Influenza virus mutants and uses thereof

Abstract

To provide mutant viruses not requiring a concern about reversion.SOLUTION: Disclosed herein are compositions and methods related to mutant viruses, and in particular, mutant influenza viruses. The mutant viruses disclosed herein include a mutant M2 sequence, and are useful in immunogenic compositions, e.g., as vaccines. Also disclosed herein are methods, compositions and cells for propagating the viral mutants, and methods, devices and compositions related to vaccination.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 61 / 501,034, filed Jun. 24, 2011, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Influenza is the leading cause of death among American adults. Each year, influenza causes approximately 36,000 deaths and over 200,000 hospitalizations. Influenza is an extremely contagious disease that is transmitted by coughing, sneezing, and direct physical contact with virus-carrying objects such as doorknobs and telephones. Symptoms of influenza include fever, extreme fatigue, headache, chills, and generalized aches and pains, and approximately 50% of infected individuals are asymptomatic but contagious. Immunization is 70-95% effective in preventing influenza in healthy individuals under 65 years of age when the antigenicity of the circulating virus strain matches that of the vaccine. Summary of the Invention [Problem to be solved by the invention]

[0003] Vaccination is the primary method of influenza prevention, and both live attenuated and inactivated (killed) virus vaccines are available. Live virus vaccines, typically administered intranasally, can activate all stages of the immune system and stimulate immune responses to multiple viral antigens. Thus, the use of live viruses eliminates the problem of destruction of viral antigens that can occur during the preparation of inactivated virus vaccines. In addition, the immunity generated by live virus vaccines is generally more durable, effective, and cross-reactive than that induced by inactivated vaccines, and live virus vaccines are less expensive to produce than inactivated virus vaccines. However, mutations of attenuated viruses are often unclear, and reversion is a concern. [Means for solving the problem]

[0004] In one aspect, the disclosure provides a nucleic acid sequence comprising SEQ ID NO:1.

[0005] In one aspect, the disclosure provides a nucleic acid sequence comprising SEQ ID NO:2.

[0006] In one aspect, the disclosure provides a nucleic acid sequence comprising SEQ ID NO:3.

[0007] In one aspect, the disclosure provides a composition comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 operably linked to (i) a promoter and (ii) a transcription termination sequence.

[0008] In one aspect, the present disclosure provides a recombinant influenza virus comprising a mutation in the M gene. In some embodiments, the recombinant influenza virus comprises SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the mutation in the M gene renders the virus unable to express the M2 protein or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:4. In some embodiments, the mutation in the M gene does not revert to a wild-type or non-wild-type sequence encoding a functional M2 protein during at least 10 passages in an in vitro host cell system. In some embodiments, the virus is an influenza A virus. In some embodiments, the virus is non-pathogenic in a mammal infected with the virus. In some embodiments, the in vitro cell system comprises Chinese hamster ovary cells. In some embodiments, the in vitro cell system comprises Vero cells.

[0009] In one aspect, the present disclosure provides a cell comprising a recombinant influenza virus according to any one of claims 5 to 10. In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo.

[0010] In one aspect, the disclosure provides a composition comprising a recombinant influenza virus comprising a mutation in the M gene. In some embodiments, the composition comprises SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the mutation in the M gene renders the virus unable to express the M2 protein or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:4. In some embodiments, the virus is an influenza A virus. In some embodiments, the composition is non-pathogenic to the mammal to which the composition is administered. In some embodiments, the composition elicits a detectable immune response in the mammal within about 3 weeks after administration of the composition to the mammal.

[0011] In one aspect, the disclosure provides a vaccine comprising a recombinant influenza virus comprising a mutation in the M gene. In some embodiments, the vaccine comprises SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the mutation in the M gene renders the virus unable to express the M2 protein or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:4. In some embodiments, the virus is an influenza A virus. In some embodiments, the vaccine is non-pathogenic to the mammal to which the vaccine is administered. In some embodiments, the vaccine elicits a detectable immune response in the mammal within about 3 weeks after administration of the vaccine to the mammal. In some embodiments, the vaccine comprises at least two different influenza virus strains in addition to the recombinant virus. In some embodiments, the vaccine comprises at least one influenza B virus or influenza B virus antigen. In some embodiments, the vaccine comprises at least one influenza C virus or influenza C virus antigen. In some embodiments, the vaccine comprises one or more viruses or viral antigens, including human influenza A virus and pandemic influenza virus from a non-human species. In some embodiments, the vaccine comprises a human influenza A virus selected from the group including H1N1, H2N2, and H3N2.

[0012] In one aspect, the disclosure provides a method of propagating a recombinant influenza virus, comprising contacting a host cell with recombinant influenza virus SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, incubating the host cell under conditions suitable for viral replication for a sufficient time, and isolating progeny viral particles.

[0013] In one aspect, the disclosure provides a method of preparing a vaccine comprising placing host cells in a bioreactor, contacting the host cells with recombinant virus SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, incubating the host cells under conditions suitable for virus growth for a sufficient time, isolating the progeny virus particles, and formulating the progeny virus particles for administration as a vaccine.

[0014] In one aspect, the disclosure provides a method of immunizing a subject comprising administering a composition comprising a recombinant influenza virus comprising a mutation in the M gene that renders the virus incapable of expressing the M2 protein or that causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:4.

[0015] In one aspect, the disclosure provides a method of reducing the likelihood or severity of infection with influenza A virus in a subject, comprising administering a composition comprising a recombinant influenza virus comprising a mutation in the M gene, wherein the mutation in the M gene renders the virus unable to express M2 protein or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:4. In some embodiments, the recombinant influenza virus comprises SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. In some embodiments, the method comprises providing at least one booster dose of the composition, wherein the at least one booster dose is provided three weeks after the initial administration. In some embodiments, the method comprises administering the composition intranasally, intramuscularly or intradermally. In some embodiments, the method comprises administering intradermally. In some embodiments, the method comprises administering using a microneedle delivery device.

[0016] In one aspect, the disclosure provides a method of intradermal administration of an immunogenic composition comprising: (a) providing a microneedle delivery device comprising (i) a piercing mechanism and (ii) an immunogenic composition layer comprising a plurality of microneedles capable of piercing the skin, the immunogenic composition being capable of being administered intradermally; and (b) depressing the piercing mechanism, the immunogenic composition comprising a recombinant influenza virus comprising a mutation in the M gene that renders the virus unable to express M2 protein or that causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO: 4. In some embodiments, the recombinant influenza virus comprises SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the microneedle array is initially disposed within the device housing, and the lever actuation causes the microneedles to penetrate the bottom of the device and insert into the skin, thereby injecting the vaccine liquid into the skin.

[0017] In one aspect, the disclosure provides a recombinant influenza virus comprising a mutation in the M gene, wherein the virus does not replicate in a non-modified host cell selected from the group consisting of Chinese hamster ovary (CHO) cells, Vero cells, or Madin-Darby canine kidney cells. In some embodiments, the mutation in the M gene renders the virus incapable of expressing an M2 protein or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:4.

[0018] In one aspect, the disclosure provides a recombinant cell comprising a nucleic acid encoding an influenza virus M2 ion channel gene, wherein the nucleic acid is expressed in the cell.

[0019] In one aspect, the disclosure provides a recombinant cell comprising a 2,6-sialic acid receptor gene.

[0020] In one aspect, the disclosure provides a recombinant cell comprising an intracellular genome or expression vector expressing (i) a viral M2 ion channel gene, and (ii) a 2,6-sialic acid receptor gene. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a Chinese Hamster Ovary cell or a Vero cell. In some embodiments, the recombinant cell further comprises a human influenza virus that does not express a functional M2 protein.

[0021] In one aspect, the disclosure provides a method of producing recombinant influenza virus particles, comprising: (A) infecting a cell according to any one of claims 47-52, the cell (i) constitutively expressing a functional M2 ion channel protein or (ii) being induced after viral infection to express a functional M2 ion channel protein and successfully replicating only in the presence of the functional M2 ion channel protein expressed by the cell, with a human influenza virus particle; and (B) isolating the progeny virus particles. In some embodiments, the method further comprises incorporating the isolated virus particles into a vaccine. In some embodiments, the virus comprises a human influenza virus, and the virus does not express a functional M2 protein. [Brief description of the drawings]

[0022] [Figure 1] Diagram showing the role of M2 ion channels in the influenza virus life cycle: (1) influenza virus attaches to sialic acid receptors on the cell surface, (2) the virus is internalized into the cell, (3) M2 ion channels are expressed on the viral surface, (4) the M2 ion channels open to allow protons to enter and release viral RNA, which enters the nucleus and is replicated, synthesizing viral proteins, and (5) viral components are packaged into virions and released. [Diagram 2]Schematic diagram of wild-type and mutant M2 genes. The M2 gene was deleted in the A / Puerto Rico / 8 / 1934 (PR8) M segment by inserting two stop codons downstream of the open reading frame of the M1 protein followed by a deletion of 51 nucleotides within the transmembrane domain to prevent expression of the full-length M2 protein. [Diagram 3] The nucleotide sequences of unprocessed M1 and M2 are shown. [Figure 4] Figure 1 shows the growth kinetics of M2KO(ΔTM) (upper panel) and wild-type PR8 (lower panel) viruses in normal MDCK cells and MDCK cells stably expressing the M2 protein (M2CK). Cells were infected with viruses at a multiplicity of infection of 10-5. Viral titers were measured in cell supernatants. Wild-type PR8 grew to high titers in both cell types, whereas M2KO(ΔTM) grew well only in M2CK cells and did not grow at all in MDCK cells. [Diagram 5] Western blots showing that M2KO(ΔTM) virus produces viral antigens but not M2 in normal cells. Cell lysates were probed with PR8-infected mouse serum (Panel A) or anti-M2 monoclonal antibody (Panel B). Lane 1: molecular weight markers, lane 2: MDCK cells infected with PR8, lane 3: MDCK cells infected with M2KO(ΔTM), lane 4: uninfected MDCK cells. [Figure 6] 1 is a graph showing the weight change of mice after inoculation with the M2KO mutant. [Figure 7A] Graph showing antibody responses in mice inoculated with the M2KO mutant. [Figure 7B] 1 is a graph showing anti-PR8 IgG antibody titers in the serum of boosted mice 6 weeks after infection. [Figure 8] 13 is a graph showing the weight change of mice after influenza challenge following inoculation with the M2KO mutant. [Figure 9] FIG. 13 is a graph showing survival of mice following influenza challenge after inoculation with the M2KO mutant. [Figure 10]Graph showing body weight changes in mice following intranasal (IN), intradermal (ID) or intramuscular (IM) inoculation with PR8. [Figure 11A] This is a graph showing antibody titers in mouse serum collected two weeks after inoculation of PR8 at a virus concentration of 1.8 x 101 pfu (Lo) or 1.8 x 104 pfu (Hi). [Figure 11B] 1 is a graph showing antibody titers in mouse serum collected 7 weeks after inoculation with PR8 at a vaccine concentration of 1.8×10 1 pfu (Lo) or 1.8×10 4 pfu (Hi). [Figure 12] 1 is a graph showing survival of mice after influenza challenge following inoculation with PR8. [Figure 13] 1 is a graph showing changes in mouse body weight after influenza challenge following PR8 inoculation. [Figure 14] 1 is a graph showing antibody titers in serum collected from mice intradermally inoculated with 1.8×10 4 pfu of PR8 7 weeks after inoculation. [Figure 15] 1 is a graph showing changes in body weight of mice intradermally inoculated with 1.8×10 4 pfu of PR8. [Figure 16] FIG. 1 is a graph showing % survival after challenge of mice infected with heterosubtypic viruses. [Figure 17] FIG. 1 is a graph showing ELISA titers of mice from different vaccination groups. [Figure 18] Graph showing % survival of mice following homosubtypic virus infection. [Figure 19] FIG. 1 is a graph showing % survival of mice following heterosubtypic virus challenge. [Figure 20] Figure 1 shows the weight change in inoculated ferrets. Ferrets were inoculated with 107TCID50 of M2KO(ΔTM) virus (Panel A) or 107TCID50 of A / Brisbane / 10 / 2007(H3N2) influenza A virus (Panel B). Weight was monitored for 3 days after inoculation. [Figure 21]Figure 1 shows the temperature profile of inoculated ferrets. Ferrets were inoculated with 107TCID50 of M2KO(ΔTM) virus (Panel A) or 107TCID50 of A / Brisbane / 10 / 2007(H3N2) influenza A virus (Panel B). Temperature was monitored for 3 days after inoculation. [Figure 22] Graph showing weight changes in ferrets after vaccination. Ferrets were inoculated with 107TCID50 of M2KO(ΔTM) virus [G1 and G3], 107TCID50 of FM#6 virus [G2 and G4], or OPTI-MEM™ [G5]. Weight changes were monitored for 14 days after prime vaccination (Panel A) and after receiving boost vaccination (Panel B). [Figure 23] Figure 1 is a graph showing weight changes in ferrets after challenge. Ferrets were challenged with 107 TCID50 of A / Brisbane / 10 / 2007 (H3N2) influenza A virus. Weight was monitored for 14 days after inoculation. [Figure 24] Graph showing temperature fluctuations of ferrets after vaccination. Ferrets were inoculated with 107TCID50 of M2KO(ΔTM) virus [G1 and G3], 107TCID50 of FM#6 virus [G2 and G4], or OPTI-MEM™ [G5]. Temperature fluctuations were monitored for 14 days after prime vaccination (Panel A) and after receiving boost vaccination (Panel B). [Diagram 25] Figure 1 is a graph showing the temperature profile of ferrets after challenge. Ferrets were challenged with 107 TCID50 of A / Brisbane / 10 / 2007 (H3N2) influenza A virus. Temperatures were monitored for 14 days after challenge. [Figure 26]Graph showing weight fluctuations in ferrets after virus inoculation. Donor ferrets were inoculated with 107TCID50 of M2KO(ΔTM) virus (panel A) or 107TCID50 of A / Brisbane / 10 / 2007(H3N2) virus (panel B) on day 0. 24 hours after inoculation (day 1), donors were placed in a cage with a direct contactor (DC) adjacent to a cage housing an aerosol contactor (AC). Weight fluctuations were monitored for 14 days after donor inoculation. [Figure 27] Graph showing ferret body temperature fluctuations after virus inoculation. Donor ferrets were inoculated with 107TCID50 of M2KO(ΔTM) virus (panel A) or 107TCID50 of A / Brisbane / 10 / 2007(H3N2) virus (panel B) on day 0. 24 hours after inoculation (day 1), donors were placed in a cage with a direct contactor (DC) adjacent to a cage housing an aerosol contactor (AC). Body temperature fluctuations were monitored for 14 days after donor inoculation. [Figure 28] Graphs showing that M2KO(ΔTM) vaccine induces humoral and mucosal responses. Panel A shows serum IgG and IgA titers after administration of PR8, M2KO(ΔTM), inactivated PR8 (IN, IM), or PBS. Panel B shows lung lavage IgG and IgA titers after administration of PR8, M2KO(ΔTM), inactivated PR8 (IN, IM), or PBS. [Figure 29] Graphs showing that M2KO(ΔTM) vaccine protects mice from lethal homosubtypic and heterosubtypic virus challenge. Panel A shows mouse weight changes after homologous PR8 (H1N1) challenge. Panel B shows mouse survival after heterologous Aichi (H3N2) challenge. [Diagram 30] Graphs showing that M2KO(ΔTM) controls challenge virus replication in the airways. Panel A shows viral titers after PR8(H1N1) challenge. Panel B shows viral titers after Aichi(H3N2) challenge. [Diagram 31]1 is a graph showing the kinetics of antibody responses to the M2KO(ΔTM) vaccine in serum. [Diagram 32] Graph showing mucosal antibody responses to M2KO(ΔTM) vaccine in serum and airway. [Diagram 33] FIG. 13 is a graph showing the kinetics of anti-HA IgG in mice in response to M2KO(ΔTM) vaccine. [Diagram 34] Graphs showing that M2KO(ΔTM) vaccine induces immune responses similar to FluMist® and IVR-147. Panel A shows serum viral titers in animals administered FluMist® H3, M2KO(ΔTM) H3, IVR-147, and PBS. Panel B shows lung lavage viral titers in animals administered FluMist® H3, M2KO(ΔTM) H3, IVR-147, and PBS. Panel C shows nasal turbinate viral titers in animals administered FluMist® H3, M2KO(ΔTM) H3, IVR-147, and PBS. [Diagram 35] Graphs showing that M2KO(ΔTM) vaccine protects against Aichi challenge. Panel A shows weight loss after Aichi challenge in animals administered FluMist® H3, M2KO(ΔTM) H3, IVR-147, and PBS. Panel B shows % survival after Aichi challenge in animals administered FluMist® H3, M2KO(ΔTM) H3, IVR-147, and PBS. [Diagram 36] 1 is a graph showing that the H5N1 M2KO(ΔTM) vaccine induces IgG antibody titers against HA. [Figure 37] Graph showing body weight following administration of M2KO(ΔTM) CA07, WT CA07, and FluMist® CA07 vaccines. [Figure 38] 1 is a graph showing that M2KO(ΔTM) virus does not replicate in the respiratory tract of mice. [Figure 39]13 is a graph showing that the M2KO(ΔTM) vaccine exhibits rapid antibody kinetics. [Diagram 40] 1 is a graph showing that the M2KO(ΔTM) vaccine protects against heterologous challenge with H3N2 virus, A / Aichi / 2 / 1968. [Diagram 41] 13 is a graph showing that M2KO(ΔTM) vaccine stimulates recalled cellular responses upon challenge. [Diagram 42] 1 is a graph showing that M2KO(ΔTM) virus produces similar mRNA levels as wild-type M2 virus. [Diagram 43] Agarose gel showing restriction digests of pCMV-PR8-M2 expression plasmid: Lanes 1 and 5: 1 Kb DNA Ladder (Promega, Madison, WI, USA), Lanes 2-4: pCMLV-PR8-M2 digested with Eco R1: 0.375 μg (lane 2), 0.75 μg (lane 3), and 1.5 μg (lane 4). [Figure 44A] 1 is a graph showing the sequence alignment of pCMLV-PR8-M2 to the open reading frame of the influenza M2 gene. [Figure 44B] 1 is a graph showing the sequence alignment of pCMLV-PR8-M2 to the open reading frame of the influenza M2 gene. [Figure 44C] 1 is a graph showing the sequence alignment of pCMLV-PR8-M2 to the open reading frame of the influenza M2 gene. [Fig.44D] 1 is a graph showing the sequence alignment of pCMLV-PR8-M2 to the open reading frame of the influenza M2 gene. [Diagram 45] 1 is a graph showing M2KO(ΔTM) and FluMist® virus replication in the respiratory tract of ferrets. [Diagram 46]Graph showing M2KO(ΔTM) and FluMist® viral titers in nasal washes following nasal challenge with A / Brisbane / 10 / 2007 (H3N2) virus. [Figure 47] 1 is a graph showing IgG titers in ferrets following vaccination with M2KO(ΔTM) and FluMist® prime only groups. [Figure 48] 1 is a graph showing IgG titers in ferrets following vaccination with M2KO(ΔTM) and FluMist® prime-boost groups. [Figure 49] Graph showing a summary of ELISA IgG titers in ferret sera from vaccination to post-challenge with M2KO(ΔTM) or FluMist®. [Figure 50] 1 is a graph showing viral titers in nasal washes from ferrets in a transmission study. The M2KO(ΔTM) virus did not transmit (no virus detected), but the control Brisb / 10 virus did. [Figure 51] Graph showing IgG titers in subjects vaccinated intranasally (IN), intramuscularly (IM) and intradermally (ID FGN) with A / California, A / Perth and B / Brisbane viruses. [Figure 52] FIG. 1 is a graph showing IgG titers in subjects administered a priming or priming plus booster dose of the A / Perth (H3N2) vaccine intramuscularly (IM) or intradermally (ID FGN). [Diagram 53] Graph showing virus titers in guinea pigs inoculated with FluLaval:A / California / 7 / 2009, NYMC X-181, A / Victoria / 210 / 2009NYMC X-187 (A / Perth / 16 / 2009-like virus), and B / Brisbane / 60 / 2008 via intramuscular (IM) and intradermal (ID) delivery at 0, 30, and 60 days post-inoculation. [Figure 54]FIG. 13 is a graph showing % survival of H5N1 M2KO(ΔTM) vaccinated subjects challenged with Vietnam / 1203 / 2004 virus 5 months after immunization. [Figure 55] FIG. 13 is a graph showing % survival of H5N1 M2KO(ΔTM) vaccinated subjects challenged with Vietnam / 1203 / 2004 virus 4 weeks after immunization. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] I. Definition The following terms are used herein and their definitions are provided by way of guide.

