A virus described herein can be assessed by any method known in the art or described herein (e.g., in cell culture (e.g., cultures of chicken embryonic kidney cells or cultures of chicken embryonic fibroblasts (CEF)). Viral titer may be determined by inoculating serial dilutions of a recombinant Influenza A virus described herein into cell cultures (e.g., CEF, MDCK (e.g., MDCK-NS1 cells), EFK-2 cells, Vero cells, primary human umbilical vein endothelial cells (HUVEC), H292 human epithelial cell line or HeLa cells), chick embryos, or live animals (e.g., avians)). After incubation of the virus for a specified time, the virus is isolated using standard methods.

[0175] A hemagglutinin (HA) assay may be conducted. A hemagglutinin (HA) assay may be carried out in V-bottom 96-well plates. Serial twofold dilutions of each sample in PBS are incubated for 1 h on ice with an equal volume of a 0.5% suspension of chicken erythrocytes in PBS. Positive wells contain an adherent, homogeneous layer of erythrocytes; negative94NAI-5003686295vlwells contain a nonadherent pellet. Physical quantitation of the virus titer can be performed using PCR applied to viral supernatants (Quinn & Trevor, 1997; Morgan et al., 1990), hemagglutination assays, tissue culture infectious doses (TCID50) or egg infectious doses (EID50).

[0176] Antibodies generated or identified in accordance with the methods described herein may be characterized in a variety of ways well-known to one of skill in the art (e.g., ELISA, Surface Plasmon resonance display (BIAcore), Western blot, immunofluorescence, immunostaining and / or microneutralization assays). For example, antibodies generated or identified in accordance may be assayed for the ability to specifically bind to an antigen of the recombinant Influenza A virus or an antigen encoded by a recombinant Influenza A virus described herein. Such an assay may be performed in solution (e.g., Houghten, 1992, Bio / Techniques 13:412 421), on beads (Lam, 1991, Nature 354:82 84), on chips (Fodor, 1993, Nature 364:555 556), on bacteria (U.S. Patent No. 5,223,409), on spores (U.S. Patent Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89: 1865 1869) or on phage (Scott and Smith, 1990, Science 249:386 390; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378 6382; and Felici, 1991, J. Mol. Biol. 222:301 310) (each of these references is incorporated herein in its entirety by reference). Antibodies that specifically bind to an antigen of a recombinant Influenza A virus can then be assayed for their specificity to said antigen.

[0177] Antibodies generated or identified in accordance with the methods described herein may be assayed for specific binding to an antigen of a recombinant A virus described herein and cross-reactivity with other antigens by any method known in the art. Immunoassays which can be used to analyze immunospecific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety).

[0178] The binding affinity of an antibody to an antigen and the off-rate of an antibodyantigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled95NAI-5003686295vlantigen (e.g.,3H or125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody can be determined from the data by Scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, a recombinant Influenza A virus of described herein or an antigen thereof is incubated with an antibody against the antigen conjugated to a labeled compound (e.g.,3H or125I) in the presence of increasing amounts of an unlabeled second antibody.

[0179] BIAcore kinetic analysis can be used to determine the binding on and off rates of an antibody to an antigen of a recombinant influenza virus described herein. BIAcore kinetic analysis comprises analyzing the binding and dissociation of polypeptide comprising the antigen of interest from chips with immobilized antibodies generated or identified in accordance with methods described herein on their surface.

[0180] Antibodies generated or identified in accordance with the methods described herein can also be assayed for their ability to inhibit the binding of an antigen of a recombinant influenza virus to a cell using techniques known to those of skill in the art. For example, cells expressing receptors known to bind to influenza virus can be contacted with influenza virus in the presence or absence of an antibody generated or identified in accordance with the methods described herein and the ability of the antibody to inhibit the binding can be measured by, for example, flow cytometry or a scintillation assay. The antigen or the antibody can be labeled with a detectable compound such as a radioactive label (e.g.,32P,35S, and125I) or a fluorescent label (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine) to enable detection of an interaction between the influenza virus and a cell.

[0181] Antibodies described herein or compositions including such antibodies can be assessed in vitro for antiviral activity. In one embodiment, the antibodies or compositions including such antibodies are tested in vitro for their effect on growth of an influenza virus (e.g., Influenza A virus). Growth of influenza virus (e.g., Influenza A virus) can be assessed by any method known in the art or described herein (e.g., in cell culture). In a specific embodiment, cells are infected at a MOI of 0.0005 and 0.001, 0.001 and 0.01, 0.01 and 0.1, 0.1 and 1, or 1 and 10, or a MOI of 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 or 10 and incubated with serum free media supplemented. Viral titers are determined in the supernatant by hemagglutinin plaques, or any other viral assay described herein. Cells in which viral titers can be assessed include, but are not limited to, EFK-2 cells, Vero cells, primary human umbilical vein endothelial cells (HUVEC), H292 human epithelial cell line and HeLa cells.96NAI-5003686295vlIn vitro assays include those that measure altered viral replication (as determined, e.g., by plaque formation) or the production of viral proteins (as determined, e.g., by Western blot analysis) or viral RNAs (as determined, e.g., by RT-PCR or northern blot analysis) in cultured cells in vitro using methods which are well known in the art or described herein.

[0182] In one non-limiting example, a monolayer of the target mammalian cell line is infected with different amounts (e.g., multiplicity of 3 plaque forming units (pfu) or 5 pfu) of influenza and subsequently cultured in the presence or absence of various dilutions of antibodies (e.g., 0.1 pg / ml, 1 pg / ml, 5 pg / ml, or 10 pg / ml). Infected cultures are harvested 48 hours or 72 hours post infection and titered by standard plaque assays known in the art on the appropriate target cell line (e.g., Vero cells).