[0024] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless clearly indicated to refer to the singular only.

[0025] In general, the term "about" and its use in ranges, whether the term "about" is qualified or not, means that the numbers included are not limited to the exact numbers set forth herein, but are substantially within the ranges stated without departing from the scope of the present invention. As used herein, "about" is understood by those of ordinary skill in the art and may vary somewhat depending on the context in which it is used. If a term is used that is not clear to those of ordinary skill in the art in the context in which it is used, "about" means ±10% of the particular term.

[0026] As used herein, "subject" and "patient" are used interchangeably and refer to an animal, for example, a member of any vertebrate species. The subject methods and compositions disclosed herein are particularly useful for warm-blooded vertebrates, such as mammals and birds. Exemplary subjects can include mammals, such as humans, and mammals and birds that are important because they are endangered, economically important to humans (animals raised on farms for human consumption), and / or socially important (animals raised as pets or in zoos). In some embodiments, the subject is a human. In some embodiments, the subject is not a human.

[0027] The term "effective amount" or "therapeutically effective amount" or "pharmaceutical effective amount" as used herein refers to an amount sufficient to achieve the desired therapeutic and / or prophylactic effect, e.g., an amount that causes the prevention of a disease, condition, and / or symptom. In the context of therapeutic or prophylactic applications, the amount of the composition administered to a subject depends on the type and severity of the disease, as well as the individual's characteristics, such as general health, age, sex, weight, and tolerance to the composition drug. It also depends on the extent, severity, and type of the disease or condition. Those skilled in the art will be able to determine the appropriate dosage depending on these and other factors. In some embodiments, multiple doses are administered. Additionally or alternatively, in some embodiments, multiple therapeutic compositions or compounds (e.g., immunogenic compositions such as vaccines) are administered.

[0028] As used herein, the terms "isolated" and / or "purified" refer to an in vitro preparation, isolation and / or purification of a nucleic acid (e.g., a vector or plasmid), a polypeptide, a virus or a cell such that it is free from undesirable in vivo substances or substantially purified from undesirable in vivo substances with which it normally occurs. For example, in some embodiments, an isolated virus preparation is obtained by in vitro culture and propagation and is substantially free of other infectious substances. As used herein, "substantially free" means that a particular compound, e.g., undesirable nucleic acid, protein, cell, virus, infectious substance, etc., is below the detection level using standard detection methods for that compound or substance.

[0029] As used herein, the term "recombinant virus" refers to a virus that has been engineered in vitro, e.g., using recombinant nucleic acid techniques, to introduce a viral genome and / or introduce changes to viral proteins. For example, in some embodiments, a recombinant virus may contain both wild-type, endogenous nucleic acid sequences and mutant and / or foreign nucleic acid sequences. Additionally or alternatively, in some embodiments, a recombinant virus may contain modified protein components, e.g., mutant or variant matrix, hemagglutinin, neuraminidase, nucleoprotein, nonstructural proteins, and / or polymerase proteins.

[0030] As used herein, the term "recombinant cell" or "modified cell" refers to a cell that has been engineered in vitro, e.g., using recombinant nucleic acid techniques, to introduce a nucleic acid into the cell and / or to modify intracellular nucleic acid. Examples of recombinant cells include prokaryotic or eukaryotic cells that contain exogenous plasmids, expression vectors, etc., and / or cells that contain modifications to intracellular nucleic acid (e.g., substitutions, mutations, insertions, deletions, etc., into the cellular genome). An exemplary recombinant cell is one that has been engineered in vitro to express an exogenous protein, such as a viral M2 protein.

[0031] As used herein, the terms "mutant," "mutation," and "variant" are used interchangeably and refer to a nucleic acid or polypeptide sequence that differs from a wild-type sequence. In some embodiments, the mutant or variant sequence is naturally occurring. In other embodiments, the mutant or variant sequence is recombinantly and / or chemically introduced. In some embodiments, the nucleic acid mutation comprises a modification (e.g., addition, deletion, substitution) to the RNA and / or DNA sequence. In some embodiments, the modification comprises a chemical modification (e.g., methylation) and can also comprise a substitution or addition of natural and / or non-natural nucleotides. The nucleic acid mutation can be a silent mutation (e.g., one or more nucleic acid changes that encode the same amino acids as the wild-type sequence) or can cause a change in the encoded amino acid, provide a stop codon, or introduce a splicing defect or change. The nucleic acid mutation to the coding sequence can also cause a conservative or non-conservative amino acid change.

[0032] As used herein, the term "vRNA" refers to RNA that comprises a viral genome, including split or unfractionated viral genomes, positive and negative strand viral genomes, etc. vRNA can be entirely endogenous and "wild type" and / or can comprise recombinant and / or mutant sequences.

[0033] The term "host cell" as used herein refers to a cell in which a pathogen, such as a virus, can replicate. In some embodiments, the host cell is an in vitro cultured cell (e.g., a CHO cell, a Vero cell, an MDCK cell, etc.). Additionally or alternatively, in some embodiments, the host cell is in vivo (e.g., an infected vertebrate cell, such as an avian or mammalian cell). In some embodiments, the host cell can also be modified to enhance virus production, such as by enhancing viral infection of the host cell and / or enhancing the rate of viral growth. By way of example and not limitation, exemplary host cell modifications include recombinant expression of a 2-6 linked sialic acid receptor on the cell surface of the host cell and / or recombinant expression of a protein in the host cell that was not present or ineffective in the pathogen or virus.

[0034] As used herein, the term "infected" refers to carrying a disease or pathogen, such as a virus. Infection may be intentional, for example, by administration of a virus or pathogen (e.g., by vaccination), or may be unintentional, for example, by natural transfer of a pathogen from one organism to another, or from a contaminated surface to an organism.

[0035] The term "attenuated" as used herein in conjunction with viruses refers to a virus that has reduced virulence or pathogenicity compared to its non-attenuated counterpart, but is viable or alive. Typically, attenuation renders an infectious agent, such as a virus, less virulent or pathogenic to an infected subject compared to a non-attenuated virus. This is in contrast to a killed or fully inactivated virus.

[0036] The term "type" or "strain" used herein with viruses is used interchangeably and generally refers to viruses with different characteristics.For example, influenza A virus is a different type of virus from influenza B virus.Similarly, influenza A H1N1 is a different type of virus from influenza A H2N1, H2N2 and H3N2.In addition or alternatively, in some embodiments, different types of viruses, such as influenza A H2N1, H2N2 and H3N2, may be referred to as "subtypes".

[0037] As used herein, "M2KO" or "M2KO(ΔTM)" refers to SEQ ID NO:1, a virus comprising SEQ ID NO:1, or a vaccine comprising a virus comprising SEQ ID NO:1, depending on the context in which it is used. For example, in describing the mutations in the M2 gene presented herein, "M2KO" or "M2KO(ΔTM)" refers to SEQ ID NO:1. When describing the viral component of a vaccine, "M2KO" or "M2KO(ΔTM)" refers to a recombinant influenza virus that has the internal 6 genes of PR8 (nucleoprotein (NP), polymerase genes (PA, PB1, PB2), nonstructural proteins (NS), matrix (M)) but does not express a functional M2 protein. When describing a vaccine, "M2KO" or "M2KO(ΔTM)" refers to a vaccine that includes the M2KO(ΔTM) recombinant virus.

[0038] As used herein, "M2KO(ΔTM) virus" encompasses recombinant influenza viruses that contain the internal six genes of PR8 (nucleoprotein (NP), polymerase genes (PA, PB1, PB2), nonstructural proteins (NS), matrix (M)) alone or in combination with genes encoding other viral components and / or other viral components, but do not express a functional M2 protein. In some embodiments, the M2KO(ΔTM) virus contains genes from other influenza viruses. In some embodiments, the virus contains the HA and NA genes of influenza A / Brisbane / 10 / 2007-like A / Uruguay / 716 / 2007 (H3N2). In some embodiments, the M2KO(ΔTM) virus contains the HA and NA genes of A / Vietnam / 1203 / 2004 (H5N1) virus. In some embodiments, the M2KO(ΔTM) virus comprises the HA and NA genes of the A / California / 07 / 2009 (CA07) (H1N1pdm) virus.

[0039] II. Influenza A Virus A. Overview Influenza is the leading cause of death among adults in the U.S. The causative agents of influenza are viruses of the Orthomyxoviridae family, such as influenza A, influenza B, and influenza C, with influenza A being the most common and most virulent in humans.

[0040] Influenza A viruses are enveloped negative-stranded RNA viruses. The genome of influenza A viruses contains complements of 8 single-stranded (unpaired) RNAs that code for 11 proteins (HA, NA, NP, M1, M2, NS1, NEP, PA, PB1, PB1-F2, PB2). The total genome size is about 14,000 bases. The segmented nature of the genome allows for whole gene exchange between different viral strands during intracellular cohabitation. There are eight RNA segments: 1) HA codes for hemagglutinin (about 500 molecules of hemagglutinin are required to generate one virion); 2) NA codes for neuraminidase (about 100 molecules of neuraminidase are required to generate one virion); and 3) NP codes for nucleoprotein. 4) M encodes two proteins (M1 and M2) by using different reading frames of the same RNA segment (approximately 3000 M1 molecules are required to produce one virion). 5) NS encodes two proteins (NS1 and NEP) by using different reading frames of the same RNA segment. 6) PA encodes RNA polymerase. 7) PB1 encodes RNA polymerase and PB1-F2 protein by using different reading frames of the same RNA segment (induces apoptosis). 8) PB2 encodes RNA polymerase.

[0041] There are multiple subtypes of influenza A, named according to the H number (for the type of hemagglutinin) and N number (for the type of neuraminidase). Currently, there are 16 different recognized H antigens (H1-H16) and 9 different recognized N antigens (N1-N9). Each viral subtype mutates into various viral strains with different pathogenicity profiles, some pathogenic for one species but not others, and some pathogenic for multiple species. Exemplary influenza A virus subtypes identified in humans include, but are not limited to, H1N1, which caused the "Spanish Flu" and 2009 Swine Flu pandemics, H2N2, which caused the "Asian Flu" in the late 1950s, H3N2, which caused the Hong Kong Flu in the late 1960s, H5N1, which was feared to be the harbinger of a global influenza pandemic in the mid-2000s, H7N7, H1N2, which is currently endemic in humans and pigs, as well as H9N2, H7N2, H7N3, H5N2, and H10N7.

[0042] Several influenza A variants have been identified and are named according to the known isolate to which they are most similar, i.e., assumed to share a common lineage (e.g., Fujian strain), according to their typical host (e.g., human influenza virus), according to their subtype (e.g., H3N2), and according to their pathogenicity (e.g., LP, or low pathogenicity). Thus, influenza arising from viruses similar to the A / Fujian / 411 / 2002 (H3N2) isolate can be referred to as Fujian influenza, human influenza, and H3N2 influenza.

[0043] In addition, influenza variants may be named according to the species (host) to which the virus strain is unique or adapted. The main variants named using this convention include avian influenza, human influenza, swine influenza, equine influenza, and canine influenza. Variants are also named according to their pathogenicity in poultry, particularly chickens (e.g., low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI)).

[0044] B. Life Cycle and Structure The influenza virus life cycle generally involves attachment to cell surface receptors, entry into the cell, and uncoating of the viral nucleic acid followed by replication of the viral genes inside the cell. After synthesizing new copies of the viral proteins and genes, these components assemble into progeny virus particles that then exit the cell. Different viral proteins play a role in each of these steps.

[0045] Influenza A particles consist of a lipid envelope that encapsulates the viral core. The inside of the envelope is lined by matrix protein (M1), and the outer surface is characterized by two types of spike glycoproteins, hemagglutinin (HA) and neuraminidase (NA). M2, a transmembrane ion channel protein, is also part of the lipid envelope. See, for example, Figure 1.

[0046] The HA protein, a trimeric type I membrane protein, is responsible for binding to sialyloligosaccharides (oligosaccharides containing terminal sialic acid linked to galactose) on host cell surface glycoproteins or glycolipids and is also responsible for fusion of the virus with the host cell membrane following internalization of the virion by endocytosis.

[0047] Neuraminidase (NA), a tetrameric type II membrane protein, is a sialidase that cleaves the terminal sialic acid residues of HA and NA as well as host cell glycoconjugates, and is therefore recognized as a receptor-destroying enzyme. This sialidase activity is required for efficient release of progeny virions from the host cell surface and to prevent progeny aggregation due to the binding activity of viral HA with other glycoproteins. Thus, the receptor-binding activity of HA and the receptor-destroying activity of NA likely act as a counterbalancing force to allow efficient influenza replication.

[0048] The genome segments are packaged in the core of the viral particle. The RNP (RNA + nucleoprotein NP) adopts a helical conformation with three viral polymerase polypeptides that bind to each segment.

[0049] The influenza virus life cycle begins with the binding of HA to sialic acid-containing receptors on the surface of the host cell, followed by receptor-mediated endocytosis. Figure 1. The low pH in late endosomes induces a conformational shift in HA, exposing the N-terminus of the HA2 subunit (the so-called fusion peptide). The fusion peptide initiates fusion of the virus with the endosomal membrane, and a complex of matrix protein (M1) and RNP is released into the cytoplasm. RNP consists of the nucleoprotein (NP), which encapsidates the vRNA, and the viral polymerase complex, which is formed by the PA, PB1, and PB2 proteins. RNP is transported to the nucleus, where transcription and replication occur. The RNA polymerase complex then converts the RNA into three different reactions: (1) mRNA with a 5' cap and 3' polyA structure, (2) full-length complementary RNA (cRNA), and (3) cRNA. It catalyzes the synthesis of genomic vRNA using DNA as a template. The newly synthesized vRNA, NP and polymerase proteins are then assembled into RNPs, exported from the nucleus and transported to the cell membrane, where budding of progeny virus particles occurs. The neuraminidase (NA) protein plays a key role in the late stages of infection by removing sialic acid from sialyloligosaccharides, thus releasing newly assembled virions from the cell surface and preventing self-aggregation of virus particles. Virus assembly involves protein-protein and protein-vRNA interactions, but the nature of these interactions, for the most part, remains unknown.

[0050] C. Role of M2 protein As mentioned above, three proteins, hemagglutinin (HA), neuraminidase (NA), and M2, span the viral membrane. The extracellular domains (ectodomains) of HA and NA are highly variable, whereas the ectodomain of M2 is essentially invariant among influenza A viruses. Without wishing to be bound by theory, it is believed that the M2 protein, which has ion channel activity in influenza A viruses, functions in the early stages of the viral life cycle during the penetration of the host cell and uncoating of the viral RNA. Once the virion is endocytosed, the virion-associated M2 ion channel, a hemotetrameric helix bundle, is believed to channel protons from the endosome into the virion interior, disrupting the acid-labile M1 protein-ribonucleoprotein complex (RNP) interaction, thereby facilitating RNP release into the cytoplasm. In addition, in some influenza strains in which HA is cleaved intracellularly (e.g., A / fowl plagues / Rostock / 34), the M2 ion channel appears to increase the pH of the trans-Golgi network, preventing conformational changes in HA due to the low pH conditions in this compartment. It has also been shown that the M2 transmembrane domain itself can function as an ion channel. Amantadine hydrochloride, which blocks M2 ion channel activity, has been shown to inhibit viral replication, so the M2 protein ion channel activity appears to be essential in the influenza virus life cycle. However, the requirement for this activity in influenza A virus replication has not been directly demonstrated. The structure of the M2 protein is shown in Figure 2. The nucleic acid sequence of the M2 protein, along with the M1 sequence, is shown in Figure 3.

[0051] Influenza B and C viruses are structurally and functionally similar to influenza A viruses, but with some differences. For example, influenza B viruses do not have an M2 protein with ion channel activity. Instead, the NB protein, which is the product of the NA gene, likely has ion channel activity and thus a similar function to the M2 protein of influenza A viruses. Similarly, influenza C viruses do not have an M2 protein with ion channel activity. However, the CM1 protein of influenza C viruses may have this activity.

[0052] III.M2 virus mutants In one aspect, influenza A viruses with mutant M2 vRNA sequences are disclosed. Typically, such mutants lack M2 ion channel activity, exhibit attenuated growth characteristics in vivo, are unable to generate infectious progeny, and are non-pathogenic or exhibit low pathogenicity in infected subjects. The mutant viruses are immunogenic and, when used as vaccines, confer protection against infection with comparable wild-type and / or other pathogenic viruses. In addition, the M2 mutants disclosed herein are stable and do not mutate to express functional M2 polypeptides, regardless of the host cell used. Additionally or alternatively, in some embodiments, these mutant M1 proteins are produced without detectable changes to function. In some embodiments, viruses with mutant M2 nucleic acid sequences are unable to replicate in host cells in which the corresponding wild-type virus can grow. For example, and not by way of limitation, in some embodiments, a wild-type virus is capable of growing, replicating and replicating in cultured MDCK, CHO and / or Vero cells, whereas a corresponding virus having a mutant M2 sequence is unable to grow, replicate or propagate in the same types of cells.

[0053] As mentioned above, in some embodiments, M2 mutant virus is stable and does not mutate or revert to wild type or non-wild type sequence encoding functional M2 protein in host cell.For example, in some embodiments, M2 mutant virus is stable for 2, 3, 5, 10, 12, 15, 20, 25 or more passages in host cell.In some embodiments, host cell is non-modified host cell.In another embodiment, host cell is modified host cell, for example MDCK cell expressing M2 protein.

[0054] In some embodiments, the M2 mutant comprises one or more nucleic acid substitutions and / or deletions. In some embodiments, the mutations are localized in the nucleic acid encoding one or more of the extracellular domain of the M2 protein, the transmembrane domain of the M2 protein, and / or the cytoplasmic tail of the M2 protein. Additionally or alternatively, in some embodiments, the one or more nucleic acid mutations provide a splice variant of the M2 peptide, one or more stop codons, and / or one or more amino acid deletions. In some embodiments, the virus carrying the mutant M2 nucleic acid produces a non-functional M2 polypeptide. In some embodiments, the virus carrying the mutant M2 nucleic acid does not produce an M2 polypeptide. In some embodiments, the virus carrying the mutant M2 nucleic acid produces a truncated M2 polypeptide. In some embodiments, the truncated M2 polypeptide has the amino acid sequence MSLLTEVETPIRNEWGCRCNGSSD.

[0055] Three exemplary, non-limiting M2 virus mutants (M2-1, M2-2, and M2-3) are shown in Tables 1-3 below. In the tables, lowercase letters correspond to the M2 sequence, uppercase letters correspond to the M1 sequence, and mutant sequences (e.g., stop codons, splice defects) are underlined and in bold. The underlined (boxed below) bases in the M2-2 mutant indicate the deleted region in the M2-1 and M2-3 mutants. [Table 1]

[0056] The M2 polypeptide sequence generated from this mutant is as follows: MSLLTEVETPIRNEWGCRCNGSSD (sequence number 4). [Table 2]

[0057] No M2 polypeptide sequence is produced from this variant. [Table 3]

[0058] No M2 polypeptide sequence is produced from this variant.

[0059] Additionally or alternatively, in some embodiments, M2 mutations are introduced into the cytoplasmic tail. See FIG. 2. The cytoplasmic tail of the M2 protein is a mediator of infectious virus production. In some embodiments, truncation of the M2 cytoplasmic tail reduces infectious virus titers, reduces the amount of packaged viral RNA, reduces budding events, and reduces the efficiency of budding. It has been shown that the 5' sequence is more important than the 3' sequence for genome packaging, and that longer 5' sequences are more important for genome packaging. In addition, studies have shown that while nucleotide length is important, the actual sequence is less important (random sequences are sufficient to generate virus). Developing stable M2 cytoplasmic tail mutants has been challenging, and the literature provides numerous examples of reverting mutants.

[0060] For example, Pekosz et al JVI, 2005;79(6):3595-3605 replaced two codons at amino acid position 70 with a stop codon, but the virus immediately reverted. Another exemplary M2 cytoplasmic tail mutation is called M2del11. In the M2del11 mutant, 11 amino acid residues are deleted from the carboxyl end of the cytoplasmic tail. This truncation is due to the introduction of two stop codons and does not produce a full-length M2 polypeptide. This mutant is stable when passaged in M2-expressing MDCK cells (M2CK), but reverts to full-length M2 during passage in normal MDCK cells (J Virol. 2008 82(5):2486-92). Without wishing to be bound by theory, it is possible that reversion occurs under selective pressure in MDCK cells.