[0183] In a non-limiting example of a hemagglutination assay, cells are contacted with an antibody and are concurrently or subsequently infected with the influenza virus (e.g., at an MOI of 1) and the virus is incubated under conditions to permit virus replication (e.g., 20-24 hours). The antibodies are preferably present throughout the course of infection. Viral replication and release of viral particles is then determined by hemagglutination assays using 0.5% chicken red blood cells. See, e.g., Kashyap et al., PNAS USA 105: 5986-5991.

[0184] Many assays well-known in the art can be used to assess viability of cells (infected or uninfected) or cell lines following exposure to a recombinant Influenza A virus, an antibody described herein, or a composition described herein, and, thus, determine the cytotoxicity thereof. For example, cell proliferation can be assayed by measuring Bromodeoxyuridine (BrdU) incorporation (see, e.g., Hoshino et al., 1986, Int. J. Cancer 38, 369; Campana et al., 1988, J. Immunol. Meth. 107:79), (3H) thymidine incorporation (see, e.g., Chen, J., 1996, Oncogene 13: 1395-403; Jeoung, J., 1995, J. Biol. Chem. 270: 18367 73), by direct cell count, or by detecting changes in transcription, translation or activity of known genes such as proto-oncogenes (e.g., fos, myc, etc.) or cell cycle markers (e.g., Rb, cdc2, cyclin A, DI, D2, D3, E, etc). The levels of such protein and mRNA and activity can be determined by any method well known in the art. For example, protein can be quantitated by known immunodiagnostic methods such as ELISA, Western blotting or immunoprecipitation using antibodies, including commercially available antibodies. mRNA can be quantitated using methods that are well known and routine in the art, for example, using northern analysis, RNase protection, or polymerase chain reaction in connection with reverse transcription. Cell viability can be assessed by using trypan-blue staining or other cell death or viability markers known in the art. In a specific embodiment, the level of cellular ATP is measured to determined cell viability.97NAI-5003686295vl

[0185] In some embodiments, cell viability is measured in three-day and seven-day periods using an assay standard in the art, such as the CellTiter-Glo Assay Kit (Promega) which measures levels of intracellular ATP. A reduction in cellular ATP is indicative of a cytotoxic effect. In some embodiments, cell viability can be measured in the neutral red uptake assay. In some embodiments, visual observation of cells for morphological changes assessed and may include enlargement, granularity, cells with ragged edges, a filmy appearance, rounding, detachment from the surface of the well, or other changes. These changes are given a designation of T (100% toxic), PVH (partially toxic-very heavy-80%), PH (partially toxic- heavy-60%), P (partially toxic-40%), Ps (partially toxic-slight-20%), or 0 (no toxicity-0%), conforming to the degree of cytotoxicity seen. A 50% cell inhibitory (cytotoxic) concentration (ICso) is determined by regression analysis of these data.

[0186] In some embodiments, the cells used in the cytotoxicity assay are animal cells, including primary cells and cell lines. In some embodiments, the cells are human cells. In some embodiments, cytotoxicity is assessed in one or more of the following cell lines: U937, a human monocyte cell line; primary peripheral blood mononuclear cells (PBMC); Huh7, a human hepatoblastoma cell line; 293 T, a human embryonic kidney cell line; and THP-I, monocytic cells. In some embodiments, cytotoxicity is assessed in one or more of the following cell lines: MDCK (e.g., MDCK-NS1 cells), MEF, Huh 7.5, Detroit, or human tracheobronchial epithelial (HTBE) cells.

[0187] A recombinant Influenza A virus, an antibody described herein, or a composition described herein can be tested for in vivo toxicity in animal models. For example, animal models known in the art can also be used to determine the in vivo toxicity of to test the activities of a recombinant influenza virus, an antibody described herein, or a composition described herein. In addition, a recombinant Influenza A virus, an antibody described herein, or a composition described herein can be tested for efficacy in an animal model (e.g., a cancer model, such as described in Example 2. For example, animals are administered a range of concentrations of a recombinant Influenza A virus, an antibody described herein, or a composition described herein to test the activities of the recombinant Influenza A virus, the antibody, or the composition. Subsequently, the animals are monitored over time for lethality, weight loss or failure to gain weight, tumor size, and / or levels of serum markers that may be indicative of tissue damage (e.g., creatine phosphokinase level as an indicator of general tissue damage, level of glutamic oxalic acid transaminase or pyruvic acid transaminase as indicators for possible liver damage).98NAI-5003686295vl6. SEQUENCES

[0188] cDNA of Non-Coding Region (NCR; italics) with PR8 partial NS1 ORF (Start codon of NS1 ORF, i.e., ATG, changed to TTG), splicing donor motif (double underlined), and splicing donor site (bold) 5’-A GCAAAAGCA GGGTGA GA A A GAGA T^TTGGATCC A A AC ACTGTGTC A AGCTTTCAGGTAGATTG-3’ (SEQ ID NO: 1)

[0189] Influenza Virus A / Puerto Rico / 8 / 34 Segment 8 cDNA 5’-AGCAAAAGCAGGGTGACAAAGACATAATGGATCCAAACACTGTGTCAAGCTTTC AGGTAGATTGCTTTCTTTGGCATGTCCGCAAACGAGTTGCAGACCAAGAACTAGG TGATGCCCCATTCCTTGATCGGCTTCGCCGAGATCAGAAATCCCTAAGAGGAAGG GGCAGCACCCTCGGTCTGGACATCGAGACAGCCACACGTGCTGGAAAGCAGATA GTGGAGCGGATTCTGAAAGAAGAATCCGATGAGGCACTTAAAATGACCATGGCC TCTGTACCTGCGTCGCGTTACCTAACTGACATGACTCTTGAGGAAATGTCAAGGG ACTGGTCCATGCTCATACCCAAGCAGAAAGTGGCAGGCCCTCTTTGTATCAGAAT GGACCAGGCGATCATGGATAAGAACATCATACTGAAAGCGAACTTCAGTGTGAT TTTTGACCGGCTGGAGACTCTAATATTGCTAAGGGCTTTCACCGAAGAGGGAGCA ATTGTTGGCGAAATTTCACCATTGCCTTCTCTTCCAGGACATACTGCTGAGGATG TCAAAAATGCAGTTGGAGTCCTCATCGGGGGACTTGAATGGAATGATAACACAG TTCGAGTCTCTGAAACTCTACAGAGATTCGCTTGGAGAAGCAGTAATGAGAATG GGAGACCTCCACTCACTCCAAAACAGAAACGAGAAATGGCGGGAACAATTAGGT CAGAAGTTTGAAGAAATAAGATGGTTGATTGAAGAAGTGAGACACAAACTGAAG ATAACAGAGAATAGTTTTGAGCAAATAACATTTATGCAAGCCTTACATCTATTGC TTGAAGTGGAGCAAGAGATAAGAACTTTCTCGTTTCAGCTTATTTAATAATAAAA AACACCCTTGTTTCTACT-3’ (SEQ ID NO: 2)

[0190] cDNA of Influenza Virus A / Puerto Rico / 8 / 34 Modified Genomic Segment 8 Lacking NS1 That Encodes IL-25’-AGCAAAAGCAGGGTGACAAAGACATATTGGATCCAAACACTGTGTCAAGCTTTC AGGTAGATTGATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCA CTTGTCACAAACAGTGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAA CTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACA AGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGC99NAI-5003686295vlCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGA AGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATC AGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATG TGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTA CCTTTTGTCAAAGCATCATCTCAACACTGACTTAACTTCTCTTCCAGGAATGGATC CAAACACTGTGTCAAGCTTTCAGGACATACTGCTGAGGATGTCAAAAATGCAGTT GGAGTCCTCATCGGGGGACTTGAATGGAATGATAACACAGTTCGAGTCTCTGAA ACTCTACAGAGATTCGCTTGGAGAAGCAGTAATGAGAATGGGAGACCTCCACTC ACTCCAAAACAGAAACGAGAAATGGCGGGAACAATTAGGTCAGAAGTTTGAAG AAATAAGATGGTTGATTGAAGAAGTGAGACACAAACTGAAGATAACAGAGAAT AGTTTTGAGCAAATAACATTTATGCAAGCCTTACATCTATTGCTTGAAGTGGAGC AAGAGATAAGAACTTTCTCGTTTCAGCTTATTTAATAATAAAAAACACCCTTGTTTCTACT-3’ (SEQ ID NO: 3)

[0191] cDNA of 3’ NCR of Influenza Virus A / Puerto Rico / 8 / 34 segment 85’-AGCAAAAGCAGGGTGACAAAGACATA-3’ (SEQ ID NO: 4)

[0192] cDNA of Partial NS1 ORF of Influenza Virus A / Puerto Rico / 8 / 34 segment 8 including;- Change in start codon of partial NS1 ORF: ATG to TTG (underlined)- Splicing donor motif (double underlined)- Splicing donor site (bold)5 ’ -TTGGATCC A A AC ACTGTGTC A AGCTTTC AGGTAGATTG-3 ’ (SEQ ID NO: 5)

[0193] Human IL-2 cDNA (ORF within GenBank Accession No. NM_000586) (signal sequence is underlined)5’-ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAA ACAGTGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATT TACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAA ACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAAT TTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCA ACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAA AGCATCATCTCAACACTGACTTAA-3’ (SEQ ID NO: 6)100NAI-5003686295vl

[0194] cDNA of Splicing acceptor motif (double underlined), Splicing acceptor site (bold)5 ’ -CTTCTCTTCC AGGA-3 ’ (SEQ ID NO: 7)

[0195] cDNA of Complete NEP ORF of Influenza Virus A / Puerto Rico / 8 / 34 segment 8 5’-ATGGATCCAAACACTGTGTCAAGCTTTCAGGACATACTGCTGAGGATGTCAAAA ATGCAGTTGGAGTCCTCATCGGGGGACTTGAATGGAATGATAACACAGTTCGAG TCTCTGAAACTCTACAGAGATTCGCTTGGAGAAGCAGTAATGAGAATGGGAGAC CTCCACTCACTCCAAAACAGAAACGAGAAATGGCGGGAACAATTAGGTCAGAAG TTTGAAGAAATAAGATGGTTGATTGAAGAAGTGAGACACAAACTGAAGATAACAGAGAATAGTTTTGAGCAAATAACATTTATGCAAGCCTTACATCTATTGCTTGAAG TGGAGCAAGAGATAAGAACTTTCTCGTTTCAGCTTATTTAA - 3’ (SEQ ID NO: 8)

[0196] cDNA of 5’ NCR of Influenza Virus A / Puerto Rico / 8 / 34 segment 8 5’-TAATAAAAAACACCCTTGTTTCTACT-3’ (SEQ ID NO: 9)

[0197] Human IL-2 Amino Acid (signal sequence underlined)MYRMOLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLE LKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT* (SEQ ID NO: 10)

[0198] cDNA of Splicing Donor Motif 5’-CAGGTAGATTG-3’ (SEQ ID NO: 11)

[0199] Human IL-2 Signal SequenceMYRMQLLSCIALSLALVTNS (SEQ ID NO: 12)

[0200] cDNA of Human IL-2 Signal SequenceATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAA ACAGT (SEQ ID NO: 13)

[0201] Human IL-15 cDNA (ORF within NM 000585.5); signal sequence is underlinedATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTTGTGTT TACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCTTCATTTTGGGC TGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAACTGGGTGAATGTAATAAGT GATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTAT ATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCT CTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACA GTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAA101NAI-5003686295vlCAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTAA (SEQ ID NO: 14)

[0202] IL2ssIL-15: Human IL-15 cDNA with IL-2 signal sequence (signal sequence is underlined)ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTAA (SEQ ID NO: 15)

[0203] Human IL-15 Amino Acid Sequence (signal sequence for IL-15 is underlined)MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS* (SEQ ID NO: 16)