[0061] Another M2 cytoplasmic tail mutant, M2Stop90ala78-81, did not reduce virus titers, whereas ala70-77 did (JVI 2006;80(16)p8178-8189). Alanine scanning experiments further demonstrated that amino acids 74-79 at the M2 terminus play a role in virion morphology and affect virus infectivity (J Virol. 2006 80(11):5233-40).

[0062] Thus, a novel cytoplasmic variant is presented herein that has different characteristics from those described above.For example, in some embodiments, the cytoplasmic variant is stable in MDCK cells (does not revert to express full-length M2 polypeptide).In some embodiments, the cytoplasmic variant is stable for 2, 3, 5, 10, 15, 20, 25 or more passages in host cells.

[0063] The wild-type M2 polypeptide is shown below in Table 4. For each sequence, the bold text indicates the transmembrane domain. The extracellular domain is listed first (left), followed by the transmembrane domain (center), and the cytoplasmic tail sequence (right). [Table 4]

[0064] M2-4 (M2del FG#1) was generated but was not passivable in normal MDCK cells, but may be passivable in modified host cells (e.g., cells expressing wild-type M2 polypeptide). M2-5 (M2del FG#2) and M2-6 (FG#3) were generated and passaged in normal MDCK cells. The nucleotide sequences of the M genes of these viruses are stable for at least 10 passages in MDCK cells. These mutants could also grow and passage in other cells (e.g., cells that support influenza replication). These mutants were also found to be non-attenuated and pathogenic.

[0065] As described in the following embodiment, the M2 mutant virus described herein does not replicate in the respiratory tract or spread to other organs in ferret models, and is not transmissible in ferret models. Vaccines containing M2 mutants induce robust immune responses in mammals and protect mammals from influenza virus challenge. M2KO viruses induce both humoral and mucosal immune responses in mice and protect mice from lethal homosubtype and heterosubtype challenge. Vaccines containing M2 mutant viruses described herein provide effective protection from influenza challenge and have the advantage of being attenuated in mammalian hosts. These findings indicate that the M2 mutant viruses described herein are useful in vaccines against influenza.

[0066] IV. Cell-based virus production systems A. Generation of "First Generation" Mutant Viruses Mutant viruses, such as viruses carrying mutant M2 nucleic acid, can be generated by plasmid-based reverse genetics as described in Neumann et al., Generation of influenza A viruses entirely from clone cDNAs, Proc. Natl. Acad. Sci. USA 96:9345-9350 (1999), which is incorporated herein by reference in its entirety. Briefly, eukaryotic host cells are transfected with one or more plasmids encoding the eight viral RNAs. Each viral RNA sequence is flanked by an RNA polymerase I promoter and an RNA polymerase I terminator. Notably, the viral RNA encoding the M2 protein contains the mutant M2 nucleic acid sequence. The host cells are additionally transfected with one or more expression plasmids encoding viral proteins, such as the wild-type M2 protein (e.g., polymerase, nucleoprotein, and structural proteins). Transfection of the host cells with the viral RNA plasmids results in the synthesis of all eight influenza virus RNAs, one of which carries the mutant M2 sequence. The cotransfected viral polymerase and nucleoproteins assemble functional vRNPs with the viral RNA, which are replicated and transcribed to ultimately form infectious influenza viruses that contain mutant M2 nucleic acid sequences and also have a functional M2 polypeptide incorporated into the viral lipid envelope.

[0067] Another method for generating "first generation" mutant viruses involves the ribonucleoprotein (RNP) transfection system described in Enami and Palese, High-efficiency formation of influenza virus transfectants, J. Virol. 65(5):2711-2713, incorporated herein by reference, which allows the exchange of influenza virus genes with in vitro generated recombinant RNA molecules.

[0068] As shown in Luytjes et al., Amplification, expression, and packaging of a foreign gene by influenza virus, Cell 59:1107-1113, incorporated herein by reference, viral RNA is synthesized in vitro and the RNA transcripts are coated with viral nucleoprotein (NP) and polymerase proteins which act as biologically active RNPs in transfected cells.

[0069] The RNP transfection method can be divided into four steps: 1) Preparation of RNA: Plasmid DNA encoding influenza virus segments is transcribed into negative-strand RNA in an in vitro transcription reaction. 2) Encapsidation of RNA: The transcribed RNA is then mixed with gradient-purified NP and polymerase proteins isolated from disrupted influenza viruses to form biologically active RNP complexes. 3) Transfection and rescue of encapsidated RNA: The artificial ribonucleocapsid is transfected into cells previously infected with a helper influenza virus containing a gene different from the rescued gene, and the helper virus amplifies the transfected RNA. 4) Selection of transfected genes: Because both the helper virus and transfectants containing the rescued gene are present in the culture supernatant, an appropriate selection system using antibodies is necessary for the isolation of viruses carrying the transfected genes.

[0070] The selection system allows the generation of novel transfectant influenza viruses with specific biological and molecular characteristics, after which selection of antibodies against the target surface proteins can be used for positive or negative selection.

[0071] For example, transfectants or mutant viruses containing an M2 gene that does not express the M2 protein can be grown in appropriate mammalian cell lines modified to stably express wild-type functional M2 protein. Antibodies to M2e can be used to prevent or inhibit replication of helper viruses that express the wild-type M2 gene, i.e., the M2e protein on the membrane surface. Such antibodies are commercially available and can inhibit helper virus replication, allowing transfectants / mutant viruses containing mutant M2 to grow to high concentrations in the supernatant. Inhibition of influenza virus replication by M2e antibodies has previously been described in Influenza A virus M2 proteinkoron monoclonal antibody restriction of virus growth and detection of M2 in virions, J Virol 62:2762-2772(1988) and Treanor et al, Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication in mice, J. Virol. 64:1375-1377(1990).

[0072] Additionally or alternatively, the same antibodies can be used to "capture" helper virus and enrich for transfectants, for example, they can be used to coat the bottom of tissue culture dishes or in column matrices to enrich for transfectants in supernatants or eluates.

[0073] The transfectant viruses can be grown in M2 expressing cells in multi-well plates by limiting dilution and then identified and cloned, for example, by replica plating. For example, half of an aliquot of a given well of a multi-well plate containing grown virus can be used to infect MDCK cells, and the other half can be used to infect MDCK cells expressing M2 protein. Both the transfectant virus and the helper virus will grow in MDCK cells expressing M2 protein. However, only the helper virus will grow in standard MDCK cells to identify wells in the multi-well plate containing transfectants. The transfectant viruses can be plaque purified in cells expressing M2 protein.

[0074] B. Growth of viral mutants In some embodiments, the virus variants described herein are maintained and passaged in host cells. Exemplary host cells suitable for the growth of influenza virus variants, such as, but not limited to, influenza A virus variants, include any number of eukaryotic cells, such as, but not limited to, Madin-Darby canine kidney cells (MDCK cells), monkey cells such as African green monkeys (e.g., Vero cells), CV-1 cells and rhesus kidney cells (e.g., LLcomk.2 cells), bovine cells (e.g., MDBK cells), porcine cells, ferret cells (e.g., mink lung cells) BK-1 cells, rodent cells (e.g., Chinese hamster ovary cells), human cells, such as avian cells, including embryonic human retinal cells (e.g., PER-C6®), 293T human embryonic kidney cells and embryonic fibroblast cells.

[0075] Additionally or alternatively, in some embodiments, eukaryotic host cells are modified to promote virus production, for example, by promoting viral infection of the host cells and / or promoting the rate of viral growth. For example, in some embodiments, host cells are modified to express or increase the expression of 2,6-linked sialic acid on the cell surface to allow more efficient and effective infection of these cells by mutant or wild-type influenza A viruses. See, for example, U.S. Patent Application Publication No. 2010-0021499 and U.S. Patent No. 7,176,021, which are incorporated herein by reference in their entirety. Thus, in some exemplary embodiments, Chinese hamster ovary cells (CHO cells) and / or Vero cells modified to express at least one copy of the 2,6-sialyltransferase gene (ST6GAL 1) are used. For example, but not limited to, the homo sapiens ST6β-galactosamide α-2,6-sialyltransferase gene sequence represented by accession number BC040009.1 is an example of an ST6Gal gene that can be integrated and expressed in CHO cells. One or more copies of a polynucleotide encoding a functional ST6Gal I gene product can be genetically engineered into the cell. That is, cells stably transformed to express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 copies of the ST6Gal I gene can be used. An expression cassette can include one or more copies of the ST6Gal I gene to be expressed, which can be operably linked to regulatory elements such as promoters, enhancers, and terminators, as well as polyadenylation signal sequences to facilitate the expression of the ST6Gal I gene or copies thereof. Alternatively, a single expression cassette can be engineered to express a single copy of the ST6Gal I gene, or multiple expression cassettes can be integrated into the host cell genome. Thus, in some embodiments, at least one ST6Gal I gene is integrated into the genome of a host cell such that the cell expresses the ST6Gal I gene and its enzymatic protein product.Depending on the copy number, one host cell can also express many functional ST6Gal I gene proteins.

[0076] Suitable vectors for cloning and transfecting to generate stable transformed cell lines are well known in the art. One non-limiting example is the pcDNA3.1 vector (Invitrogen).

[0077] Additionally or alternatively, in some embodiments, eukaryotic host cells are modified to produce wild-type variants of mutant viral genes, thereby providing genes in trans to the virus. For example, a viral strain having a mutant M2 protein can exhibit a high growth rate (e.g., greater virus yield) when passaged in a host cell that produces a wild-type M2 protein. In some embodiments, a viral strain having a mutant M2 protein cannot grow or replicate in cells that do not express the wild-type M2 gene. In addition, such host cells can slow or prevent viral reversion to functional M2 sequences, for example, because there is no selective pressure for reversion in such a host.

[0078] Methods for producing both expression vectors and modified host cells are well known in the art. For example, an M2 expression vector can be produced by placing the M2 nucleic acid sequence (M2 ORF sequence, from the start codon to the stop codon of "wild type" M2 (Table 5)) in a eukaryotic expression vector as follows: [Table 5]

[0079] Host cells (e.g., MDCK cells) can then be transfected by methods known in the art using commercially available reagents and kits, such as, for example, TransIT® LT1 (Mirus Bio, Madison, Wis.). For example, but not limited to, cells can be selected and examined for M2 expression by co-transfection with a detectable or selectable marker and / or by screening, for example, indirect immunostaining with an M2 antibody. M2 expression can be measured by indirect immunostaining, flow cytometry, or ELISA.

[0080] For example, but not limited to, 293T human embryonic kidney cells and Madin-Darby canine kidney (MDSK) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and minimum essential medium (MEM) containing 5% newborn calf serum, respectively. All cells were maintained at 37°C in 5% CO2. Hygromycin-resistant MDCK cells stably expressing the P2 protein from A / Puerto Rico / 8 / 34 (H1N1) were established by co-transfection with a 1:1 ratio of plasmid pRHyg containing the hygromycin resistance gene and plasmid pCAGGS / M2 expressing the full-length M2 protein. Stable MDCK cell clones expressing M2 (M2CK) were selected in medium containing 0.15 mg / mL hygromycin (Roche, Mannheim, Germany) by screening with indirect immunostaining using anti-M2 (14C2) monoclonal antibody (Iwatsuki et al., JVI, 2006, vol. 80, No. 1, p. 5233-5240). M2CK cells were cultured in MEM supplemented with 10% fetal bovine serum and 0.15 mg / mL hygromycin. In M2CK cells, the expression level and localization of M2 were similar to those of virus-infected cells (data not shown). M2-expressing Vero cells can be generated in a similar manner.

[0081] In some embodiments, cells and virus mutants are cultured and grown by methods well known in the art. For example, but not limited to, in some embodiments, host cells are grown in the presence of MEM supplemented with 10% fetal bovine serum. M2 expressing cells are infected at an MOI of 0.001 by washing with PBS followed by virus adsorption at 37° C. In some embodiments, virus growth medium containing trypsin / TPCK is added and cells are incubated for 2-3 days until cytopathic effects are observed.

[0082] In this way, disposable bioreactor systems have been developed for mammalian cells, with or without viruses, the advantages of which include faster facility set-up and lower risk of cross-contamination. For example, the cells described herein can be cultured in disposable bags, such as those from Stedim, Bioeaze bags from SAFC Biosciences, HybridBag™ from Cellexus Biosytems, or single-use bioreactors from HyClone, or Celltainers from Lonza. Bioreactors can be in 1L, 10L, 50L, 250L, 1000L size formats. In some embodiments, cells are kept suspended in an optimized serum-free medium that is free of animal products. The system can be a fed-batch system, for example, where cultures can be grown in single bags of 1L to 10L, or a perfusion system that allows a constant supply of nutrients while at the same time avoiding the accumulation of potentially toxic by-products in the medium.

[0083] For long term storage, the mutant viruses can be stored as frozen stocks.

[0084] V. Vaccines and Methods of Administration A. Immunogenic Compositions / Vaccines There are various different types of vaccines that can be made from the cell-based virus production system disclosed herein. The present disclosure includes, but is not limited to, attenuated live virus vaccines, inactivated virus vaccines, whole particle virus vaccines, split virus vaccines, virosome virus vaccines, viral surface antigen vaccines, and combinations thereof. In other words, there are numerous vaccines that can generate immune responses, such as systemic immune responses, specific for different influenza viruses, by appropriate combination of any of these vaccine types. Attenuated live virus vaccines also have the advantage of stimulating local mucosal immunity in the respiratory tract.

[0085] In some embodiments, the vaccine antigens used in the compositions described herein are "direct" antigens, i.e., they are not administered as DNA but are antigens in themselves. Such vaccines may comprise whole viruses or just parts of viruses, such as, but not limited to, viral polysaccharides, whether alone or conjugated to carrier elements, such as carrier proteins, live attenuated whole microorganisms, inactivated microorganisms, recombinant peptides and proteins, glycoproteins, glycolipids, lipopeptides, synthetic peptides, or, in the case of vaccines referred to as "split" vaccines, disrupted microorganisms.

[0086] In some embodiments, a whole virion vaccine is provided. The whole virion vaccine can be concentrated by ultrafiltration and then purified by zonal centrifugation or chromatography. Typically the virion is inactivated before or after purification, for example with formalin or beta-propiolactone.

[0087] In some embodiments, subunit vaccines are provided that contain purified glycoproteins. Such vaccines may be prepared as follows: a virus suspension fragmented by treatment with a detergent is used to purify the surface antigens, for example by ultracentrifugation. Thus, subunit vaccines contain mainly HA proteins, but also NA. The detergents used may be, for example, cationic detergents, such as hexadecyltrimethylammonium bromide, anionic detergents, such as ammonium deoxycholate, or non-ionic detergents, such as those sold under the name TRITON X100. Hemagglutinin may be isolated after treatment of the virion with a protease, for example bromelain, followed by purification by standard methods.

[0088] In some embodiments, split vaccines are provided that contain virions that have been treated with lipid-dissolving agents. Split vaccines can be prepared as follows: an aqueous suspension of inactivated or non-inactivated purified virus obtained as described above is treated with a lipid solvent, such as ethyl ether or chloroform, under stirring, and associated with a detergent. Dissolution of the viral envelope lipids causes the viral particles to fragment. The aqueous phase containing the split vaccine, composed mainly of hemagglutinin and neuraminidase, and core or its degradation products, with the original lipid environment removed, is recovered. The remaining infectious particles are then inactivated if they have not already been inactivated.

[0089] In some embodiments, inactivated influenza virus vaccines are provided. In some embodiments, inactivated vaccines are made by inactivating the virus using known methods, such as but not limited to formalin or β-propiolactone treatment. The types of inactivated vaccines that can be used in the present invention can include whole virus (WV) vaccines or subviral (SV) (split) vaccines. WV vaccines contain intact inactivated virus, and SV vaccines contain purified virus that has been disrupted with detergents that solubilize the lipid-containing viral envelope, followed by chemical inactivation of residual virus.

[0090] Additionally or alternatively, in some embodiments there is provided a live attenuated influenza virus vaccine, which can be used to prevent or treat influenza virus infection by known method steps.

[0091] In some embodiments, attenuation is achieved in a single step by introducing attenuation genes from an attenuated donor virus into the isolate or reassorted virus by known methods (see, e.g., Murphy, Infect. Dis. Clin. Pract. 2,174 (1993)). In some embodiments, the virus is attenuated by mutation of one or more viral nucleic acid sequences to obtain a mutant virus. For example, in some embodiments, the mutant viral nucleic acid sequence encodes a defective protein product. In some embodiments, the protein product has reduced or no function. In other embodiments, no protein product is produced from the mutant viral nucleic acid.

[0092] Thus, the virus can be attenuated or inactivated by known methods, formulated, and administered as an immunogenic composition (e.g., as a vaccine) that elicits an immune response in animals, such as birds and / or mammals. Methods for determining whether such attenuated or inactivated vaccines retain similar antigenicity to clinical isolates or highly viable strains derived therefrom are well known in the art. Such known methods include the use of antisera or antibodies to eliminate viruses expressing antigenic determinants of the donor virus, chemical selection (e.g., amantadine or rimantidine); HA and NA activity and inhibition; and DNA screening (such as probe hybridization or PCR) to confirm that donor genes encoding antigenic determinants (e.g., HA or NA genes) or other mutant sequences (e.g., M2) are not present in the attenuated virus. See, e.g., Robertson et al., Giornale di Igiene e Medicina Preventiva, 29, 4 (1988); Kilbourne, Bull. M2 World Health Org., 41, 643 (1969); and Robertson et al., Biologicals, 20, 213 (1992).

[0093] In some embodiments, the vaccine comprises an attenuated influenza virus that does not express a functional M2 protein, hi some embodiments, the mutant virus expresses a viral protein that replicates well in cells that express the M2 protein, but does not produce infectious progeny virions in corresponding wild-type cells.

[0094] Pharmaceutical compositions of the invention suitable for intradermal, inoculation, or parenteral or oral administration comprise attenuated or inactivated influenza viruses and may optionally further comprise sterile aqueous or non-aqueous solutions, suspensions and emulsions. The compositions may further comprise auxiliary agents or excipients as known in the art. For example, Berkow et al., The Merck Manual, 15th edition, Merck and Co., Rahway, NJ (1987); Goodman et al., eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, Eighth Edition, Pergamon Press, Inc., Elmsford, NY (1990); Avery's Drug Treatment Principles and Practice of Clinical Pharmacology and See Therapeutics, Third Edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. (1987); and Katzung, ed., Basic and Clinical Pharmacology, Fifth Edition, Appleton and Lange, Norwalk, Conn. (1992).

[0095] In some embodiments, preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and / or emulsions, and may further include auxiliary agents or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and promote antigen absorption. Liquid dosage forms for oral administration may generally include liposome solutions containing liquid dosage forms. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs that include inert diluents commonly used in the art, such as water. In addition to inert diluents, such compositions may also include adjuvants, wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, or perfuming agents.

[0096] When the composition of the present invention is used for administration to an individual, the composition may further include salts, buffers, adjuvants, or other substances that are desirable to improve the efficacy of the composition. In the case of a vaccine, an adjuvant, a substance that enhances a specific immune response, can be used. Usually, the adjuvant and the composition are mixed before presentation to the immune system, or presented separately but at the same organ site that is immunized.

[0097] In some embodiments, the immunogenic compositions (e.g., vaccines) disclosed herein comprise multiple different types of viruses or viral antigens, at least one of which comprises a mutant M2 gene (e.g., a virus comprising an M2KO(ΔTM) (SEQ ID NO:1) mutation) and / or a corresponding mutation in the M2 functional equivalent of the virus (e.g., the NB protein of influenza B, or the CM1 protein of influenza C). In another embodiment, the immunogenic compositions comprise a single type of virus or viral antigen that comprises a mutant M2 gene (e.g., a virus comprising an M2KO(ΔTM) (SEQ ID NO:1) mutation) and / or a corresponding mutation in the M2 functional equivalent of the virus (e.g., the NB protein of influenza B, or the CM1 protein of influenza C). For example, in some embodiments, the main component of an immunogenic composition, such as a vaccine composition, comprises one or more influenza viruses of type A, type B, or type C, or any combination thereof, or any combination of antigens from these viruses, where at least one of the viruses comprises a mutant M2 gene (e.g., a virus comprising the M2KO(ΔTM) (SEQ ID NO:1) mutation), and / or a corresponding mutation in the M2 functional equivalent of the virus (e.g., the NB protein of influenza type B, or the CM1 protein of influenza type C). For example, in some embodiments, at least two of three types, at least two of different subtypes, at least two of the same type, at least two of the same subtype, or different isolate(s) or reassortant(s) are provided in the immunogenic composition (e.g., vaccine). For example, but not limited to, human influenza virus type A includes H1N1, H2N2, and H3N2 subtypes. In some embodiments, an immunogenic composition (e.g., a vaccine) comprises a virus containing a mutant M2 gene (e.g., a virus containing an M2KO(ΔTM) (SEQ ID NO:1) mutation) and / or a corresponding mutation in the M2 functional equivalent of that virus (e.g., the NB protein of influenza B, or the CM1 protein of influenza C), and about 0.1-200 μg, e.g., 10-15 μg, of hemagglutinin from each of the viral strains entering the composition.Heterogeneity in the vaccine is provided by mixing replicated influenza viruses for at least 2 strains, for example 2-50 strains, or any range or number therein of influenza virus strains. In some embodiments, influenza A or B virus strains having recent antigenic compositions are used. In addition, immunogenic compositions (e.g., vaccines) can be made to induce variation within a single strain of influenza virus using techniques known in the art.

[0098] In some embodiments, the vaccine comprises a virus comprising the M2KO(ΔTM) (SEQ ID NO:1) mutation along with other viral components and / or genes expressing other viral components. In some embodiments, the vaccine (e.g., a virus comprising the M2KO(ΔTM) (SEQ ID NO:1) mutation) comprises genes from other viral strains, such as, but not limited to, HA and NA genes from other viral strains. In some embodiments, the vaccine comprises HA and NA genes from human influenza virus type A subtypes H5N1, H1N1, H2N2 or H3N2. In some embodiments, the vaccine comprises HA and NA genes from, for example, PR8xBrisbane / 10 / 2007, A / Vietnam / 1203 / 2004, or A / California / 07 / 2009 (CA07) viruses.

[0099] The pharmaceutical compositions according to the invention may further or additionally comprise at least one chemotherapeutic compound, immunosuppressant, anti-inflammatory or immunostimulant, or antiviral agent, e.g., for gene therapy, such as, but not limited to, gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon alpha, interferon beta, interferon gamma, tumor necrosis factor alpha, thiosemicarbazones, methisazone, rifampin, ribavirin, pyrimidine analogues, purine analogues, fascarnet, phosphonoacetic acid, acyclovir, dideoxynucleosides, protease inhibitors, or ganciclovir.

[0100] The compositions may contain variable but small amounts of endotoxin-free formaldehyde and preservatives that have been found to be safe and not contribute to undesirable effects in the organism to which the compositions of the invention are administered.

[0101] B. Administration The immunogenic compositions (e.g., vaccines) disclosed herein may be administered via either parenteral or mucosal routes, which are routes traditionally used or recommended for vaccines, and may be in a variety of forms, such as injectable or sprayable liquids, freeze-dried, or spray-dried, or air-dried formulations. Vaccines may be administered using a syringe for intramuscular, subcutaneous, or intradermal injection, or using a needleless injector. Vaccines may be administered by a nebulizer capable of delivering a dry powder or liquid spray to mucous membranes, whether to the nose, lungs, vagina, or rectum.

[0102] The vaccines disclosed herein can confer resistance to one or more influenza strains by either passive or active immunization. In active immunization, an inactivated or live attenuated vaccine composition is administered prophylactically to a host (e.g., a mammal) such that the host's immune response to the administration protects against infection and / or disease. For passive immunization, the induced antisera can be collected and administered to a recipient suspected of being infected by at least one influenza virus strain.

[0103] Thus, the invention encompasses methods of preventing or attenuating a disease or disorder, such as infection by at least one influenza virus strain. As used herein, a vaccine is said to prevent or attenuate a disease if its administration results in total or partial attenuation (i.e., suppression) of a symptom or condition of the disease, or total or partial immunity of an individual to the disease.

[0104] At least one inactivated or attenuated influenza virus of the present invention or a composition thereof can be administered by any means that achieves the intended purpose using the above-mentioned pharmaceutical composition.For example, administration of such a composition can be by various parenteral routes, such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, nasal, oral or transdermal routes.Parenteral administration can be by bolus injection or by slow perfusion over time.In some embodiments, the immunogenic composition disclosed herein is by intramuscular or subcutaneous application.

[0105] In some embodiments, a regimen for preventing, inhibiting, or treating influenza virus-associated pathology comprises administration of an effective amount of a vaccine composition described herein administered as a single treatment or repeated as reinforcement or booster doses for a period ranging from one week to about 24 months, or any range or value therein, hi some embodiments, the influenza vaccines disclosed herein are administered once a year.

[0106] According to the present invention, an "effective amount" of a vaccine composition is an amount sufficient to achieve a desired biological effect. It will be understood that in some embodiments, an effective dosage will depend on the age, sex, health and weight of the recipient, the type of concomitant treatment, if applicable, the frequency of treatment, and the nature of the desired effect. The effective dose ranges provided below are not intended to limit the invention, but represent exemplary dosage ranges. Thus, in some embodiments, dosages are tailored to individual subjects, as will be understood and determinable by those skilled in the art. A dosage of an attenuated virus vaccine for an adult mammal (e.g., human) is approximately 10 mg / kg. 3 ~10 7 The dose may be in plaque forming units (PFU), or any range or value therein. The dose of the inactivated vaccine may be in the range of about 0.1 to 200 μg of hemagglutinin protein, e.g., 50 μg. However, the dose should be a safe and effective amount, as conventionally determined using existing vaccines as a starting point.

[0107] C. Intradermal delivery Live influenza vaccines are traditionally delivered to the nasal cavity to mimic the natural route of infection and promote immune responses similar to natural virus infection. Instead, disclosed herein is an intradermal delivery method that includes the use of a novel microneedle device that utilizes the immunological advantages of intradermal delivery. In some embodiments, attenuated viruses (e.g., M2 virus mutants) are used in vaccine compositions for intradermal administration. In some embodiments, M2 virus mutants that do not produce infectious progeny viruses are provided in the vaccine. Thus, any possibility of reassortment with wild-type circulating influenza viruses is virtually eliminated.

[0108] In embodiments disclosed herein, the vaccine is administered to the skin by intradermal delivery (intradermal). In some embodiments, intradermal delivery is performed using a microneedle delivery device. As disclosed herein, intradermal delivery has numerous advantages. For example, the immunogenicity of the vaccine is enhanced by triggering the immunological capacity of the skin immune system. The vaccine has direct access to the skin's potent antigen-presenting dendritic cells, i.e., epidermal Langerhans cells and dermal dendritic cells. The skin cells generate proinflammatory signals that promote an immune response to antigens introduced through the skin. Furthermore, the skin immune system generates antigen-specific antibodies and intracellular immune responses. Intradermal delivery can conserve vaccine dose, i.e., lower doses of antigens can be effective when delivered intradermally, taking into account the above factors.

[0109] Also, because the vaccine is delivered to the skin through the microneedle array of the device, the risk of an unintentional needle stick is reduced, and intradermal vaccine delivery via a microneedle array is relatively painless compared to intramuscular injection with a conventional needle and syringe.

[0110] Microneedle devices are known in the art, such as those described in U.S. Patent Application Publication Nos. 2012 / 0109066, 2011 / 0172645, 2011 / 0172639, 2011 / 0172638, 2011 / 0172637, and 2011 / 0172609. Microneedle devices can be fabricated, for example, from the fabrication of stainless steel sheets (e.g., Trinity Brand Industries, Geogia; SS 304; 50 μm thick) by wet etching. In some embodiments, the individual microneedles have a length of about 500 μm to 1000 μm, e.g., about 750 μm, and a width of about 100 μm to 500 μm, e.g., about 200 μm. The vaccine can then be applied to the microneedles as a coating. For example, but not by way of limitation, the coating solution may contain 1% (w / v) carboxymethylcellulose sodium salt (low viscosity, USP grade; Carbo-Mer, San Diego, CA), 0.5% (w / v) Lutrol F-68 NF (BASF, Mount Olive, NJ) and antigen (e.g., soluble HA protein at 5 ng / ml; live attenuated virus such as the M2 mutant virus described herein). To reach higher vaccine concentrations, the coating solution may be allowed to evaporate at room temperature (~23°C) for 5-10 minutes. Coating may be performed by dip coating. The amount of vaccine per row of microneedles may be determined by immersing the microneedles in 200 μl of phosphate buffered saline (PBS) for 5 minutes and assaying for antigen by methods known in the art.

[0111] In some embodiments, a microneedle device is used that is made primarily of polypropylene and stainless steel first-cut parts that are fitted together by simple snap-fit ​​and heat seal. In some embodiments, the device is fully self-contained and includes the vaccine, pump mechanism, activation mechanism, and microneedle unit. These components are hidden in a plastic cover. Pressing the activation button initiates the injection of vaccine using the device. The microneedle is inserted into the skin at the same time as pressing the button, starting the pump mechanism that applies pressure to the main drug container. When the spring mechanism applies sufficient pressure to the vaccine reservoir, the vaccine begins to flow through the microneedle array and into the skin. In some embodiments, the delivery of the vaccine dose is completed within about 2 minutes after the device is activated. After the injection is completed, the device is gently removed from the skin.

[0112] In some embodiments, a method of administering an immunogenic composition (e.g., a vaccine) into the skin using a microneedle device is provided. In some embodiments, the microneedle device includes a piercing mechanism and an immunogenic composition layer, and includes a plurality of microneedles capable of piercing the skin, allowing the immunogenic composition to be administered into the skin. In some embodiments, the method includes depressing the piercing mechanism. In some embodiments, the immunogenic composition (e.g., a vaccine) includes a virus including a nucleic acid sequence encoding an expressed mutant M2 protein or a non-expressed mutant M2 protein, and the expressed mutant M2 protein includes or consists of the amino acid sequence of SEQ ID NO: 4. In some embodiments, the microneedle array is initially disposed inside the device housing, and the lever action can extend and insert the microneedles into the skin through the device button, thereby injecting the vaccine liquid into the skin.

[0113] The delivery devices described herein may be utilized to deliver any desired substance. In one embodiment, the substance to be delivered is a drug, and the delivery device is a drug delivery device configured to deliver the drug to a subject. As used herein, the term "drug" is intended to include any substance (e.g., vaccines, medicines, nutrients, functional foods, etc.) delivered to a subject for any therapeutic, prophylactic or medicinal purpose. In one such embodiment, the drug delivery device is a vaccine delivery device configured to deliver a dose of a vaccine to a subject. In one embodiment, the delivery device is configured to deliver an influenza vaccine. The embodiments discussed herein primarily relate to devices configured to deliver a substance transdermally. In some embodiments, the device may be configured to deliver a substance directly to an organ other than the skin. EXAMPLES

[0114] Although the following examples are presented with influenza A, it should be understood that the mutations and methods described herein are equally applicable to other viruses that express M2, M2-like proteins, or proteins with the same or similar function as the influenza A M2 protein.

[0115] Example 1: Generation of M2 virus mutants The M2 mutant was constructed as follows. a) M2-1: M2 ectodomain + 2 stop codons + TM deletion (PR8 M segment + 2 stop codons (786-791) not including 792-842 (TM))

[0116] The partial wild type M gene from PR8 was amplified by PCR using oligo set 1 and oligo set 2 shown below. [Table 6]

[0117] The PCR product was then digested with BsmBI. The expression vector (pHH21) was also digested with BsmBI, and the digested PCR product was then ligated into the vector using T4 DNA ligase. E. coli cells were transformed with the vector, and after appropriate incubation, the vector was isolated and purified by methods known in the art. The mutant M2 portion of the vector was characterized by nucleic acid sequencing. b) M2-2: M2 ectodomain + 2 stop codons + splice deletion (PR8 M segment + 2 stop codons (786-791) + splice deletion nt51)

[0118] The partial wild-type M gene from PR8 was amplified by PCR using the primer set shown below. [Table 7]

[0119] The PCR product was then digested with BsmBI. The expression vector (pHH21) was also digested with BsmBI. A double-stranded DNA fragment was then generated by annealing the two nucleotides shown below. [Table 8]

[0120] The digested vector, PCR product and double-stranded fragment were then ligated using T4 DNA ligase. E. coli cells were transformed with the vector, and after appropriate incubation, the vector was isolated and purified by methods known in the art. The mutant M2 portion of the vector was characterized by nucleic acid sequencing. c) M2-3: M2 ectodomain + 2 stop codons + TM deletion (PR8 M segment + 2 stop codons (786-791) not including 792-842 (TM) + splice deletion nt51)

[0121] The partial M2-1 mutant (M2 ectodomain + two stop codons + TM deletion (PR8 M segment + two stop codons (786-791) not including 792-842 (TM))) was amplified from PR8 by PCR with the following primers: [Table 9]

[0122] The PCR product was then digested with BsmBI. The expression vector (pHH21) was also digested with BsmBI. A double-stranded DNA fragment was then generated by annealing the two nucleotides shown below. [Table 10]

[0123] The digested vector, PCR product and double-stranded fragment were then ligated using T4 DNA ligase. E. coli cells were transformed with the vector, and after appropriate incubation, the vector was isolated and purified by methods known in the art. The mutant M2 portion of the vector was characterized by nucleic acid sequencing.

[0124] The sequences of each of the three M2 mutant constructs are shown in Tables 1-3.

[0125] Example 2: Generation and cultivation of M2 mutant viruses This example shows the cultivation of PR8 virus containing the M2KO(ΔTM) (SEQ ID NO:1) mutation. The mutant virus was generated with some modifications from that reported in Neumann et al., Generation of influenza A viruses entirely from clone cDNAs, Proc. Natl. Acad. Sci. USA 96:9345-9350 (1999). Briefly, 293T cells were transfected with 17 plasmids: 8 PolI constructs for 8 RNA segments, one of which has the mutant M2 sequence, and 9 protein expression constructs for 5 structural proteins, as follows: NP (pCAGGS-WSN-NP0 / 14) of A / Puerto Rico / 8 / 34 (H1N1) virus; M2 (pEP24c); PB1 (pcDNA774); PB2 (pcDNA762); and PA (pcDNA787).

[0126] The plasmid was mixed with transfection reagent (2 μL of Trans IT® LT-1 (Mirus, Madison, WI) per μg of DNA) and incubated at room temperature for 15-30 min to give a total of 1 × 10 6 The virus was added to 293T cells. After 48 hours, the virus in the supernatant was serially diluted and inoculated into M2CK cells. Two to four days after inoculation, the virus in the supernatant of the final dilution well, where the cells showed obvious cytopathic effect (CPE), was inoculated into M2CK cells for generation of virus stock. The M2 gene of the generated virus was sequenced to confirm the presence of the gene and the intended mutations and to ensure the absence of inappropriate mutations.

[0127] Mutant M2 viruses were grown and passaged as follows: M2CK host cells were grown in the presence of MEM supplemented with 10% fetal bovine serum. Cells were infected at an MOI of 0.001 by washing with PBS and then allowing virus to adsorb at 37°C. Virus growth medium containing trypsin / TPCK was added and cells were incubated for 2-3 days until cytopathic effects were observed.

[0128] Example 3: M2KO Replication is Restricted in Normal Cells The growth kinetics of PR8 virus containing the M2KO(ΔTM) (SEQ ID NO: 1) mutation and wild-type PR8 were analyzed in both normal MDCK cells and MDCK cells stably expressing the M2 protein (M2CK). -5 The cells were infected with the virus at a multiplicity of infection of 100 μg / mL. The virus titers in the cell supernatants were determined in MDCK or M2CK cells. Wild-type PR8 grew to high titers in both cell types, whereas M2KO grew well only in M2CK cells and did not grow at all in MDCK cells (Fig. 4).

[0129] Example 4: M2KO virus produces viral antigens in normal cells but does not produce M2 This example shows that PR8 virus containing the M2KO(ΔTM) (SEQ ID NO:1) mutation produces viral antigens in normal cells but not M2 protein. Viral protein expression was assessed by infecting wild-type MDCK cells with wild-type PR8 or M2KO at a multiplicity of infection (MOI) of 0.5 in trypsin-free medium to ensure that the virus completed only one life cycle. Viral proteins in cell lysates were separated on 4-12% SDS-PAGE gels and detected by Western blot with PR8-infected mouse serum (panel A) or anti-M2 monoclonal antibody (14C2, Santa Cruz Biotechnology) (panel B). Figure 3A shows that antisera to PR8 detect similar levels of protein expression for both PR8 and M2KO. When lysates were probed with anti-M2 monoclonal antibody (panel B), M2 expression was detected only in PR8-infected cells and not in M2KO. These results indicate that the M2KO virus expresses all viral proteins except the M2 protein to levels similar to those of the PR8 virus (Fig. 5 ).

[0130] Example 5: M2 mutants are attenuated in vivo An experiment was performed to demonstrate that the M2 mutant virus is attenuated in vivo. Six-week-old female BALB / c mice (23 per group) were inoculated intranasally with one of the mutants M2KO(yk);M2-1(TM del M2KO aka M2KO(ΔTM)) and M2-2(Splice def M2KO) (collectively referred to as "M2KO mutants") described in J. Virol (2009) 83:5947-5950. The mutants were administered at a concentration of 1.2×10 4 pfu / mouse. Mice in the control group received PBS. Mice were observed for 14 days after inoculation for any weight changes and symptoms of infection. In addition, virus titers were obtained from the lungs and nasal turbinates (NT) of three mice from each group three days after inoculation.

[0131] As shown in Figure 6, mice inoculated with the M2KO mutant and PBS showed no clinical symptoms of infection or weight loss over 14 days. Weight fluctuations between groups were comparable over 14 days. In addition, no virus was detected in titers collected from lungs and NT. Taken together, the absence of clinical symptoms, weight loss, and virus indicates that the M2 virus is attenuated and non-pathogenic in mice.

[0132] Example 6: M2 mutant induces antibodies to influenza virus and protects mice from lethal virus challenge Tests were also performed to measure antibody titers from mice and survival after challenge with a lethal viral dose as described in Example 5 above. Serum samples were collected 3 weeks after inoculation and anti-viral IgG antibody titers from the serum samples were determined by enzyme-linked immunosorbent assay (ELISA). The humoral responses are shown in Figure 7 and show that all three M2 mutants raised anti-influenza virus antibodies higher than the control PBS group.

[0133] In addition, half of the mice in each group were boosted 28 days after inoculation with the same amount of M2 mutant virus. Serum was then collected 6 weeks after the first inoculation to determine IgG antibody titers to the virus. As shown in Figure 7B, mice boosted with M2 mutant virus had higher levels of anti-influenza virus antibodies than non-boosted mice.

[0134] Forty-nine days after the first inoculation (three weeks after the boost), mice were administered a lethal dose (50% lethal dose (MLD) of 40 mice). 50 )) PR8 virus. As shown in Figure 8 and Figure 9, all mice vaccinated with the M2KO mutant survived the challenge and did not lose weight. However, the control mice given only PBS lost weight and did not survive 8 days after the challenge date. Three days after challenge, lungs and NTs were obtained and virus titers were determined in MDCK cells by plaque assay. As shown in Table 11, the lung virus titers of the M2KO mutant were at least 1 log lower than those in the naive control PBS. Also, almost none of the virus was detected in the nasal turbinates in the M2KO mutant group, but more than 100,000 PFU / g was detected in the naive control PBS group, indicating that the M2 mutant vaccine confers protection and limits the replication of the challenge virus. [Table 11]

[0135] In another experiment, 6 weeks after immunization, the M2KO(ΔTM) group was challenged with homosubtypic or heterosubtypic influenza viruses. Mice were challenged with Aichi (H3N2) virus and assessed for survival over 14 days. The results of heterosubtypic challenge are shown in Figure 16.

[0136] Example 7: Intradermal Vaccine Delivery An experiment was conducted to demonstrate that intradermal vaccine delivery / immunization protects subjects against influenza. Six to seven week old female BALB / c mice (n = 5 per group) (Harland Laboratories) were administered 1.8 × 10 1 , 1.8×10 2 , 1.8×10 3 or 1.8 x 10 4 PR8 virus at a concentration of 3.5 × 10 pfu (50 μl) / animal 1 pfu) were inoculated either intranasally (IN), intramuscularly (IM) or intradermally (ID). Control mice also received PBS by three different routes of administration. Body weight and survival were monitored for 14 days after inoculation. In mouse experiments, an allergy syringe with an intradermal beveled needle was used.