[0204] Human IL-15 Signal Sequence Amino Acid SequenceMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEA (SEQ ID NO: 17)

[0205] cDNA of Human IL-15 Signal SequenceATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTTGTGTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCC (SEQ ID NO: 18)

[0206] Human IL-2 Amino Acid Sequence without the signal sequenceAPTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 19)

[0207] Human IL-2 cDNA (ORF within GenBank Accession No. NM 000586) without the signal sequenceGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTA102NAI-5003686295vlATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATG AGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCAT CATCTCAACACTGACTTAA (SEQ ID NO: 20)

[0208] Human IL-15 Amino Acid Sequence without the signal sequenceNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS* (SEQ ID NO: 21)

[0209] Human IL-15 cDNA (ORF within NM 000585.5) without the signal sequenceAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTAA (SEQ ID NO: 22)

[0210] cDNA of Influenza Virus A / Puerto Rico / 8 / 34 modified genomic segment 8 lacking NS1 encoding IL-15AGCAAAAGCAGGGTGACAAAGACATATTGGATCCAAACACTGTGTCAAGCTTTCAGGTAGATTGATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTTGTGTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTAACTTCTCTTCCAGGAATGGATCCAAACACTGTGTCAAGCTTTCAGGACATACTGCTGAGGATGTCAAAAATGCAGTTGGAGTCCTCATCGGGGGACTTGAATGGAATGATAACACAGTTCGAGTCTCTGAAACTCTACAGAGATTCGCTTGGAGAAGCAGTAATGAGAATGGGAGACCTCCACTCACTCCAAAACAGAAACGAGAAATGGCGGG AACAATTAGGTCAGAAGTTTGAAGAAATAAGATGGTTGATTGAAGAAGTGAGAC ACAAACTGAAGATAACAGAGAATAGTTTTGAGCAAATAACATTTATGCAAGCCT103NAI-5003686295vlTACATCTATTGCTTGAAGTGGAGCAAGAGATAAGAACTTTCTCGTTTCAGCTTAT TTAATAATAAAAAACACCCTTGTTTCTACT (SEQ ID NO: 23)

[0211] cDNA of Influenza Virus A / Puerto Rico / 8 / 34 modified genomic segment 8 lacking NS1 encoding IL2ssIL-15AGCAAAAGCAGGGTGACAAAGACATATTGGATCCAAACACTGTGTCAAGCTTTC AGGTAGATTGATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCA CTTGTCACAAACAGTAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAA GATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCA CCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATT TCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCC TAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAG AATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTAC ATATTGTCCAAATGTTCATCAACACTTCTTAACTTCTCTTCCAGGAATGGATCCAA ACACTGTGTCAAGCTTTCAGGACATACTGCTGAGGATGTCAAAAATGCAGTTGG AGTCCTCATCGGGGGACTTGAATGGAATGATAACACAGTTCGAGTCTCTGAAACT CTACAGAGATTCGCTTGGAGAAGCAGTAATGAGAATGGGAGACCTCCACTCACT CCAAAACAGAAACGAGAAATGGCGGGAACAATTAGGTCAGAAGTTTGAAGAAA TAAGATGGTTGATTGAAGAAGTGAGACACAAACTGAAGATAACAGAGAATAGTT TTGAGCAAATAACATTTATGCAAGCCTTACATCTATTGCTTGAAGTGGAGCAAGA GATAAGAACTTTCTCGTTTCAGCTTATTTAATAATAAAAAACACCCTTGTTTCTAC T (SEQ ID NO: 24)