[0137] The majority of vaccines are administered by intramuscular or subcutaneous injection using a conventional needle and syringe. However, recent studies have shown that intradermal vaccine delivery achieves better immunogenicity than intramuscular or subcutaneous administration. Intradermal vaccination delivers antigens directly to the abundant skin immune system and has been shown to be effective for a range of vaccines, including rabies, Hepatitis B and influenza. Intradermal delivery also results in dose savings, allowing the same immune response to be achieved with less vaccine than would be required for intramuscular injection. The current state of the art for intradermal delivery (using a conventional needle and syringe) is the Mantoux technique, but this method requires significant training, is difficult to perform, and often results in incorrect (subcutaneous) or incomplete dosing. Even though superior immune responses have been demonstrated with this route of administration, the lack of a suitable delivery device has hindered research and product development in intradermal vaccination.

[0138] As shown in Table 12, mice inoculated IN received 1.8 x 10 3 and 1.8 × 10 4 At the highest dose of pfu / animal, death from influenza infection occurred, and complete survival was observed at the lowest dose of 1.8 × 10 1However, mice inoculated IM and ID survived all doses. Table 13 shows the median lethal dose (MLD) for mice in the IN inoculation group. 50 ) Figure 10 shows the virus at 1.8 × 10 4 pfu inoculated IM and ID mice did not exhibit any weight fluctuations, and the virus was administered at 1.8 × 10 4 1 shows that pfu inoculated IN-vaccinated mice did not survive. [Table 12] [Table 13]

[0139] Sera were collected at 2 weeks (Fig. 11A) and 7 weeks (Fig. 11B) after inoculation and assessed for anti-PR8 IgG antibodies determined by ELISA. "Hi" was 1.8 × 10 4 "Lo" represents the pfu inoculum and "Lo" is 1.8 x 10 1 pfu are shown. The responses of IM and ID inoculated mice at both time periods are similar. At each time period, IN inoculated mice displayed the greatest number of antibodies. By this time, the IN inoculated mice receiving the higher dose had died, resulting in a total of 1.8 × 10 1 Only IN-inoculated mice inoculated with pfu were identified (i.e., "Lo"). Although IM- and ID-inoculated mice displayed lower levels of antibody than IN-inoculated mice, mice inoculated with the higher doses displayed greater amounts of antibody compared to control mice given PBS only. In addition, over time, the intradermal route of administration produced more antibody than the intramuscular route, as shown by the higher titer levels shown in FIG. 11B.

[0140] In a separate experiment, groups of IN, IM and ID inoculated mice (1.8 × 10 3 All mice (except for group 4, 5 mice / group) were challenged 8 weeks after vaccination. 1 IN-inoculated mice, 1.8 × 10 3 IM inoculated mice, 1.8 × 10 4IM inoculated mice, 1.8 × 10 3 I.D. inoculated mice, and 1.8 x 10 4 ID inoculated mice were challenged and mice that lost more than 25% body weight were euthanized.

[0141] As shown in Figure 12, a dose of 1.8 x 10 3 Of the mice vaccinated IM, 100% did not survive 8 days after challenge. The survival rate of all ID-vaccinated mice was between 40% and 60%. However, 1.8 × 10 4 The survival rate of the IM-inoculated mice was 100%. 4 Mice in the ) group had an initial average weight loss but ended up with a relatively low weight loss from the day of challenge.

[0142] ID inoculated mice (1.8 × 10 4 From the evaluation of the IM vaccination group (1.8 × 10 4 ) mice showed some symptoms and at least 10% weight loss.

[0143] Example 8: Stability of M2KO mutants To examine the stability of the M2 gene of the M2KO mutants in wild-type cells, the M2KO mutants were passaged in wild-type MDCK cells that do not show M2 protein expression, together with M2CK cells, which are MDCK cells that express M2 protein. All M2KO mutants could be passaged in M2CK cells without any mutations for at least 10 passages. M2-1 (TM del M2KO), M2-2 (Splice def M2KO), and M2-3 (TM del+Splice def M2KO) could not be passaged in wild-type MDCK cells (no cytopathic effect (CPE) was seen in wild-type MDCK cells), but M2KO(yk) showed CPE even after the fourth passage in MDCK cells. M segment RNA was extracted from the fourth passage of M2KO(yk) in wild-type MDCK, and cDNA was sequenced. As shown in Table 14, the two inserted stop codons of M2KO(yk) were edited, and the fourth passage of M2KO(yk) in wild-type MDCK had the full-length M2 protein gene. [Table 14]

[0144] Example 9: M2KO Vaccination To demonstrate that the M2KO vaccine can stimulate an immune response similar to natural influenza infection, vaccine experiments were performed. Natural influenza infection was represented by a low-dose inoculation of PR8 virus, and standard inactivated influenza vaccine was represented by inactivated PR8 virus (Charles River) delivered via the standard intramuscular route and intranasally.

[0145] Six to seven week old BALB / c mice were transfected with live virus (10 pfu PR8), PR8 virus containing M2KO(ΔTM) (10 4 M2KO(ΔTM) was immunized intranasally with 10 pfu of infectious particles or 1 μg of inactivated PR8 virus was delivered both intranasally and intramuscularly. 4Mice given PR8 intranasally did not lose any weight or show any signs of infection. Furthermore, the lungs of mice treated with M2KO(ΔTM) contained no detectable infectious particles 3 days after inoculation. Serum was obtained from immunized mice on day 21, and antibody titers against hemagglutinin were determined by standard ELISA assay. Figure 17 shows that anti-HA IgG titers were highest in the live virus and M2KO(ΔTM) groups versus the inactivated vaccine group. Mucosal IgA antibodies against influenza were detected only in the serum of live PR8 or M2KO vaccinated mice.

[0146] Six weeks after immunization, all groups were challenged with homosubtypic (PR8 H1N1) or heterosubtypic (Aichi, H3N2) influenza viruses. Both M2KO and inactivated vaccination protected mice from homosubtypic virus infection (Figure 18). However, only M2KO-vaccinated mice were protected from heterosubtypic virus challenge (Figure 19). Mice immunized with inactivated vaccine succumbed to infection similar to naive mice.

[0147] Example 10 M2KO(ΔTM) virus does not replicate in the airways or other organs Summary - This example shows that M2KO(ΔTM) virus does not replicate in the respiratory tract or spread to other organs in a ferret model. M2KO(ΔTM) virus was injected at 1×10 7 TCID 50 As a control, a second group of three male ferrets was administered 1 × 10 doses of A / Brisbane / 10 / 2007 (H3N2) influenza A virus intranasally. 7 TCID 50 The virus was administered intranasally at a dose of 0.01 mg / kg / day. After virus inoculation, ferrets were observed for mortality until day 3 post-inoculation, and body weight, temperature, and clinical signs were observed once daily. Necropsy was performed on all animals 3 days after inoculation. Organs were harvested for histopathological examination and virus titers.

[0148] The control group receiving A / Brisbane / 10 / 2007(H3N2) showed a transient weight loss and an increase in body temperature 2 days after inoculation that was not observed in the M2KO(ΔTM) group. Activity levels were also reduced in the A / Brisbane / 10 / 2007 group, with sneezing observed 2-3 days after infection. No changes in activity levels or clinical signs related to virus exposure were observed in the M2KO(ΔTM) group. Histopathological analysis revealed changes in the nasal turbinates in animals exposed to influenza A / Brisbane / 10 / 2007(H3N2) that were not seen in ferrets exposed to the M2KO(ΔTM) virus. Exposure to A / Brisbane / 10 / 2007 resulted in atrophy of the respiratory epithelium, neutrophil infiltration, and edema of the nasal turbinates. No other organs were affected by the virus inoculation. Under the conditions of the experiment, the M2KO(ΔTM) virus did not induce clinical signs of infection or result in histological variations in the organs analyzed.

[0149] material and method A. Vaccine material and control virus: M2KO(ΔTM) virus is a recombinant virus that harbors the six internal genes of PR8 (nucleoprotein (NP), polymerase genes (PA, PB1, PB2), nonstructural proteins (NS), matrix (M)) but does not express a functional M2 protein, as well as the HA and NA genes of influenza A / Brisbane / 10 / 2007-like A / Uruguay / 716 / 2007(H3N2). A / Brisbane / 10 / 2007(H3N2) wild-type virus served as the control virus and was provided by IITRI. Viruses were stored frozen at -65°C until use.

[0150] B. Test substance and positive control formulation: 1×1010 TCID 50 Dilute 8 µL of 10 / mL into 2.528 mL of PBS to obtain 1 x 10 per 316 µL. 7 TCID 50 A M2KO(ΔTM) dosing solution was prepared at 1 × 10 per 316 μL. 7 TCID 50A / Brisbane / 10 / 2007(H3N2) at a titer of 10 / mL was used undiluted.

[0151] C. Animals and Animal Husbandry: Eight male ferrets were purchased from Triple F Farm, and six ferrets were enrolled in the study. Animals were approximately 4 months of age at the start of the study. Animals were certified by the supplier to be healthy and free of antibodies to infectious diseases. Upon arrival, animals were housed individually in hanging wire cages with open bottoms and paper-lined litter trays. Animal rooms and cages were cleaned and sanitized prior to receipt of animals according to accepted animal care procedures and associated standard operating procedures. Certified Teklad Global Ferret Diet #2072 (Teklad Diets, Madison, WI) and Chicago tap water were provided ad libitum and were refreshed at least once daily. Fluorescent lighting in the animal room was maintained on a 12-hour light / dark cycle. Temperature and relative humidity in the animal room were within protocol limits and ranged from 22.0 to 25.0°C and 33 to 56%, respectively, during the study.

[0152] D. Animal Quarantine and Randomization: Ferrets were quarantined for 5 days and then randomized and observed daily. Ferrets were released from quarantine for randomization and testing based on daily observations indicating the animals' general health. After quarantine, ferrets were weighed and assigned to treatment groups using a weight-based computerized randomization procedure that produced similar group means (ToxData® version 2.1.E.11 (PDS Pathology Data Systems, Inc., Basel, Switzerland)). Within a group, the weights of all animals were within 20% of the mean. Animals selected for the study received permanent identification numbers by ear tag and transponder, and individual cage cards also identified the study animals by individual number and group. The assigned identification numbers were for that study only.

[0153] E. Experimental Design: All animal procedures were performed in an animal biosafety level 2 facility following protocols approved by the IIT Research Institute Animal Care and Use Committee. Six male ferrets (Triple F Farms, Sayre, PA), 4 months of age at the start of the study, were used in the study. Prior to infection, ferrets were monitored for 3 days for weight measurement and determination of base body temperature. Temperature measurements were recorded once daily with a transponder (BioMedic data systems, Seaford, DE) implanted subcutaneously in each ferret. Prior to the start of the study, blood was collected from the jugular vein and serum was tested for influenza antibodies. Test animals without influenza antibodies were randomized and divided into two groups (3 animals / group) as shown in Table 15. One group of 3 animals was anesthetized and injected with 1 x 10 6 of M2KO(ΔTM) virus. 7 TCID 50 A single dose of 316 μL was inoculated intranasally. The control group (3 mice) received 1 × 10 7 TCID 50 316 μL was inoculated. Ferrets were observed once a day to monitor body weight, temperature and clinical symptoms. On the third day after inoculation, ferrets (3 per group) were euthanized and subjected to necropsy. Tissue samples of nasal turbinates, trachea, lungs, kidneys, pancreas, olfactory bulbs, brain, liver, spleen, small intestine and large intestine were collected. Part of the collected samples was fixed in buffered neutral formalin for histological evaluation, and the other part of the samples was stored at -65°C for virus titer determination. [Table 15]

[0154] F. Viral inoculation: Ferrets were inoculated with either M2KO(ΔTM) virus or wild-type A / Brisbane / 10 / 2007(H3N2) influenza A virus. Vials of frozen stock were thawed and diluted to the appropriate concentrations in phosphate-buffered saline solution. Ferrets were anesthetized with ketamine / xylazine and virus doses were administered intranasally in a volume of 316 μL for M2KO(ΔTM) virus and 316 μL for A / Brisbane / 10 / 2007(H3N2) virus. To confirm the inoculum titer of A / Brisbane / 10 / 2007(H3N2) virus, TCID 50 Assays were performed at IITRI with aliquots of the prepared virus challenge solution. Viral titer assays were performed according to standard operating procedures at the Illinois Institute of Technology Research Institute (IITRI).

[0155] G. Moribund / Lethality Observations: After challenge, all animals were observed twice daily for evidence of mortality or moribundity. Animals were observed for 3 days after challenge. Animals were euthanized with an overdose of 150 mg / kg sodium pentobarbital administered intravenously.

[0156] H. Body Weight and Body Weight Fluctuations: Animal body weights were recorded at the time of receipt (10% randomized sample), at randomization (days 3-0 pre-study), and once daily after virus inoculation.

[0157] I. Clinical Observations: Temperature (in degrees Celsius) fluctuations were measured once a day for each ferret. Clinical signs of anorexia, respiratory signs such as dyspnea, sneezing, coughing, and rhinorrhea, and activity levels were assessed once a day. Activity levels were assessed using a grading system based on that described by Reuman, et al., “Assessment of signs of influenza illness in the ferret model,” J. Virol, Methods 24:27-34 (1989): 0, alert and playful; 1, alert but playful only when stimulated; 2, alert but not very playful when stimulated; 3, not alert and not very playful even when stimulated. Relative inactivity index (RII) was calculated as the mean score for each group of ferrets per observation (day) during the study period.

[0158] J. Euthanasia: Test animals were euthanized with an intravenous dose of 150 mg / kg sodium pentobarbital. Death was confirmed by the absence of observable heartbeat and respiration. Necropsy was performed on all test animals.

[0159] K. Necropsy: The nasal turbinates, trachea, lungs, kidneys, pancreas, olfactory bulb, brain, liver, spleen, small intestine, and large intestine were collected. A portion of each tissue was fixed in formalin and the other portion was given to IITRI personnel for freezing and storage. Tissues collected for titer were the right nasal turbinate, upper third of the trachea, right posterior lobe of the lung, right kidney, right side of the pancreas (near the duodenum), right olfactory bulb, right brain, right lateral lobe of the liver, right half of the spleen (the end of the spleen visible when the abdominal cavity is opened), small intestine, and large intestine.

[0160] L. Histopathological Analysis: Tissues were processed into paraffin blocks, sectioned at approximately 5 micron thickness, and stained with hematoxylin and eosin (H&E).

[0161] M. Serum Collection: Pre-vaccination (day 3 pre-dose) serum was collected from all ferrets. Ferrets were anesthetized with a mixture of ketamine (25 mg / kg) and xylazine (2 mg / kg). A blood sample (approximately 0.5-1.0 mL) was collected from the vena cava of each ferret and processed into serum. Blood was collected into Serum Gel Z / 1.1 tubes (Sarstedt Inc., Newton, NC) and stored at room temperature for no more than 1 hour before serum was collected. Serum was collected by centrifugation of the Serum Gel Z / 1.1 tubes at 10,000 x g for 3 minutes. Individual pre-vaccination serum samples were collected and two aliquots were made from each sample. One aliquot was tested prior to the start of the study to ensure that the ferrets did not have antibodies to influenza A virus, and the other aliquot of serum was stored at -65°C.

[0162] N. Hemagglutination Inhibition (HI) Assay: Serum samples were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) to eliminate inhibitors of nonspecific hemagglutination. RDE was regenerated according to the manufacturer's instructions. Serum was diluted 1:3 in RDE and incubated in a water bath at 37°C ± 2°C for 18–20 h. After adding an equal volume of 2.5% (v / v) sodium citrate, the samples were incubated in a water bath at 56°C ± 2°C for 30 ± 5 min. 0.85% NaCl was added to each sample after RDE treatment to a final serum dilution of 1:10. The diluted samples were then diluted in duplicate four times (1:10–1:80) in phosphate-buffered saline (PBS) and then incubated with 4 hemagglutination units of A / Brisbane / 10 / 2007 (H3N2) influenza A virus. After incubation, 0.5% chicken red blood cells were added to each sample and incubated, after which the presence or absence of hemagglutination was assessed.

[0163] O. Viral titer: The infectious virus concentration in samples before and after challenge virus inoculation was measured as TCID 50The cellular activity was determined by the method described above. Briefly, samples stored at -65°C were thawed and centrifuged to remove cell debris. The resulting supernatants were diluted 10-fold in triplicate in 96-well microtiter plates in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA) containing penicillin / streptomycin, 0.1% gentamicin, 3% NaCO3, 0.3% BSA fraction V (Sigma, St. Louis, MO), 1% MEM vitamin solution (Sigma) and 1% L-glutamine (Mediatech, Manassas, VA, USA). After making 10-fold serial dilutions, 100 L was transferred to each well of a 96-well plate containing a monolayer of MDCK cells. The plates were incubated at 37°C ± 2°C in 5 ± 2% CO2 and 70% humidity. After 48 hours, the wells were observed for cytopathic effects (CPE). Supernatants (50 μl) from each well were transferred to a 96-well plate to measure and record hemagglutination (HA) activity. HA activity of the supernatants was assessed by HA assay using 0.5% turkey packed red blood cells (cRBC). TCID 50 Titers were calculated using the method of Reed LJ and Muench H, "A simple method for estimating 50% endpoints," Am. J. Hygiene 27: 493-497 (1938).

[0164] P. Data Analysis: Body weight and body weight gain (loss) and body temperature fluctuations were measured for each individual animal and expressed as the mean and standard deviation of the mean for each test group.

[0165] result After inoculation with either M2KO(ΔTM) virus or A / Brisbane / 10 / 2007(H3N2) influenza A virus, ferrets were monitored for survival and clinical signs of infection. The results are presented in Tables 16A and 16B. All ferrets survived infection with M2KO(ΔTM) virus and A / Brisbane / 10 / 2007(H3N2). Ferrets inoculated with A / Brisbane / 10 / 2007 showed respiratory signs (sneezing) on ​​days 2 and 3. Ferrets inoculated with A / Brisbane / 10 / 2007 had a relative sluggishness index of 0.67, while ferrets inoculated with M2KO(ΔTM) did not show any reduction in activity level, with a relative sluggishness index of 0.0.

[0166] The variation in body weight and temperature after virus inoculation is shown in Figures 20 and 21. After inoculation with A / Brisbane / 10 / 2007(H3N2), a 2-3% weight loss was observed in all animals at 2 days post-inoculation. Minimal to zero weight loss was observed in ferrets inoculated with M2KO(ΔTM) virus. One ferret inoculated with M2KO(ΔTM) showed a 1% weight loss at 2 days post-inoculation. High body temperatures of 40.3-40.7°C were observed in ferrets inoculated with A / Brisbane / 10 / 2007 at 2 days post-inoculation. Body temperatures returned to normal range by day 3. Body temperatures in ferrets inoculated with M2KO(ΔTM) remained within normal range throughout the entire study period. To determine whether the M2KO(ΔTM) virus replicates and induces pathogenicity in the respiratory tract or other organs, ferret tissues were examined histologically 3 days after inoculation and compared with tissues from ferrets inoculated with A / Brisbane / 10 / 2007. In ferrets inoculated with A / Brisbane / 10 / 2007, pathogenicity was observed only in the nasal turbinates. Respiratory epithelial atrophy, neutrophil infiltration, and edema were observed in the nasal turbinates. No histopathological changes associated with viral infection were observed in ferrets inoculated with M2KO(ΔTM) virus. The concentrations of the virus administration solutions before and after challenge were 10 7.5 TCID50 / mL and 10 7.75 TCID 50 / mL, indicating good stability of the challenge material throughout dosing. [Table 16] [Table 17]

[0167] conclusion This example shows that the M2KO(ΔTM) virus does not induce clinical signs or histopathological changes associated with wild-type virus infection 3 days after inoculation, demonstrating that the M2KO(ΔTM) virus of the present technology is useful for a nasal influenza vaccine.

[0168] Example 11: Immune responses and protective effects of M2KO(ΔTM) virus versus other vaccines Overview - This example demonstrates the immune response elicited by and protective efficacy of the M2KO(ΔTM) vaccine in a ferret model. The M2KO(ΔTM) virus was administered at 1×10 7 TCID 50 As a control, a second group of 12 male ferrets was administered FM#6 virus intranasally at a dose level of 1 × 10 7 TCID 50A third group of ferrets received OPTI-MEM™ as a placebo control. Prime-only or prime-boost vaccination regimes were used for each treatment group. Ferrets that received the prime-boost vaccination regime were administered the prime vaccine (day 0) and boost vaccination 28 days later (day 28). Ferrets that received the prime vaccine only received a single vaccination on the same day that the boost vaccine was given to the prime-boost ferrets. After each vaccination, ferrets were observed for mortality for 14 days after vaccination, and weights, temperatures, and clinical signs were measured once a day. Nasal washes were collected from ferrets 1, 3, 5, 7, and 9 days after prime vaccination to probe for viral shedding. Nasal washes and serum were collected weekly from all ferrets after vaccination to assess antibody levels over time.