[0212] See Table 2, infra, for SEQ ID NOs: 25-38.7. EMBODIMENTS

[0213] Provided herein are exemplary embodiments.1. An NS nucleotide sequence comprising in 5’ to 3’ order: (a) a nucleotide sequence of a 3’ non-coding region (NCR) of segment 8 of an Influenza A virus, (b) a nucleotide sequence of a partial NS1 open reading frame with the ATG changed to TTG, (c) a nucleotide sequence of a splicing acceptor motif, (d) a nucleotide sequence of a nuclear export protein (NEP) open reading frame of segment 8 of an Influenza A virus, and (e) a nucleotide sequence of 5’ NCR of segment 8 of an Influenza A virus, wherein the partial NS1 open reading frame comprises a splicing donor motif.2. The NS nucleotide sequence of embodiment 1, wherein the nucleotide sequence of the splicing donor motif comprises the nucleotide sequence of SEQ ID NO: 11.104NAI-5003686295vl3. The NS nucleotide sequence of embodiment 1 or 2, wherein the nucleotide sequence of the splicing acceptor motif comprises the nucleotide sequence of SEQ ID NO: 7.4. The NS nucleotide sequence of any one of embodiments 1 to 3, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides 27 to 64 of segment 8 of an Influenza A virus.5. The NS nucleotide sequence of any one of embodiments 1 to 3, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides of an Influenza A virus segment 8 that correspond to nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934.6. The NS nucleotide sequence of any one of embodiments 1 to 3, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934.7. The NS nucleotide sequence of any one of embodiments 1 to 3, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotide sequence of SEQ ID NO: 5.8. The NS nucleotide sequence of any one of embodiments 1 to 7, wherein the NEP open reading frame comprises the NEP open reading frame of Influenza A virus A / Puerto Rico / 8 / 1934 NS1 open reading frame.9. The NS nucleotide sequence of embodiment 5, wherein the Influenza A virus segment 8 is the segment 8 of Influenza A virus A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northem shovel er / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / 10BM01929 / 10, A / Jiangxi-Donghu / 346- 1 / 2013 , A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14.10. The NS nucleotide sequence of any one of embodiments 1 to 3, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of Influenza A virus A / Califomia / 04 / 09, A / Vietnam / 1203 / 04, A / Hong Kong / 1 / 68, or A / PR / 8 / 34.11. The NS nucleotide sequence of any one of embodiments 1 to 3, 9, or 10, wherein the NEP open reading frame comprises the NEP open reading of Influenza A virus105NAI-5003686295vlA / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interi or Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14.12. The NS nucleotide sequence of any one of embodiments 1 to 11, wherein the nucleotide sequence of the NEP open reading frame comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 8.13. The NS nucleotide sequence of any one of embodiments 1 to 8, wherein the nucleotide sequence of the NEP open reading frame comprises the nucleotide sequence of SEQ ID NO: 8.14. The NS nucleotide sequence of any one of embodiments 1 to 13, which further comprises a transgene.15. The NS nucleotide sequence of embodiment 14, wherein the transgene comprises a nucleotide sequence encoding a biomolecule.16. The NS nucleotide sequence of embodiment 15, wherein the transgene is between the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG and the nucleotide sequence of the splicing acceptor motif.17. The NS nucleotide sequence of embodiment 15 or 16, wherein the biomolecule comprises a secretory protein.18. The NS nucleotide sequence of embodiment 15 or 16, wherein the biomolecule comprises a cytokine, a chemokine, or an antigen.19 The NS nucleotide sequence of any one of embodiments 1 to 18, which is cDNA.20. The NS nucleotide sequence of any one of embodiments 1 to 19, which is isolated.21. A vector comprising the NS nucleotide sequence of any one of embodiments 1 to 19.22. The vector of embodiment 21, which is a plasmid.23. An NS genomic segment comprising the corresponding negative-sense RNA sequence of the NS nucleotide sequence of any one of embodiments 1 to 14.24. An NS genomic segment comprising the corresponding negative-sense RNA sequence of the NS nucleotide sequence of any one of embodiments 15 to 18.25. A vector comprising the NS genomic segment of embodiment 23 or 24.106NAI-5003686295vl26. A recombinant Influenza A virus comprising the NS genomic segment of embodiment23.27. The recombinant Influenza A virus of embodiment 26, which comprises PB1, PB2, PA, NP, M, HA and NA genomic segments.28. The recombinant Influenza A virus of embodiment 27, wherein the HA and NA genomic segments are from a seasonal Influenza A virus.29. The recombinant Influenza A virus of embodiment 27, wherein the PB 1, PB2, PA, NP, M, HA and NA genomic segments are from the same Influenza A virus.30. The recombinant Influenza A virus of embodiment 27 or 28, wherein the PB1, PB2, PA, NP, and M genomic segments are from the same Influenza A virus.31. A recombinant Influenza A virus comprising the NS genomic segment of embodiment24.32. The recombinant Influenza A virus of embodiment 31, which comprises PB1, PB2, PA, NP, M, HA and NA genomic segments.33. The recombinant Influenza A virus of embodiment 32, wherein the HA and NA genomic segments are from a seasonal Influenza A virus.34. The recombinant Influenza A virus of embodiment 32, wherein the PB 1, PB2, PA, NP, M, HA and NA genomic segments are from the same Influenza A ...

Claims

What is claimed is:

1. An NS nucleotide sequence comprising in 5’ to 3’ order: (a) a nucleotide sequence of a 3’ non-coding region (NCR) of segment 8 of an Influenza A virus, (b) a nucleotide sequence of a partial NS1 open reading frame with the ATG changed to TTG, (c) a nucleotide sequence of a splicing acceptor motif, (d) a nucleotide sequence of a nuclear export protein (NEP) open reading frame of segment 8 of an Influenza A virus, and (e) a nucleotide sequence of 5’ NCR of segment 8 of an Influenza A virus, wherein the partial NS1 open reading frame comprises a splicing donor motif.

2. The NS nucleotide sequence of claim 1, wherein the nucleotide sequence of the splicing donor motif comprises the nucleotide sequence of SEQ ID NO: 11.

3. The NS nucleotide sequence of claim 1 or 2, wherein the nucleotide sequence of the splicing acceptor motif comprises the nucleotide sequence of SEQ ID NO: 7.

4. The NS nucleotide sequence of any one of claims 1 to 3, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides 27 to 64 of segment 8 of an Influenza A virus.

5. The NS nucleotide sequence of any one of claims 1 to 3, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides of an Influenza A virus segment 8 that correspond to nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934.

6. The NS nucleotide sequence of any one of claims 1 to 3, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934.

7. The NS nucleotide sequence of any one of claims 1 to 3, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotide sequence of SEQ ID NO: 5.

8. The NS nucleotide sequence of any one of claims 1 to 7, wherein the NEP open reading frame comprises the NEP open reading frame of Influenza A virus A / Puerto Rico / 8 / 1934 NS1 open reading frame.129NAI-5003686295vl9. The NS nucleotide sequence of claim 5, wherein the Influenza A virus segment 8 is the segment 8 of Influenza A virus A / Califomia / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14.

10. The NS nucleotide sequence of any one of claims 1 to 3, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, A / Hong Kong / 1 / 68, or A / PR / 8 / 34.

11. The NS nucleotide sequence of any one of claims 1 to 3, 9, or 10, wherein the NEP open reading frame comprises the NEP open reading of Influenza A virus A / Califomia / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14.

12. The NS nucleotide sequence of any one of claims 1 to 11, wherein the nucleotide sequence of the NEP open reading frame comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 8.

13. The NS nucleotide sequence of any one of claims 1 to 8, wherein the nucleotide sequence of the NEP open reading frame comprises the nucleotide sequence of SEQ ID NO: 8.

14. The NS nucleotide sequence of any one of claims 1 to 13, which further comprises a transgene.130NAI-5003686295vl15. The NS nucleotide sequence of claim 14, wherein the transgene comprises a nucleotide sequence encoding a biomolecule.

16. The NS nucleotide sequence of claim 15, wherein the transgene is between the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG and the nucleotide sequence of the splicing acceptor motif.

17. The NS nucleotide sequence of claim 15 or 16, wherein the biomolecule comprises a secretory protein.

18. The NS nucleotide sequence of claim 15 or 16, wherein the biomolecule comprises a cytokine, a chemokine, or an antigen.19 The NS nucleotide sequence of any one of claims 1 to 18, which is cDNA.

20. The NS nucleotide sequence of any one of claims 1 to 19, which is isolated.

21. A vector comprising the NS nucleotide sequence of any one of claims 1 to 19.

22. The vector of claim 21, which is a plasmid.

23. An NS genomic segment comprising the corresponding negative-sense RNA sequence of the NS nucleotide sequence of any one of claims 1 to 14.

24. An NS genomic segment comprising the corresponding negative-sense RNA sequence of the NS nucleotide sequence of any one of claims 15 to 18.

25. A vector comprising the NS genomic segment of claim 23 or 24.

26. A recombinant Influenza A virus comprising the NS genomic segment of claim 23.

27. The recombinant Influenza A virus of claim 26, which comprises PB1, PB2, PA, NP, M, HA and NA genomic segments.

28. The recombinant Influenza A virus of claim 27, wherein the HA and NA genomic segments are from a seasonal Influenza A virus.

29. The recombinant Influenza A virus of claim 27, wherein the PB1, PB2, PA, NP, M, HA and NA genomic segments are from the same Influenza A virus.131NAI-5003686295vl30. The recombinant Influenza A virus of claim 27 or 28, wherein the PB1, PB2, PA, NP, and M genomic segments are from the same Influenza A virus.

31. A recombinant Influenza A virus comprising the NS genomic segment of claim 24.

32. The recombinant Influenza A virus of claim 31, which comprises PB1, PB2, PA, NP, M, HA and NA genomic segments.

33. The recombinant Influenza A virus of claim 32, wherein the HA and NA genomic segments are from a seasonal Influenza A virus.

34. The recombinant Influenza A virus of claim 32, wherein the PB1, PB2, PA, NP, M, HA and NA genomic segments are from the same Influenza A virus.

35. The recombinant Influenza A virus of claim 32 or 33, wherein the PB1, PB2, PA, NP, and M genomic segments are from the same Influenza A virus.

36. A pharmaceutical composition comprising the recombinant Influenza A virus of any one of claims 26 to 30, and a pharmaceutically acceptable carrier.

37. A pharmaceutical composition comprising the recombinant Influenza A virus of any one of claims 31 to 35, and a pharmaceutically acceptable carrier.

38. A method for delivering a biomolecule to a cell(s), comprising contacting the cell(s) with the recombinant Influenza A virus of any one of claims 31 to 35, or the composition of claim 37.

39. The method of claim 38, wherein the cell(s) is a cell line.

40. The method of claim 38, wherein the cell(s) is in vitro or ex vivo.

41. A method for delivering a biomolecule to a subject, comprising administering to the subject the recombinant Influenza A virus of any one of claims 31 to 35, or the composition of claim 37.

42. A method of inducing an immune response to Influenza A virus in a subject, comprising administering to the subject the recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37.132NAI-5003686295vl43. A method of inducing an immune response to an antigen in a subject, comprising administering to the subject the recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37.

44. A method of immunizing a subject against Influenza A virus, comprising administering to the subject the recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37.

45. A method of immunizing a subject against an antigen in a subject, comprising administering to the subject the recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37.

46. A method for treating cancer in a subject in need thereof, comprising administering to the subject the recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37.

47. The method of claim 46, wherein the cancer is a thoracic cancer.

48. The method of claim 47, wherein the thoracic cancer is non-small lung cancer, or a carcinoma that has metastasized to the lungs.

49. The method of any one of claims 41 to 48, wherein the subject is human.

50. The recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37, for use in a method of inducing an immune response to Influenza A virus in a subject, wherein the method comprises administering to the subject the recombinant Influenza A virus or the composition.

51. The recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37, for use in a method of inducing an immune response to an antigen in a subject, wherein the method comprises administering to the subject the recombinant Influenza A virus or the composition.

52. The recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37, for use in a method of immunizing a subject against Influenza A virus, wherein the method comprises administering to the subject the recombinant Influenza A virus or the composition.133NAI-5003686295vl53. The recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37, for use in a method of immunizing a subject against an antigen in a subject, wherein the method comprises administering to the subject the recombinant Influenza A virus or the composition.

54. The recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37, for use in a method for treating cancer in a subject in need thereof, wherein the method comprises administering to the subject the recombinant Influenza A virus or the composition.

55. The recombinant Influenza A virus or the composition of claim 54, wherein the cancer is a thoracic cancer.

56. The recombinant Influenza A virus or the composition of claim 55, wherein the thoracic cancer is non-small lung cancer, or a carcinoma that has metastasized to the lungs.

57. The recombinant Influenza A virus or the composition of any one of claims 50 to 56, wherein the subject is a human.

58. Use of the recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37, in the preparation of a medicament for inducing an immune response to Influenza A virus.

59. Use of the recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37, in the preparation of a medicament for inducing an immune response to an antigen.

60. Use of the recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37, in the preparation of a medicament for immunizing a subject against Influenza A virus.

61. Use of the recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37, in the preparation of a medicament for immunizing a subject against an antigen.

62. Use of the recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37, in the preparation of a medicament for treating cancer.134NAI-5003686295vl63. The use according to claim 62, wherein the cancer is a thoracic cancer.

64. The use according to claim 63, wherein the thoracic cancer is non-small lung cancer, or a carcinoma that has metastasized to the lungs.

65. The use according to claim 60 or 61, wherein the subject is a human.

66. A kit comprising a container containing the NS nucleotide sequence of any one of claims 1 to 20.

67. The kit of claim 66, which further comprises one or more containers containing a nucleotide sequence of a PB1 genomic segment, a nucleotide sequence of a PB2 genomic segment, a nucleotide sequence of a PA genomic segment, a nucleotide sequence of an NP genomic segment, a nucleotide sequence of an M genomic segment, a nucleotide sequence of an HA genomic segment, and a nucleotide sequence of an NA genomic segment.