[0169] All animals were given 1 × 10 7 TCID 50 were challenged intranasally on day 56. After challenge, ferrets were observed for mortality for 14 days post-inoculation, and body weight, temperature, and clinical signs were measured daily. Nasal washes were collected from each group 1, 3, 5, 7, 9, and 14 days after challenge for virus titers. In addition, serum was collected from surviving ferrets post-challenge (day 70) for analysis. Necropsies were performed on 3 ferrets per group 3 days after challenge. Organs were harvested for histopathology and virus titers.

[0170] No vaccine-related adverse events were observed in any of the five groups. After challenge, the placebo control group showed an increase in body temperature on the second day after challenge, and a loss in body weight. A loss in body weight was also observed in the M2KO(ΔTM) and FM#6 vaccinated groups, but the loss was less than that observed in the OPTI-MEM™ group. Activity levels were not reduced in any of the groups, but sneezing was observed in all groups after challenge. Histopathological analysis revealed a higher severity of mixed cell infiltrates in the lungs of vaccinated ferrets compared to pulmonary infiltrates in the OPTI-MEM™ group. In the nasal turbinates, animals receiving either the M2KO(ΔTM) or FM#6 prime or prime+boost regimen had a less severe atrophy of the respiratory epithelium compared to the OPTI-MEM™ control group. Vaccination with the M2KO(ΔTM) virus appeared to confer protection against viral challenge similar to that of the FM#6 virus.

[0171] material and method A. Vaccine material: M2KO(ΔTM) virus is a recombinant virus that contains the six internal genes of PR8 (nucleoprotein (NP), polymerase genes (PA, PB1, PB2), nonstructural proteins (NS), and matrix (M)) but does not express a functional M2 protein, as well as the HA and NA genes of influenza A / Brisbane / 10 / 2007-like A / Uruguay / 716 / 2007 (H3N2). FM#6 virus is clone #6 of A / Uruguay / 716 / 2007 (H3N2) influenza A virus in FluMist® (2009-2010 formulation). M2KO(ΔTM) and FM#6 viruses were cultured at 1×10 7 TCID 50 Animals were administered a 316 μL dose (50% tissue culture infectious dose) intranasally.

[0172] B. Test substance and positive control formulations: 1 x 10 9 TCID 50 Dilute 120 µL of 10 / mL into 3.680 mL of PBS to obtain 1 x 10 per 316 µL. 7TCID 50 A M2KO(ΔTM) virus administration solution was prepared at 1 × 10 9 TCID 50 Dilute 120 µL of PBS into 3.680 mL of PBS to obtain 1 x 10 per 316 µL. 7 TCID 50 FM#6 was prepared at a titer of 100 / mL.

[0173] C. Animals and Animal Husbandry: Thirty-six male ferrets were purchased from Triple F Farm, and 30 ferrets were enrolled in the study. Animals were approximately 4 months old at the start of the study. Animals were guaranteed by the supplier to be healthy and free of antibodies to infectious diseases. Upon arrival, animals were housed individually in open-bottom hanging wire cages with paper-lined litter trays. Animal rooms and cages were cleaned and sanitized prior to receiving animals according to accepted animal care procedures and associated standard operating procedures. Certified Teklad Global Ferret Diet #2072 (Teklad Diets, Madison, WI) and Chicago tap water were provided ad libitum and were refreshed at least once daily. Fluorescent lighting in the animal room was maintained on a 12-hour light / dark cycle. Temperature and relative humidity in the animal room were within protocol limits and ranged from 22.0 to 25.0 °C and 30 to 63%, respectively, during the study.

[0174] D. Animal Quarantine and Randomization: Ferrets were quarantined for 7 days and then randomized and observed daily. Ferrets were released from quarantine for randomization and testing based on daily observations indicating the animals' general health. After quarantine, ferrets were weighed and assigned to treatment groups using a weight-based computerized randomization procedure that produced similar group means (ToxData® version 2.1.E.11 (PDS Pathology Data Systems, Inc., Basel, Switzerland)). Within a group, the weights of all animals were within 20% of the mean. Animals selected for the study received permanent identification numbers by ear tag and transponder, and individual cage cards also identified the study animals by individual number and group. The assigned identification numbers were for that study only.

[0175] E. Experimental Design: To evaluate the efficacy of the M2KO(ΔTM) vaccine, ferrets were immunized with M2KO(ΔTM) virus, cold-adapted live attenuated virus (FM#6), or sham-immunized with culture medium (OPTI-MEM™). Animal weight, temperature, and clinical signs were monitored to evaluate immunological responses. Thirty male ferrets (Triple F Farms, Sayre, PA), 4 months old at the start of the study, were used in the study. All animal procedures were performed in an animal biosafety level 2 or biosafety level 3 facility according to protocols approved by the IIT Research Institute Animal Care and Use Committee. Prior to inoculation, ferrets were monitored for 3 days for weight measurement and determination of base body temperature. Temperature measurements were recorded once daily with a transponder (BioMedic data systems, Seaford, DE) implanted subcutaneously in each ferret. Prior to the start of the study, blood was collected and serum was tested for influenza antibodies. Only ferrets with an HAI (hemagglutination inhibition) titer of 40 to A / Brisbane / 10 / 2007 (H3N2) were deemed seronegative and used in the study. Test animals were randomized into two groups (6 animals / group) as shown in Table 17. Two groups (1 and 3) received M2KO(ΔTM) virus and two groups (2 and 4) received FM#6 virus. One group (5) was sham immunized with OPTI-MEM™. Within each vaccine group, ferrets were divided into two regimens, with six receiving prime vaccination only (prime only) and six receiving prime vaccination followed by a boost vaccination 28 days after prime vaccination (prime / boost). Prime / boost group: Ferrets were administered 1×10 immunizations of M2KO(ΔTM) virus on days 0 and 28. 7 TCID 50 The control group was inoculated intranasally with a single dose of 316 μL. 7 TCID 50Ferrets were either inoculated intranasally with 316 μL (same dose as M2KO(ΔTM)) or sham-immunized with 316 μL OPTI-MEM™ on days 0 and 28. Ferrets were monitored daily for temperature, weight, and clinical signs for 14 days after inoculation. Nasal washes were collected from all ferrets, including the OPTI-MEM™ control group, on days 1, 3, 5, 7, 9, and 14 after prime vaccination for virus titer in cells, and on days 21 and 49 for antibody titers. Nasal wash samples were kept at -65°C. Blood was collected pre-inoculation (days 3-5) and on days 7, 21, 35, 42, and 49 and kept at -65°C until antibody titers were measured by ELISA and HI assays.

[0176] Prime-only group: Ferrets were inoculated with 1 × 10 M2KO(ΔTM) virus on day 28. 7 TCID 50 The control group received 1 × 10 FM#6 on day 28. 7 TCID 50 Ferrets were inoculated intranasally with a single dose of 316 μL (same dose as M2KO(ΔTM)) or sham inoculated with 316 μL OPTI-MEM™. Ferrets were monitored for temperature, weight, and clinical signs once daily for 14 days post-inoculation. Nasal washes were collected from all ferrets on days 29, 31, 33, 35, 37, and 42 for virus titer in cells, and on day 49 for antibody titer. Nasal wash samples were kept at -65°C. Blood was collected pre-inoculation (days 23-25) and on days 35, 42, and 49, and serum was kept at -65°C until antibody titer was measured by ELISA and HI assay. All ferrets were inoculated with 1 × 10 wild-type A / Brisbane / 10 / 2007 (H3N2) influenza virus on day 56, 4 weeks after prime / boost vaccination. 7 TCID 50The animals were challenged with a dose of 316 μL / mL. The weight, temperature and clinical symptoms of the ferrets were monitored for 14 days after challenge, and nasal washes and organs were collected. On days 1, 3, 5, 7, 9 and 14 (days 57, 59, 61, 63, 65 and 70) after challenge, nasal washes were collected from the challenged ferrets and samples were kept at −65°C in cells for virus titration. On day 3 after challenge (day 59), the animals (3 / group, total of 15 animals) were euthanized and the following tissue samples were collected: nasal turbinates, trachea, and lungs. Part of the collected samples was fixed in buffered neutral formalin for histological evaluation, and the other part of the samples was stored at −65°C for virus titration. Blood was collected 14 days after challenge (day 70) and all surviving animals were euthanized. [Table 18]

[0177] F. Viral inoculation: Ferrets were inoculated with either M2KO(ΔTM) or FM#6 influenza A viruses. Vials of frozen stock were thawed and diluted to the appropriate concentrations in phosphate buffered saline solution. Ferrets were anesthetized with ketamine / xylazine and virus doses were administered intranasally in volumes of 316 μL for M2KO(ΔTM) virus and 316 μL for FM#6. To confirm inoculum titers of M2KO(ΔTM) and FM#6 viruses, aliquots of the dosing solution were taken before (pre-dose) and after (post-dose) dosing. Aliquots were stored at -65°C for virus titer determination.

[0178] G. Challenge Virus: Influenza A virus, strain A / Brisbane / 10 / 2007, serotype H3N2, was used to challenge ferrets. Virus was stored at approximately -65°C prior to use. The challenge virus level used was 1 x 10 in a volume of 316 μL. 7 TCID 50 The quantitative viral infection assay, TCID 50Assays were performed at IITRI with aliquots of the prepared virus challenge solutions. Viral titer assays were performed according to IITRI standard operating procedures.

[0179] H. Moribund / Lethality Observations: After challenge, all animals were observed twice daily for evidence of mortality or moribundity. Animals were observed for 14 days post-vaccination and 14 days post-challenge.

[0180] I. Body Weight and Body Weight Variability: Animal body weights were recorded within 2 days of receipt and at randomization. All study animals were weighed pre-inoculation and once daily for 14 days after each vaccination and evaluated once daily for 14 days after challenge. Ferrets were monitored for 3-5 days prior to inoculation to ascertain basal body temperature. Temperature measurements were recorded via a transponder (BioMedic data systems, Seaford, DE) implanted subcutaneously in each ferret once daily for 14 days after each vaccination and once daily for 14 days after challenge. Body temperature (in degrees Celsius) variability was calculated for each ferret at each time point.

[0181] J. Clinical Observations: Temperature (in degrees Celsius) fluctuations were measured once a day for each ferret. Clinical signs of anorexia, respiratory signs such as dyspnea, sneezing, coughing, and rhinorrhea, and activity levels were assessed once a day. Activity levels were assessed using a grading system based on that described by Reuman, et al., “Assessment of signs of influenza illness in the ferret model,” J. Virol, Methods 24:27-34 (1989): 0, alert and playful; 1, alert but playful only when stimulated; 2, alert but not very playful when stimulated; 3, not alert and not very playful even when stimulated. Relative inactivity index (RII) was calculated as the mean score for each group of ferrets per observation (day) during the study period.

[0182] K. Survival Checks: Two survival checks were performed daily on all test animals throughout the study. Two survival checks were performed at the same time as clinical observations. The second survival check was performed later on the same day.

[0183] L. Nasal wash: Ferrets were anesthetized with a mixture of ketamine (25 mg / kg) and xylazine (2 mg / kg) and 0.5 ml of sterile PBS containing penicillin (100 U / ml), streptomycin (100 μg / ml), and gentamicin (50 μg / ml) was injected into each nostril and collected in a specimen cup once the ferret had deflated. Nasal washes were collected in cryovials and the volume recovered recorded.

[0184] M. Euthanasia: Test animals were euthanized with an intravenous dose of 150 mg / kg sodium pentobarbital. Death was confirmed by the absence of observable heartbeat and breathing.

[0185] N. Necropsy: The autopsy was performed by Charles River Laboratories, Pathology Associates (PAI). The PAI team included the Chief Pathology Officer and two autopsy technicians. The nasal turbinates, trachea, and lungs were harvested. A portion of each tissue was fixed in formalin, and the other portion was given to IITRI personnel for freezing and storage. Tissues harvested for titer were the right nasal turbinate, upper third of the trachea, and right posterior lobe of the lung.

[0186] O. Histopathological Analysis: After each necropsy, tissues were transported to the PAI Chicago facility. Upon receipt, partial tissues from all 15 ferrets were processed into paraffin blocks, sectioned at approximately 5 micron thickness, and stained with hematoxylin and eosin (H&E). All paraffin H&E slides were evaluated microscopically.

[0187] P. Serum Collection: Serum was collected from ferrets prior to vaccination (days 3-5 pre-dose for groups 3, 4, and 5, and days 23-25 ​​post-dose for groups 1 and 2). Serum was collected on days 7, 14, 21, 35, 42, 49, and 70 post-vaccination from groups 3, 4, and 5. Serum was collected on days 35, 42, 49, and 70 from groups 1 and 2. Ferrets were anesthetized with a mixture of ketamine (25 mg / kg) and xylazine (2 mg / kg). Blood samples (approximately 0.5-1.0 mL) were collected from the vena cava of each ferret and processed into serum. Blood was collected into Serum Gel Z / 1.1 tubes (Sarstedt Inc., Newton, NC) and stored at room temperature for no more than 1 hour before serum was collected. Serum was collected by centrifugation of the Serum Gel Z / 1.1 tubes at 10,000 × g for 3 min.

[0188] Q. Hemagglutination inhibition (HI) assay: Serum samples were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) to eliminate inhibitors of nonspecific hemagglutination. RDE was regenerated according to the manufacturer's instructions. Serum was diluted 1:3 in RDE and incubated in a water bath at 37°C ± 2°C for 18–20 h. After adding an equal volume of 2.5% (v / v) sodium citrate, the samples were incubated in a water bath at 56°C ± 2°C for 30 ± 5 min. 0.85% NaCl was added to each sample after RDE treatment to a final serum dilution of 1:10. The diluted samples were then diluted in duplicate four times (1:10–1:80) in phosphate-buffered saline (PBS) and then incubated with 4 hemagglutination units of A / Brisbane / 10 / 2007 (H3N2) influenza A virus. After incubation, 0.5% chicken red blood cells were added to each sample and incubated for 30±5 minutes, after which the presence or absence of hemagglutination was assessed.

[0189] R. Viral titers: The infectious virus concentration in samples before and after challenge was measured as TCID 50Assays were performed to determine the cytopathic effect of MDCK cells. Briefly, samples stored at -65°C were thawed and centrifuged to remove cell debris. The resulting supernatants were diluted 10-fold in triplicate in 96-well microtiter plates in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA) containing penicillin / streptomycin, 0.1% gentamicin, 3% NaCO3, 0.3% BSA fraction V (Sigma, St. Louis, MO), 1% MEM vitamin solution (Sigma) and 1% L-glutamine (Mediatech, Manassas, VA, USA). After making 10-fold serial dilutions, 100 μL was transferred to each well of a 96-well plate containing a monolayer of MDCK cells. Plates were incubated at 37°C ± 2°C in 5 ± 2% CO2 and 70% humidity. After 48 hours, wells were observed for cytopathic effect (CPE). Supernatants (50 μl) from each well were transferred to a 96-well plate to measure and record hemagglutination (HA) activity. HA activity in the supernatants was assessed by HA assay using 0.5% turkey packed red blood cells (tRBC). TCID 50 Titers were calculated using the method of Reed LJ and Muench H, "A simple method for estimating 50% endpoints," Am. J. Hygiene 27:493-497(1938).

[0190] S. Data Analysis: Body weight and body weight gain (loss) and body temperature fluctuations were measured for each individual animal and expressed as the mean and standard deviation of the mean for each test group.

[0191] result After nasal inoculation with either M2KO(ΔTM) or FM#6 virus, ferrets were monitored daily for clinical signs of infection. Nasal washes were collected after prime vaccination to monitor viral shedding, and serum was collected to measure serum antibody titers. The results are presented in Table 18A, Table 18B, and Table 18C. [Table 19] [Table 20] [Table 21]

[0192] All ferrets survived after vaccination with M2KO(ΔTM) and FM#6 viruses. After prime vaccination, two ferrets inoculated with M2KO(ΔTM) virus showed respiratory signs (sneezing) on ​​day 8. After boost vaccination, a ferret inoculated with FM#6 virus showed respiratory signs (sneezing) 7 days after vaccination. Sneezing was also observed in OPTI-MEM™ ferrets on day 4 after boost. After prime vaccination, the relative inactivity indexes of ferrets inoculated with M2KO(ΔTM) and FM#6 viruses were 0.07 and 0.27, respectively. This decrease in activity was observed in only one group per virus after prime vaccination. No decrease in activity level was observed after boost vaccination. The changes in body weight and temperature after virus vaccination are shown in Figures 22 and 23. No weight loss was observed after vaccination, but vaccination seemed to have an effect on weight gain. After vaccination, the body weight of OPTI-MEM™ control ferrets increased by 20% over the 14-day observation period, while the weight gain of M2KO(ΔTM) or FM#6 vaccinated ferrets was in the range of 6-15% after prime and 4-6% after boost. No increase in body temperature was observed in either group after vaccination. The changes in body weight and body temperature after challenge are shown in Figures 24 and 25, and the clinical signs are summarized in Table 19. [Table 22]

[0193] After challenge with A / Brisbane / 10 / 2007(H3N2), a 2-4% weight loss was observed in all animals on day 2 post-challenge. Animal weights remained below initial weight throughout the 14-day observation period. OPTI-MEM™ ferrets experienced the greatest weight loss (8%). Weight loss in vaccinated ferrets was dependent on the vaccine regimen. Ferrets receiving the prime-only regimen of M2KO(ΔTM) or FM#6 lost up to 5% and 4%, respectively. Booster-receiving ferrets lost up to 3% in the FM#6 group and up to 2% in the M2KO(ΔTM) group. A post-challenge increase in body temperature was observed on day 2 in OPTI-MEM™ ferrets and on day 1 in ferrets receiving the prime-only regimen of M2KO(ΔTM) or FM#6 (Figure 25). The body temperature of booster-receiving ferrets remained within the normal range.

[0194] To determine whether vaccination protects against challenge virus replication in the respiratory tract and reduces organ pathology, the pathology of challenged ferrets was examined histologically 3 days after inoculation. Lung changes in animals receiving the M2KO(ΔTM) prime only or prime / boost regimens were associated with increased severity of mixed cell infiltrates in the lungs compared to the OPTI-MEM™ group. A slight difference in the incidence of pulmonary infiltrates was observed between the M2KO(ΔTM) prime and M2KO(ΔTM) prime / boost groups. Increased severity of mixed cell infiltrates in the lungs was also observed between the FM#6 prime only and FM#6 prime / boost groups compared to the OPTI-MEM™ group. A slight increase in severity in pulmonary mixed cell infiltrates was observed in the FM#6 prime only group compared to the FM#6 prime / boost group. In the nasal turbinates, animals receiving the M2KO(ΔTM) virus prime or prime / boost had less severe respiratory epithelial atrophy compared to the OPTI-MEM™ group. There was no difference in nasal turbinate atrophy when comparing the M2KO(ΔTM) prime and prime / boost groups. A slightly increased severity of respiratory epithelial atrophy was observed in animals receiving the FM#6 prime / boost regimen compared to animals receiving the FM#6 prime only regimen, and the severity of respiratory epithelial atrophy in FM#6 animals was less than that observed in the OPTI-MEM™ group. The incidence of neutrophil infiltration into the nasal cavity (luminal) was reduced in the M2KO(ΔTM) prime and prime / boost groups compared to the OPTI-MEM™ group. Luminal infiltration of neutrophils in the M2KO(ΔTM) prime only group was interpreted as different from the OPTI-MEM™ group. The severity of luminal neutrophil infiltration in the FM#6 prime only and FM#6 prime / boost groups was slightly increased compared to the OPTI-MEM™ group. The concentrations of the virus administration solutions before and after challenge were 10 7 .83TCID 50 / mL and 10 7 .25TCID 50 / mL, indicating good stability of the challenge material throughout dosing.

[0195] FIG. 45 shows M2KO(ΔTM) and FluMist® virus replication in the respiratory tract of ferrets.

[0196] FIG. 46 shows M2KO(ΔTM) and FluMist® viruses in nasal washes following nasal challenge with A / Brisbane / 10 / 2007 (H3N2) virus.

[0197] FIG. 47 shows IgG titers in ferrets following vaccination with M2KO(ΔTM) and FluMist® prime only groups.

[0198] FIG. 48 shows IgG titers in ferrets following vaccination with M2KO(ΔTM) and FluMist® prime-boost groups.

[0199] FIG. 49 shows a summary of ELISA IgG titers in ferret sera vaccinated with M2KO(ΔTM) or FluMist® after challenge.

[0200] conclusion This example shows that nasal administration of the M2KO(ΔTM) virus was not associated with any vaccine-related adverse events (elevated body temperature, weight loss, or clinical signs). These results indicate that the M2KO(ΔTM) virus of the present technology is useful for use in a nasal influenza vaccine.

[0201] Example 12: M2KO(ΔTM) virus is not transmitted in the ferret model Summary - This example shows that M2KO(ΔTM) virus is not transmitted in the ferret model. M2KO(ΔTM) virus was injected at 1×10 7 TCID 50 As a control, a second group of three female ferrets was administered the A / Brisbane / 10 / 2007 (H3N2) virus intranasally at a dose level of 1 × 10 7 TCID 50The donor ferrets were administered intranasally at a dose of 0.01 mg / kg / day. Twenty-four hours after inoculation, each donor ferret was introduced into a transmission chamber along with two naive ferrets (direct and aerosol contact). After inoculation, the ferrets were observed for mortality for 14 days after inoculation, and body weight, temperature, and clinical signs were measured once daily. Nasal washes were collected from all inoculated donor ferrets on days 1, 3, 5, 7, and 9, and from all contact (direct and aerosol) ferrets on days 2, 4, 6, 8, and 10 to probe for virus shedding. Nasal washes and serum were collected from all ferrets at the time of inoculation of the study (day 14) to assess antibody levels. No clinical signs of infection were observed in the M2KO(ΔTM) group, whereas the A / Brisbane / 10 / 2007(H3N2) group had weight loss, elevated body temperature, and sneezing. After inoculation with Brisbane / 10, donor ferrets showed elevated body temperature and weight loss 2 days after challenge. Activity levels did not decrease in either group. Ferrets in direct contact with donor ferrets showed a gradual increase in body weight until day 4 after inoculation. A similar trend was observed in aerosol-contact ferrets, which began on day 6 after inoculation. Weight loss in contact ferrets correlated with elevated body temperature. Inoculation with M2KO(ΔTM) virus did not induce clinical signs of infection in inoculated animals. Transmission to contact ferrets is unlikely.

[0202] material and method A. Vaccine material: The M2KO(ΔTM) virus is a recombinant virus that contains six internal genes of PR8 (nucleoprotein (NP), polymerase genes (PA, PB1, PB2), nonstructural proteins (NS), and matrix (M)) but does not express a functional M2 protein, nor the HA and NA genes of influenza A / Brisbane / 10 / 2007-like A / Uruguay / 716 / 2007 (H3N2). The M2KO(ΔTM) virus was administered at 1×107 TCID 50 Animals were administered a 316 μL dose (50% tissue culture infectious dose) intranasally.

[0203] B. Test substance administration formulation: 1 x 10 9 TCID50 Dilute 45 µL of 10 / mL into 1.377 mL of PBS to obtain 1 x 10 per 316 µL. 7 TCID 50 1 / mL M2KO(ΔTM) virus dosing solution was prepared.

[0204] C. Animals and Animal Husbandry: Twenty-two female ferrets were purchased from Triple F Farm, and 18 ferrets were enrolled in the study. Animals were approximately 4 months old at the start of the study. Animals were guaranteed by the supplier to be healthy and free of antibodies to infectious diseases. Upon arrival, animals were housed individually in open-bottom hanging wire cages with paper-lined litter trays. Animal rooms and cages were cleaned and sanitized prior to receiving animals according to accepted animal care procedures and associated standard operating procedures. Certified Teklad Global Ferret Diet #2072 (Teklad Diets, Madison, WI) and Chicago tap water were provided ad libitum and were refreshed at least once daily. Fluorescent lighting in the animal room was maintained on a 12-hour light / dark cycle. Temperature and relative humidity in the animal room were within protocol limits and ranged from 23.0 to 25.0 °C and 36 to 50%, respectively, during the study.

[0205] D. Animal Quarantine and Randomization: Ferrets were quarantined for 7 days and then randomized and observed daily. Ferrets were released from quarantine for randomization and testing based on daily observations indicating the animals' general health. After quarantine, ferrets were weighed and assigned to treatment groups using a weight-based computerized randomization procedure that produced similar group means (ToxData® version 2.1.E.11 (PDS Pathology Data Systems, Inc., Basel, Switzerland)). Within a group, the weights of all animals were within 20% of the mean. Animals selected for the study received permanent identification numbers by ear tag and transponder, and individual cage cards also identified the study animals by individual number and group. The assigned identification numbers were for that study only.

[0206] E. Experimental Design: To evaluate the transmissibility of M2KO(ΔTM) virus, ferrets were inoculated with M2KO(ΔTM) virus or A / Brisbane / 10 / 2007(H3N2) virus. Animals were monitored for weight, temperature, clinical signs, and viral shedding to evaluate immune responses. Eighteen female ferrets (Triple F Farms, Sayre, PA), 4 months old at the start of the study, were used in the study. All animal procedures were performed in an animal biosafety level 2 or live level 3 facility. Prior to inoculation, ferrets were monitored for 3 days for weight measurement and determination of base body temperature. Temperature measurements were recorded once daily with a transponder (BioMedic data systems, Seaford, DE) implanted subcutaneously in each ferret. Prior to the start of the study, blood was collected and serum was tested for influenza antibodies. Only ferrets with an HI titer of 40 to the A / Brisbane / 10 / 2007(H3N2) virus were deemed seronegative and included in the study. Test animals were randomized into two groups (9 animals / group, 3 / transmission chamber) as shown in Table 20. Ferrets in group 1 (chambers A-C) were assigned to receive M2KO(ΔTM) virus. Ferrets in group 2 (chambers A-C) were assigned to receive A / Brisbane / 10 / 2007(H3N2) virus. Within each group, ferrets were assigned to be inoculated or naive donors. [Table 23]

[0207] Each group was housed in a separate room, with a strict flow pattern after individual work with the animals to prevent cross-contamination between the two groups. In each group, one donor ferret was administered 1 × 10 7 TCID 50 (group 1) or 1 × 10 7 TCID 50(Group 2) were inoculated intranasally with a single dose of 316 μl. Twenty-four hours after inoculation, each donor was placed in the same cage as one naive ferret (direct contact), housed two per wire cage. An additional ferret (aerosol contact) was placed in a separate adjacent wire cage in the transmission chamber, separated from the donor cage by a distance of 10–12 cm (individual housing). The temperature, weight, and clinical signs of the ferrets were monitored once daily for 14 days after inoculation. For virus titer determination in cells, nasal washes were collected from all inoculated donor ferrets on days 1, 3, 5, 7, and 9, and from all contact (direct and aerosol) ferrets on days 2, 4, 6, 8, and 10. Nasal wash samples were collected from all ferrets on day 14 for antibody titer determination. Nasal wash samples were kept at -65°C.

[0208] F. Transmission chambers: Each transmission chamber was 2 m3. A computer-controlled air conditioner was used for HEPA filtration as well as for monitoring and controlling the environmental conditions within the transmission chambers. To provide directional airflow, HEPA-filtered air was supplied through an inlet located at one end of the chamber and exhausted through an outlet at the opposite end of the chamber, HEPA-filtered, and exhausted back into the room. The air exchange rate was 20 complete air changes per hour in each chamber, and airflow was maintained at <0.1 m / sec. The chambers were maintained at a negative pressure of -0.15 inches of water. Ferrets were housed in open-bottom wire cages with paper-lined waste trays. Ferrets were housed two per 32 x 24 x 14 cage or individually in 24 x 24 x 14 wire cages housed inside each HEPA-filtered transmission chamber.

[0209] G. Viral inoculation: Ferrets were inoculated with M2KO(ΔTM) virus. A vial of frozen stock was thawed and diluted to the appropriate concentration in phosphate buffered saline solution. Ferrets were anesthetized with ketamine / xylazine and the virus dose was administered intranasally in a volume of 316 μL. To confirm the inoculum titer of M2KO(ΔTM) virus, aliquots of the dosing solution were taken before (pre-dose) and after (post-dose) dosing. Aliquots were stored at -65°C for virus titer determination.

[0210] H. Challenge Virus: Influenza A virus strain A / Brisbane / 10 / 2007, serotype H3N2, was used to inoculate control ferrets. Virus was stored at approximately -65°C prior to use. The dose level of challenge virus used was 1 x 107 TCID 50 The quantitative viral infection assay, TCID 50 Assays were performed at IITRI with aliquots of the prepared virus challenge solutions. Viral titer assays were performed according to IITRI standard operating procedures.

[0211] I. Moribund / Lethality Observations: After challenge, all animals were observed twice daily for evidence of mortality or moribundity. Animals were observed for 14 days post-vaccination and 14 days post-challenge.

[0212] J. Body Weight and Body Weight Variability: Animal body weights were recorded within 2 days of receipt and at randomization. All study animals were weighed pre-inoculation and once daily for 14 days after each vaccination and evaluated once daily for 14 days after challenge. Ferrets were monitored for 3-5 days prior to inoculation to ascertain basal body temperature. Temperature measurements were recorded via a transponder (BioMedic data systems, Seaford, DE) implanted subcutaneously in each ferret once daily for 14 days after each vaccination and once daily for 14 days after challenge. Body temperature (in degrees Celsius) variability was calculated for each animal at each time point.

[0213] K. Clinical Observations: Temperature (in degrees Celsius) fluctuations were measured once a day for each ferret. Clinical signs of anorexia, respiratory signs such as dyspnea, sneezing, coughing, and rhinorrhea, and activity levels were assessed once a day. Activity levels were assessed using a grading system based on that described by Reuman, et al., “Assessment of signs of influenza illness in the ferret model,” J. Virol, Methods 24:27-34 (1989): 0, alert and playful; 1, alert but playful only when stimulated; 2, alert but not very playful when stimulated; 3, not alert and not very playful even when stimulated. Relative inactivity index (RII) was calculated as the mean score for each group of ferrets per observation (day) during the study period.

[0214] L. Survival Checks: Two survival checks were performed on all test animals once per day throughout the study. Two survival checks were performed at the same time as clinical observations. The second check was performed later on the same day.

[0215] M. Nasal wash: Ferrets were anesthetized with a mixture of ketamine (25 mg / kg) and xylazine (2 mg / kg) and 0.5 ml of sterile PBS containing penicillin (100 U / ml), streptomycin (100 μg / ml), and gentamicin (50) was injected into each nostril and collected in a specimen cup once the ferret had excreted.

[0216] N. Euthanasia: Test animals were euthanized with an intravenous dose of 150 mg / kg sodium pentobarbital. Death was confirmed by the absence of observable heartbeat and respiration. Necropsy was performed on all test animals.

[0217] O. Serum Collection: Pre-vaccination serum (days 3-5) and post-vaccination serum (day 14) were collected from all ferrets. Ferrets were anesthetized with a mixture of ketamine (25 mg / kg) and xylazine (2 mg / kg). Blood samples (approximately 0.5-1.0 mL) were collected from the vena cava of each ferret and processed into serum. Blood was collected into Serum Gel Z / 1.1 tubes (Sarstedt Inc., Newton, NC) and stored at room temperature for no more than 1 hour before serum was collected. Serum was collected by centrifugation of the Serum Gel Z / 1.1 tubes at 10,000 x g for 3 minutes. Individual post-vaccination serum samples were collected and two aliquots were made from each sample. One aliquot was tested prior to the start of the study to ensure that the ferrets did not have influenza A virus antibodies, and the other aliquot of serum was stored at -65°C.

[0218] P. Hemagglutination Inhibition (HI) Assay: Serum samples were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) to eliminate inhibitors of nonspecific hemagglutination. RDE was regenerated according to the manufacturer's instructions. Serum was diluted 1:3 in RDE and incubated in a water bath at 37°C ± 2°C for 18–20 h. After adding an equal volume of 2.5% (v / v) sodium citrate, the samples were incubated in a water bath at 56°C ± 2°C for 30 ± 5 min. 0.85% NaCl was added to each sample after RDE treatment to a final serum dilution of 1:10. The diluted samples were then diluted in duplicate four times (1:10–1:80) in phosphate-buffered saline (PBS) and then incubated with 4 hemagglutination units of A / Brisbane / 10 / 2007 (H3N2) influenza A virus. After incubation, 0.5% chicken red blood cells were added to each sample and incubated for 30±5 minutes, after which the presence or absence of hemagglutination was assessed.

[0219] Q. Viral titer: The infectious virus concentration in samples before and after challenge virus inoculation was measured in TCID 50The MDCK activity was determined by the assay. Briefly, samples stored at -65°C were thawed and centrifuged to remove cell debris. The resulting supernatants were diluted 10-fold in triplicate in 96-well microtiter plates in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA) containing penicillin / streptomycin, 0.1% gentamicin, 3% NaCO3, 0.3% BSA fraction V (Sigma, St. Louis, MO), 1% MEM vitamin solution (Sigma) and 1% L-glutamine (Mediatech, Manassas, VA, USA). After making 10-fold serial dilutions, 100 flL was transferred to each well of a 96-well plate containing a monolayer of MDCK cells. The plates were incubated at 37°C ± 2°C in 5 ± 2% CO2 and 70% humidity. After 48 hours, the wells were observed for cytopathic effects (CPE). Supernatants (50 μl) from each well were transferred to a 96-well plate to measure and record hemagglutination (HA) activity. HA activity in the supernatants was assessed by HA assay using 0.5% turkey packed red blood cells (tRBC). TCID 50 Titers were calculated using the method of Reed LJ and Muench H, "A simple method for estimating 50% endpoints," Am. J. Hygiene 27:493-497(1938).

[0220] R. Data Analysis: Body weight and body weight gain (loss) and body temperature fluctuations were measured for each individual animal and expressed as the mean and standard deviation of the mean for each test group.

[0221] result After inoculation of donor ferrets with either M2KO(ΔTM) virus or A / Brisbane / 10 / 2007(H3N2) influenza A, the donor ferrets were introduced into a transmission chamber containing naive contact ferrets. The ferrets were monitored daily for clinical signs of infection, nasal washes were collected to monitor viral shedding, and serum was collected to measure serum antibody titers. All of the ferrets survived inoculation with M2KO(ΔTM) virus and A / Brisbane / 10 / 2007 (Table 21). No clinical signs of disease were observed in any of the ferrets in the M2KO(ΔTM) group. Two of three donor ferrets inoculated with A / Brisbane / 10 / 2007(H3N2) virus showed respiratory signs (sneezing) on ​​days 6 and 8. No sneezing was observed in any of the aerosol contact ferrets. No reduction in activity levels was observed. [Table 24]

[0222] The variation in body weight and temperature after virus inoculation is shown in Figure 26 and Figure 27. No significant weight loss was observed after inoculation with M2KO(ΔTM) virus. Aerosol contacts had an average of 1% weight loss per day, which is unlikely due to exposure to the virus. The weight gain of ferrets in M2KO(ΔTM) virus was 9% in donor ferrets and 10-11% in contact ferrets over the 14-day observation period (Figure 26A). The weight gain in A / Brisbane / 10 / 2007 was only 3% in donor ferrets and 6-8% in contact ferrets, indicating virus infection (Figure 26B). In the M2KO(ΔTM) group, body temperature remained at normal levels except on the third day after infection (Figure 27A). Body temperature was lower than normal in aerosol contact ferrets. This was due to a malfunction or defect in the temperature transponder, and temperatures were recorded within the normal range throughout the remainder of the study. High body temperatures were observed in A / Brisbane / 10 / 2007 donor ferrets on day 2 and in aerosol-exposed ferrets on day 7 (Figure 27B). 7.50 TCID 50 / mL and 10 7.25 TCID 50 / mL, indicating good stability of the challenge material throughout dosing.

[0223] Figure 50 shows virus titers in nasal washes from ferrets in a virus transmission study. The data show that the M2KO(ΔTM) virus does not transmit (no virus detected), but the control Brisb / 10 virus does.

[0224] conclusion This example shows that ferrets inoculated with A / Brisbane / 10 / 2007 virus showed clinical signs of infection (sneezing, weight loss and transient temperature increase), whereas ferrets inoculated with M2KO(ΔTM) virus did not show any clinical signs of disease. Therefore, inoculation of donor ferrets with M2KO(ΔTM) did not appear to induce infection or virus transmission via contact or aerosol. These findings indicate that the M2KO(ΔTM) virus of the present technology is useful for nasal influenza vaccine.

[0225] Example 13. M2KO(ΔTM) virus induces both humoral and mucosal immune responses in mice This example shows that M2KO(ΔTM) virus induces both humoral and mucosal immune responses in mice. The immunogenicity of M2KO(ΔTM) was evaluated in mice and compared with immune responses generated by other modes of vaccination. Immunogenicity studies were performed in the following groups as outlined in Table 22: 1. M2KO(ΔTM) virus, 2. PR8 virus (10 pfu) live vaccine representative, 3. Inactivated PR8 virus (Charles River Laboratories, Wilmington, MA), 1 μg, intranasal (IN), 4. Inactivated PR8 virus, 1 μg, intramuscular (IM) or PBS only. [Table 25]

[0226] To test the immunogenicity of the M2KO(ΔTM) virus, mice were infected with 1.2 × 10 4Mice were inoculated intranasally with 10 pfu of wild-type PR8, 10 pfu of inactivated whole PR8 (Charles River Laboratories, Wilmington, MA), or control PR8, and one group was given 1 μg of inactivated whole PR8 intramuscularly. Three weeks after immunization, serum and tracheal-lung lavage fluids were collected from the mice, and anti-PR8 immunoglobulin G (IgG) and IgA levels were measured by enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with inactivated whole PR8, blocked with bovine serum albumin (BSA), and samples were applied. Mouse IgG and IgA antibodies were detected with horseradish peroxidase-conjugated anti-mouse IgG and IgA goat antibodies (KPL, Inc., Gaithersburg, MD) and SureBlue TMB (KPL, Inc.) substrate.

[0227] As expected, mice in the immunized group showed a significant increase in anti-PR8 antibodies in serum and tracheal-lung lavage fluid compared to the PBS-only group (Figure 28). The anti-PR8 IgG levels in serum in the case of M2KO(ΔTM) virus were higher than those in the inactivated PR8 group and similar to those of live PR8 virus. More importantly, anti-PR8 IgA antibodies were present only in PR8 and M2KO(ΔTM) immunized mice in both serum and tracheal-lung lavage fluid. These data suggest that M2KO(ΔTM) virus induces significant humoral and mucosal immune responses in mice.

[0228] Example 14: M2KO(ΔTM) virus protects mice from lethal homosubtypic and heterosubtypic challenges These examples show that M2KO(ΔTM) virus protects mice from lethal homosubtypic and heterosubtypic challenges. Six weeks after immunization, the protective efficacy of M2KO(ΔTM) virus was evaluated by challenging immunized mice with a lethal dose of wild-type PR8 (H1N1; homosubtypic challenge) or mouse-adapted influenza A / Aichi / 2 / 68 (Aichi; H3N2; heterosubtypic challenge). None of the mice immunized with M2KO(ΔTM) or PR8 10 pfu and subsequently challenged with wild-type PR8 showed any clinical symptoms, such as weight loss (Figure 29A). In contrast, naive PBS mice died or were euthanized due to weight loss of more than 20% by day 5. Viral replication in the airways of challenged mice was measured using TCID 50 Assay was performed 3 days after challenge. As shown in Figure 30A, no virus was detected in the lungs of M2KO(ΔTM) or PR8 immunized mice (detection limit 10 2.75 TCID 50 / organ), indicating that M2KO(ΔTM) provides sterile immunity similar to PR8 infection, whereas challenge virus was not recovered from the inactivated PR8 and PBS groups.

[0229] For heterosubtypic challenge, mice were challenged with Aichi (H3N2). M2KO(ΔTM) and wild-type PR8 immunized mice survived the challenge, whereas mice receiving inactivated PR8 or PBS succumbed to infection (Figure 29). Viral titers in the mouse airways 3 days after challenge were not significantly reduced in M2KO(ΔTM) vaccinated mice compared to mice in other groups (Figure 30). These results suggest that the cross-protection observed against Aichi challenge may be due in part to T cell-mediated immune responses induced by the M2KO(ΔTM) vaccine. Hemagglutination inhibition (HI) antibodies to Aichi were undetectable in post-challenge sera from challenged mice (<1:40), suggesting that protection was not mediated by neutralizing antibodies.