68. A kit comprising a container containing the NS genomic segment of claim 23 or 24.

69. The kit of claim 68, which further comprises one or more containers containing PB2, PA, NP, M, HA and NA genomic segments.

70. A kit comprising a container containing the vector of claim 21, 22, or 25.

71. The kit of claim 70, which further comprises one or more containers containing vectors comprising nucleotide sequences encoding PB2, PA, NP, M, HA and NA genomic segments.

72. The kit of claim 70 or 71, which further comprises one or more containers containing one or more vectors comprising a nucleotide sequence encoding an Influenza A virus PB1 protein, a nucleotide sequence encoding an Influenza A virus PB2 protein, a nucleotide sequence encoding an Influenza A virus PA protein, and a nucleotide sequence encoding an Influenza A virus NP protein.

73. A kit comprising the recombinant Influenza A virus of any one of claims 26 to 35, or the composition of claim 36 or 37.

74. An NS nucleotide sequence comprising in 5’ to 3’ order: (a) a nucleotide sequence of a 3’ non-coding region (NCR) of segment 8 of an Influenza A virus, (b) a nucleotide135NAI-5003686295vlsequence of a partial NS1 open reading frame with the ATG changed to TTG, (c) a transgene comprising a nucleotide sequence encoding interleukin (IL)-2 or IL-15, (d) a nucleotide sequence of a splicing acceptor motif, (e) a nucleotide sequence of a nuclear export protein (NEP) open reading frame of segment 8 of an Influenza A virus, and (f) a nucleotide sequence of 5’ NCR of segment 8 of an Influenza A virus, wherein the partial NS1 open reading frame comprises a splicing donor motif.

75. The NS nucleotide sequence of claim 74, wherein the nucleotide sequence of the splicing donor motif comprises the nucleotide sequence of SEQ ID NO: 11.

76. The nucleotide sequence of claim 74 or 75, wherein the nucleotide sequence of the splicing acceptor motif comprises the nucleotide sequence of SEQ ID NO: 7.

77. The NS nucleotide sequence of any one of claims 74 to 76, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises nucleotides 27 to 64 of segment 8 of an Influenza A virus.

78. The NS nucleotide sequence of any one of claims 74 to 76, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides of an Influenza A virus segment 8 that correspond to nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934.

79. The NS nucleotide sequence of any one of claims 74 to 76, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides 27 to 64 of segment 8 of the Influenza A virus A / Puerto Rico / 8 / 1934.

80. The NS nucleotide sequence of any one of claims 74 to 76, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotide sequence of SEQ ID NO: 5.

81. The NS nucleotide sequence of any one of claims 74 to 80, wherein the NEP open reading comprises the NEP open reading of Influenza A virus A / Puerto Rico / 8 / 1934 NS1 open reading frame.

82. The NS nucleotide sequence of claim 78, wherein the Influenza A virus segment 8 is the segment 8 of Influenza A virus A / Califomia / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea136NAI-5003686295vlfowl / Hong Kong / WF10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interior Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14.

83. The NS nucleotide sequence of any one of claims 74 to 76, wherein the nucleotide sequence of the partial NS1 open reading frame with the ATG changed to TTG comprises the nucleotides corresponding to nucleotides 27 to 64 of segment 8 of Influenza A virus A / California / 04 / 09, A / Vietnam / 1203 / 04, A / Hong Kong / 1 / 68, or A / PR / 8 / 34.

84. The NS nucleotide sequence of any one of claims 74 to 76, 82, or 83, wherein the NEP open reading frame comprises the NEP open reading frame of Influenza A virus A / California / 04 / 09, A / Singapore / 1 / 57, A / duck / Czech / 56, A / Vietnam / 1203 / 04, A / mallard / Sweden / 81 / 02, A / mallard / Sweden / 24 / 02, A / guinea fowl / Hong Kong / WF 10 / 99, A / Northern shoveler / Netherlands / 18 / 99, A / mallard / Interi or Alaska / 7MP0167 / 07, A / black headed gull / Sweden / 1 / 99, A / black headed gull / Sweden / 5 / 99, A / Hong Kong / 1 / 68, A / mallard / Alberta / 24 / 01, A / Anhui / 1 / 13, A / mallard / Interior Alaska / IOBMO 1929 / 10, A / Jiangxi-Donghu / 346-1 / 2013, A / mallard / Gurjev / 263 / 82, A / wedge tailed shearwater / Western Australia / 2576 / 79, A / Perth / 16 / 09, A / PR / 8 / 34, or A / HK / 4801 / 14.

85. The NS nucleotide sequence of any one of claims 74 to 84, wherein the nucleotide sequence of the NEP open reading frame comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 8.

86. The NS nucleotide sequence of any one of claims 74 to 81, wherein the nucleotide sequence of the NEP open reading frame comprises the nucleotide sequence of SEQ ID NO: 8.

87. The NS nucleotide sequence of any one of claims 74 to 86, wherein the transgene comprises a nucleotide sequence encoding IL-2.

88. The NS nucleotide sequence of claim 87, wherein the IL-2 comprises the amino acid sequence of SEQ ID NO: 19.137NAI-5003686295vl89. The NS nucleotide sequence of claim 87, wherein the IL-2 comprises the amino acid sequence of SEQ ID NO: 10.

90. The NS nucleotide sequence of claim 87, wherein the nucleotide sequence encoding IL-2 comprises the nucleotide sequence of SEQ ID NO: 20.

91. The NS nucleotide sequence of claim 87, wherein the nucleotide sequence encoding IL-2 comprises the nucleotide sequence of SEQ ID NO: 6.

92. The NS nucleotide sequence of any one of claims 74 to 86, wherein the transgene comprises a nucleotide sequence encoding IL-15.

93. The NS nucleotide sequence of claim 92, wherein the IL-15 comprises the amino acid sequence of SEQ ID NO: 21.

94. The NS nucleotide sequence of claim 92, wherein the IL-15 comprises the amino acid sequence of SEQ ID NO: 16.

95. The NS nucleotide sequence of claim 92, wherein the IL-15 comprises the amino acid sequence of SEQ ID NO: 21, and wherein the IL-15 is fused to the amino acid sequence of signal peptide of IL-2.

96. The NS nucleotide sequence of claim 95, wherein the signal peptide of IL-2 comprises the amino acid sequence of SEQ ID NO: 12.

97. The NS nucleotide sequence of claim 92, wherein the nucleotide sequence encoding IL-15 comprises the nucleotide sequence of SEQ ID NO: 22.

98. The NS nucleotide sequence of claim 92, wherein the nucleotide sequence encoding IL-15 comprises the nucleotide sequence of SEQ ID NO: 14 or 15.

99. The NS nucleotide sequence of any one of claims 74 to 98, which is cDNA.

100. The NS nucleotide sequence of any one of claims 74 to 99, which is isolated.

101. A vector comprising the nucleotide sequence of any one of claims 74 to 100.

102. The vector of claim 101, which is a plasmid.138NAI-5003686295vl103. An NS genomic segment comprising the corresponding negative-sense RNA sequence of the NS nucleotide sequence of any one of claims 74 to 99.

104. A vector comprising the NS genomic segment of claim 103.

105. A recombinant Influenza A virus comprising the NS genomic segment of claim 103.

106. The recombinant Influenza A virus of claim 105, which comprises PB1, PB2, PA, NP, M, HA and NA genomic segments.

107. The recombinant Influenza A virus of claim 106, wherein the HA and NA genomic segments are from a seasonal Influenza A virus.

108. The recombinant Influenza A virus of claim 106, wherein the PB1, PB2, PA, NP, M, HA and NA genomic segments are from the same Influenza A virus.

109. The recombinant Influenza A virus of claim 106 or 107, wherein the PB1, PB2, PA, NP, and M genomic segments are from the same Influenza A virus.

110. A pharmaceutical composition comprising the recombinant Influenza A virus of any one of claims 105 to 109, and a pharmaceutically acceptable carrier.

111. A method for delivering IL-2 or IL-15 to a cell(s), comprising contacting the cell(s) with the recombinant Influenza A virus of any one of claims 105 to 109, or the composition of claim 110.

112. The method of claim 111, wherein the cell(s) is a cell line.

113. The method of claim 111, wherein the cell(s) is in vitro or ex vivo.

114. A method for delivering IL-2 or IL- 15 to a subject, comprising administering to the subject the recombinant Influenza A virus of any one of claims 105 to 109, or the composition of claim 110.

115. A method for treating cancer in a subject in need thereof, comprising administering to the subject the recombinant Influenza A virus of any one of claims 105 to 109, or the composition of claim 110.

116. The method of claim 115, wherein the cancer is a thoracic cancer.139NAI-5003686295vl117. The method of claim 116, wherein the thoracic cancer is non-small lung cancer, or a carcinoma that has metastasized to the lungs.

118. The method of any one of claims 114 to 117, wherein the subject is human.

119. The recombinant Influenza A virus of any one of claims 105 to 109, or the composition of claim 110, for use in a method for delivering IL-2 or IL- 15 to a subject, wherein the method comprises administering to the subject the recombinant Influenza A virus or the composition.

120. The recombinant Influenza A virus of any one of claims 105 to 109, or the composition of claim 110, for use in a method for treating cancer in a subject in need thereof, wherein the method comprises administering to the subject the recombinant Influenza A virus or the composition.

121. The recombinant Influenza A virus or the composition for use of claim 120, wherein the cancer is a thoracic cancer.

122. The recombinant Influenza A virus or the composition for use of claim 121, wherein the thoracic cancer is non-small lung cancer, or a carcinoma that has metastasized to the lungs.

123. The recombinant Influenza A virus or the composition for use of any one of claims 119 to 122, wherein the subject is a human.

124. Use of the recombinant Influenza A virus of any one of claims 105 to 109, or the composition of claim 110, in the preparation of a medicament for delivering IL-2 or IL- 15 to a subject.

125. Use of the recombinant Influenza A virus of any one of claims 105 to 109, or the composition of claim 110, in the preparation of a medicament for treating cancer.

126. The use according to claim 125, wherein the cancer is a thoracic cancer.

127. The use according to claim 126, wherein the thoracic cancer is non-small lung cancer, or a carcinoma that has metastasized to the lungs.

128. The use according to claim 124 , wherein the subject is a human.140NAI-5003686295vl129. A kit comprising a container containing the NS nucleotide sequence of any one of claims 74 to 100.

130. The kit of claim 129, which further comprises one or more containers containing a nucleotide sequence of a PB1 genomic segment, a nucleotide sequence of a PB2 genomic segment, a nucleotide sequence of a PA genomic segment, a nucleotide sequence of an NP genomic segment, a nucleotide sequence of an M genomic segment, a nucleotide sequence of an HA genomic segment, and a nucleotide sequence of an NA genomic segment.

131. A kit comprising a container containing the NS genomic segment of claim 103.

132. The kit of claim 131, which further comprises one or more containers containing PB2, PA, NP, M, HA and NA genomic segments.

133. A kit comprising a container containing the vector of claim 101, 102, or 104.

134. The kit of claim 133, which further comprises one or more containers containing vectors comprising nucleotide sequences encoding PB2, PA, NP, M, HA and NA genomic segments.

135. The kit of claim 133 or 134, which further comprises one or more containers containing one or more vectors comprising a nucleotide sequence encoding an Influenza A virus PB 1 protein, a nucleotide sequence encoding an Influenza A virus PB2 protein, a nucleotide sequence encoding an Influenza A virus PA protein, and a nucleotide sequence encoding an Influenza A virus NP protein.

136. A kit comprising a container containing the recombinant Influenza A virus of any one of claims 105 to 109, or the composition of claim 110.141NAI-5003686295vl

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