[0230] The M2KO(ΔTM) virus stimulates both humoral and cellular immune responses against lethal homo- and heterosubtypic challenges, conferring protective immunity to animals, as summarized in Table 23. [Table 26]

[0231] Example 15 M2KO(ΔTM) Vaccine Compared to Fluzone® and FluMist® This example shows the efficacy of M2KO(ΔTM) virus compared to live attenuated virus (FluMist®), Fluzone® inactivated influenza vaccine. Mice were immunized with M2KO(ΔTM) virus, cold-adapted live attenuated virus (FluMist®), Fluzone® inactivated influenza vaccine, or sham immunized with PBS. M2KO(ΔTM)-H3 virus was constructed by inserting the HA and NA coding sequences of influenza A / Brisbane / 10 / 2007-like A / Uruguary / 716 / 2007 (H3N2) into the M2KO(ΔTM) backbone (SEQ ID NO: 1). FluMist®-H3 internal genes derived from the cold-adapted A / AA / 6 / 60 backbone containing the HA and NA genes of influenza A / Brisbane / 10 / 2007-like A / Uruguary / 716 / 2007 (H3N2) were plaque-purified from the 2009 / 2010 trivalent vaccine formulation. The Fluzone® 2009 / 2010 formulation was used directly as the trivalent formulation.

[0232] Sera were obtained 7, 14, and 21 days after immunization to compare the kinetics of antibody responses by ELISA (Figure 31). M2KO(ΔTM)-H3 virus, a replication-deficient virus, generated antibodies earlier than FluMist®-H3, a live influenza virus that undergoes multi-cycle replication in an attenuated manner. The inactivated vaccine Fluzone® had the highest serum antibody titers due to its high antigen presentation.

[0233] The presence of anti-HA mucosal antibodies in serum, lung lavage fluid and nasal turbinates was assessed by ELISA. M2KO(ΔTM)-H3 and FluMist® (both live influenza vaccines) had higher IgA in the airways than the inactivated vaccine Fluzone® (Figure 32).

[0234] Example 16: Comparison of protection and immunogenicity induced by live viruses Six-week-old female BALB / c mice were anesthetized with isoflurane and administered 10 mL of M2KO(ΔTM)-H3 (previously described), FluMist® (2009-2010) (H3N2) IVR-147 (PR8×Brisbane / 10 / 2007) on days 0 and 28. 6 TCID 50 Mice were infected intranasally with 50 μl / mL of IVR-147. IVR-147 is the wild type of M2KO(ΔTM) virus, i.e., contains a functional M2 protein. Mock-infected control mice received 50 μl of PBS instead of virus. Serum was collected from all mice once a week and analyzed for the presence of anti-HA antibodies by ELISA. As shown in FIG. 33, M2KO(ΔTM) virus and IVR-147 rapidly produced higher antibody levels compared to FluMist®.

[0235] Animal weight was monitored for 14 days after infection. Vaccinated mice did not have any weight loss. 21 days after boost, 3 mice per group were euthanized and tracheal-lung washes, nasal washes, and serum were collected for antibody titration (Figure 34). M2KO(ΔTM) induced both humoral and mucosal antibodies in serum and airways to similar levels as FluMist® and IVR-147.

[0236] Mice were immunized with A / Aichi / 2 / 68 virus 40MLD 6 weeks after the boost. 50 Mice were monitored for weight loss and survival for 14 days (Figure 35). M2KO(ΔTM) protected mice from lethal Aichi challenge as shown by less weight loss (Panel A) and 100% survival (Panel B) in contrast to FluMist®. Three days after challenge, three mice per group were euthanized and lungs and nasal turbinates were harvested for virus titration (Table 24). M2KO(ΔTM) controlled the challenge virus better than FluMist® as shown in Table 24. [Table 27]

[0237] Example 17: Generation of M2KO(ΔTM) vaccine against highly pathogenic avian H5n1 influenza virus Overview-M2KO(ΔTM) is an influenza virus that does not express functional M2 protein. M2 protein is essential for the initiation of influenza virus infection and efficient viral RNA incorporation into progeny viruses. M2KO(ΔTM) can enter cells and express viral proteins, but cannot generate infectious progeny viruses due to deletion of the M2 gene. M2KO(ΔTM) is generated in permissive M2 protein-expressing cells, but not in non-permissive wild-type cells. M2KO(ΔTM) induces both mucosal and humoral immunity in mice and protects against both homozygous and heterozygous subtype lethal challenge.

[0238] The H5N1 M2KO(ΔTM) virus contains the HA (avirulent) and NA genes of A / Vietnam / 1203 / 2004 in an M2KO(ΔTM) backbone. "M2KO(ΔTM) backbone" refers to the sequence of PR8 containing the M2KO(ΔTM) (SEQ ID NO:1) mutation. The A / Vietnam / 1203 / 2004 (avirulent) and NA sequences used are shown below. [ka]

[0239] Generation of H5N1 M2KO(ΔTM): Avirulent HA and NA of A / Vietnam / 1203 / 2004 (H5N1) were chemically synthesized by GeneArt® Gene Synthesis based on the CDC sequences of each gene (CDC ID: 2004706280, Accession Nos: EF541467 and EF541403). The constructs were sequence verified and subcloned into appropriate vectors to generate seed viruses using standard protocols.

[0240] M2KO(ΔTM)VN1203avHA,NA (H5N1 M2KO(ΔTM)) virus was amplified in M2CK cells (MDCK cells stably expressing M2 protein), and the supernatant was cleared of cell debris and concentrated 100-fold with Centricon Plus-70 (Millipore). This virus was used as the immunogen in mouse studies.

[0241] Mouse study design: Mice (7-8 weeks old, female BALB / c) were cultured in a 10-mL tube with H5N1 M2KO(ΔTM) 6 TCID 50 / fish), M2KO(ΔTM)CA07HA,NA(10 6 TCID 50Mice were inoculated intranasally with 1.5 μg / mouse or intramuscularly with VN1203 protein (1.5 μg). Body weight and clinical signs were observed for 14 days after inoculation. Serum was collected on days 7, 14, and 21 after inoculation. On day 28, mice were boosted with a new prime-only group started at the same time.

[0242] Boost immunization and "prime only" groups: On day 28, mice pre-vaccinated with H5N1 M2KO(ΔTM) were 6 pfu / mouse. At the same time, the "prime only" group received their first dose. Body weights were taken of all groups after vaccination on day 28. Mice receiving the boost dose of M2KO(ΔTM) vaccine lost at most 5% body weight. The "prime only" group lost up to 10% body weight. [Table 28]

[0243] H5N1 M2KO(ΔTM) induces IgG antibody titers to HA: Sera were obtained from mice on days 7, 14, and 21 post-inoculation and analyzed by ELISA for antibodies to hemagglutinin. M2KO(ΔTM) produced titers at least 100-fold higher than the H5HA protein (Figure 36). Mice were boosted on day 28 and serum obtained one week later (day 35). Titers of M2KO(ΔTM) were boosted 130-fold, whereas HA protein was boosted only 13-fold. First week bleeds on day 35 in the M2KO(ΔTM) prime-only group showed high IgG titers similar to the first week of the prime-boost group.

[0244] Mice were inoculated with a lethal dose (20 MLD) of the Vietnam / 1203 / 2004 virus. 50). All mice vaccinated with H5N1 M2KO(ΔTM) (prime only and prime-boost) survived (Figures 54 and 55). The high survival rate of mice challenged 5 months after immunization suggests that the H5N1 M2KO(ΔTM) vaccine stimulates a memory response. Mice challenged 4 weeks after immunization received only one dose of vaccine, indicating that the M2KO(ΔTM) vaccine stimulates a strong immune response. Mice immunized with H1N1pdmM2KO(ΔTM) also survived 5 months after H5N1 challenge, indicating that M2KO(ΔTM) stimulates a cross-reactive immune response and provides protection against heterologous challenge.

[0245] Example 18: H1N1pdm: FluMist® CA07 vs. M2KO(ΔTM) CA07 A / California / 07 / 2009 (CA07) (H1N1pdm) HA and NA cDNA clones were generated by standard molecular biology protocols. Constructs were sequence verified and subcloned into appropriate vectors to generate seed M2KO(ΔTM) and M2WTCA07 / PR8 viruses using standard protocols. FluMist® CA07 (H1N1pdm) was plaque purified from FluMist® 2011-2012 vaccine Lot#B11K1802 in MDCK cells. The A / California / 07 / 2009 (CA07) HA and NA sequences used are shown below. [ka]

[0246] Mice (7-8 weeks old, female BALB / c) were cultured with M2KO(ΔTM) CA07(10 6 TCID 50 / fish), M2WT CA07(10 6 TCID 50 / animal), FluMist(registered trademark) CA07(10 6 TCID 50Mice were inoculated intranasally with 1000 ng / mouse) or OPTI-MEM™ as a naive control. Body weight and clinical symptoms were observed for 14 days after inoculation. Figure 37 shows that mice vaccinated with M2KO(ΔTM) and FluMist® did not lose weight, but viruses containing WT M2 lost weight and died from infection. These results indicated that deletion of the M2 gene attenuated the virus and that M2KO(ΔTM) was attenuated.

[0247] Figure 38. Titers of M2KO(ΔTM), FluMist® and M2 wild type virus in lungs and nasal tips. Lungs and nasal turbinates were harvested 3 days after vaccination for cellular virus titration. No virus was detected in either lungs or nasal turbinates in M2KO(ΔTM) immunized mice. In contrast, FluMist® caused virus replication in both lungs and nasal turbinates, but at lower levels than wild type virus.

[0248] Figure 39. M2KO(ΔTM) and FluMist® titers, as well as anti-HA IgG titers, were determined by ELISA in serum collected 7, 14, and 21 days after inoculation. M2KO(ΔTM) induced a higher response than that of FluMist®, which was detected earlier. Peak antibody levels were reached for both viruses by day 21.

[0249] Figure 40 shows the 40MLD of the heterologous virus, mouse-adapted influenza A / Aichi / 2 / 1968 (H2N2). 50 Figure 1 shows the % survival of mice challenged 12 weeks after immunization with M2KO(ΔTM) and challenged with Aichi (H3N2). Body weight fluctuations and clinical symptoms were observed for 14 days after challenge. All mice immunized with M2KO(ΔTM) (H1N1pdmHA,NA) were protected against Aichi (H3N2) challenge, whereas only 80% were protected with FluMist® (H1N1pdmHA,NA). Surviving FluMist® mice lost nearly 20% body weight, whereas M2KO(ΔTM) mice lost ~10% body weight.

[0250] Table 26 shows the viral titers in lungs and nasal turbinates harvested on day 3 post-challenge. M2KO(ΔTM) and FluMist® controlled challenge virus replication in lungs and nasal turbinates to similar levels, while naive mice showed 1 log higher viral titers in both lungs and nasal turbinates.

[0251] Intracellular staining of cells in bronchoalveolar bronchial (BAL). BAL was collected 3 days after challenge and stained with immunostaining surface markers by flow cytometry to detect CD8+CD4+, CD8+CD4-, CD8-CD4+, CD8-CD4- cell populations. Both CD4+ and CD8+ cell populations were larger in vaccinated mice than in naive mice, indicating that M2KO(ΔTM) stimulated a similar cellular response as FluMist®. M2KO(ΔTM) vaccinated mice had a larger CD8+CD4- cell population than FluMist® (49% vs. 40%) (Figure 41). [Table 29]

[0252] Example 19 M2KO(ΔTM) mRNA Expression for FluMist® and Wild-Type Virus In some embodiments, M2KO(ΔTM) virus is produced in cells that stably provide M2 ​​protein in trans, producing a virus that has a functional M2 protein in the viral membrane but does not encode M2 ​​in the genome. Therefore, the inventors hypothesize that M2KO(ΔTM) virus behaves similarly to wild-type virus in the initial infection of normal cells and the first replication cycle. The inventors suggest that the mRNA levels of viral antigens are similar to wild-type levels early in infection, stimulating a stronger immune response more quickly than attenuated replicating virus vaccines.

[0253] Human lung cancer (A549) cells were infected with M2KO(ΔTM), FluMist® and wild-type viruses at a multiplicity of infection of 0.5. Non-adsorbed virus was removed by washing five times with PBS. After adding virus growth medium, infected cells were placed in a 35° C. CO2 incubator. One round of replication of all viruses was confirmed without adding any trypsin to the growth medium. Cell monolayers were harvested and RNA was extracted 4, 9 and 22 hours post-infection.

[0254] Total RNA (100 mg) from control and infected A549 cells was used for quantitative RT-PCR analysis. cDNA was synthesized with oligo-dT primers and SuperscriptII reverse transcriptase (Invitrogen) and quantified by real-time quantitative PCR analysis using gene-specific primers for the early influenza gene M1 and the late influenza gene HA and the cytokine IP-10 gene. Reactions were performed using SYBR Green reagent (Invitrigen, Carlsbad) according to the manufacturer's instructions. Reaction efficiency was calculated by utilizing the housekeeping gene γ-actin and serial 10-fold dilutions of the sample genes. Reactions were performed on an ABI 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA) and used a thermal profile of stage 1: 50°C for 30 min, stage 2: 95°C for 15 min, stage 3: 94°C for 15 s, 55°C for 30 s, and 72°C for 30 s, repeated for 30 cycles. All quantifications (threshold cycle [CT] values) were normalized to housekeeping genes to generate ΔCT, and the difference between the ΔCT value of a sample and that of the reference (wild-type sample) was calculated as -ΔΔCT. The relative level of mRNA expression was expressed as 2-ΔΔCT.

[0255] M2KO(ΔTM) virus HA mRNA expression was similar to wild-type M2 viruses of H3 (Table 27), PR8 (Table 28) and H1N1pdm (Figure 42) at 4 hours post-infection. Cold-adapted FluMist® was lower than wild-type and M2KO(ΔTM) at early time points due to slower replication kinetics. Similar results were observed when examining M1, gene, and mRNA expression at early time points (Table 27, Figure 42). These results suggest that M2KO(ΔTM) produces similar levels of mRNA in the early infection cycle and produces de novo viral antigens that generate "danger signals" similar to wild-type viruses and induce strong immune responses. [Table 30] [Table 31]

[0256] Example 20: Generation of M2 Vero-producing cells The M2 gene of the PR8 virus was cloned into the vector pCMV-SC (Stratagene, La Jolla, Calif.) by standard molecular techniques to generate pCMV-PR8-M2. The plasmid was digested with EcoR1 to confirm the presence of the 300 bp M2 gene and the 4.5 Kb vector, as shown in Figure 43. The sequence of the plasmid containing the M2 gene insert was confirmed, as shown in Figure 44.

[0257] Generation of M2 Vero cells: The pCMV-PR8-M2 plasmid containing the neomycin resistance gene described above was transfected into Vero cells (ATCC CCL-81) by using Trans IT-LT1 transfection reagent (Mirus) according to the manufacturer's instructions. Briefly, the day before transfection, Vero cells were transfected at a concentration of 5 × 10 5Cells were seeded at 100 mm dish per 100-mm dish. On day 1, 10 μg of plasmid DNA was mixed with 20 μg of Trans IT-LT1 in 0.3 mL of OptiMEM (Invitrogen) and incubated with these cells overnight at 37°C in 5% CO2. On day 2, the transfection mixture was replaced with complete medium, which was modified Eagle's medium (MEM) supplemented with 5% newborn calf serum. The medium also contained 1 mg / ml of Geneticin (Invitrogen), a broad-spectrum antibiotic used to select mammalian cells expressing the neomycin protein. Resistant cells (Vero cells stably expressing the M2 gene) started to grow in the selection medium, the medium was replaced with fresh selection medium, and geneticin-resistant clones were isolated by limiting dilution in TC-96 plates. Surface expression of M2 protein was demonstrated by immunostaining with the M2-specific monoclonal antibody 14C2 (Santa Cruz Biotechnology).

[0258] Infection of parental and modified M2 Vero cells with M2KO(ΔTM) virus: The ability of M2 Vero cells to act as producer cells for M2KO(ΔTM) virus was examined by infection with M2KO(ΔTM)-PR8 virus. Briefly, monolayers of M2 Vero and parental Vero cells were infected with serial 10-fold dilutions (10 -1 ~10 -6 ) were used for infection. Infected cells were incubated at 35°C and observed once a day for cytopathic effect (CPE). M2 Vero cells showed CPE, indicating virus growth. The supernatant was diluted with 10 mL of PBS on day 4. -3 The TCID values ​​were calculated using MDCK cells stably expressing the M2 gene (M2CK). 50 The virus titer was determined by assay. The M2KO(ΔTM)-PR8 virus titer grown in M2 Vero cells was 10 6.75 TCID 50 / ml, indicating that M2 Vero cells can serve as producer cells for M2KO(ΔTM) vaccine production.

[0259] Example 21: Intradermal delivery of influenza vaccine This example demonstrates the immunogenicity of the seasonal influenza vaccine FluLaval (2011-2012 formulation) when administered intramuscularly (IM), intradermally (ID) and using a subcutaneous microneedle device as described in published U.S. Patent Application Publication No. 2011 / 0172609. Hairless guinea pigs were inoculated on day 0 and select groups were boosted on day 30. Serum was collected on days 0, 30 and 60 and analyzed by enzyme-linked immunosorbent assay (ELISA) for hemagglutinin-specific IgG responses.

[0260] Results are shown in Figures 51-53. The data show qualitative absorption of antibody levels against three virus strains formulated in the seasonal influenza vaccine FluLaval: A / California / 7 / 2009 NYMC X-181, A / Victoria / 210 / 2009 NYMC X-187 (A / Perth / 16 / 2009-like virus), and B / Brisbane / 60 / 2008. At day 30, IM and ID delivery produced comparable IgG responses against all virus HAs. The ID prime only group showed higher titers at day 60, suggesting that ID delivery induces long-lasting immunity to all virus HAs.

Claims

1. A nucleic acid sequence comprising SEQ ID NO:

1.

2. A nucleic acid sequence comprising SEQ ID NO:

2.

3. A nucleic acid sequence comprising SEQ ID NO:

3.

4. A composition comprising SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 operably linked to (i) a promoter and (ii) a transcription termination sequence.

5. A recombinant influenza virus containing a mutation in the M gene.

6. 6. The recombinant influenza virus of claim 5, comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:

3.

7. 7. The recombinant influenza virus of claim 5 or 6, wherein a mutation in the M gene renders the virus unable to express M2 protein or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:

4.

8. 8. The recombinant influenza virus of claim 5, wherein the mutation in the M gene does not revert to a wild-type or non-wild-type sequence encoding a functional M2 protein during at least 10 passages in an in vitro host cell system.

9. The recombinant virus according to any one of claims 5 to 8, wherein the virus is an influenza A virus.

10. The recombinant virus according to any one of claims 5 to 9, which is non-pathogenic in an infected mammal.

11. The recombinant virus of claim 8, wherein the in vitro cell system comprises Chinese hamster ovary cells.

12. The recombinant virus of claim 8 , wherein the in vitro cell system comprises Vero cells.

13. A cell comprising a recombinant influenza virus according to any one of claims 5 to 10.

14. The cell of claim 13 , wherein the cell is in vitro.

15. The cell of claim 13 , wherein the cell is in vivo.

16. A composition comprising a recombinant influenza virus containing a mutation in the M gene.

17. 17. The composition of claim 16, comprising SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

18. 18. The composition of claim 16 or 17, wherein a mutation in the M gene renders the virus unable to express M2 protein or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:

4.

19. The composition according to any one of claims 16 to 18, wherein the virus is an influenza A virus.

20. The composition according to any one of claims 16 to 19, which is non-pathogenic to a mammal to which it is administered.

21. The composition of any one of claims 16 to 20, which induces a detectable immune response in a mammal within about three weeks after administration to the mammal.

22. A vaccine comprising a recombinant influenza virus containing a mutation in the M gene.

23. 23. The vaccine of claim 22 comprising SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

24. 24. The vaccine of claim 22 or 23, wherein a mutation in the M gene renders the virus unable to express M2 protein or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:

4.

25. The vaccine of any one of claims 22 to 24, wherein the virus is an influenza A virus.

26. The vaccine according to any one of claims 22 to 25, which is non-pathogenic to a mammal to which it is administered.

27. The vaccine of any one of claims 22 to 26, which induces a detectable immune response in a mammal within about three weeks after administration to the mammal.

28. The vaccine of any one of claims 22 to 27, which comprises, in addition to the recombinant virus, at least two different influenza virus strains.

29. The vaccine of any one of claims 22 to 28, comprising at least one influenza B virus or influenza B virus antigen.

30. The vaccine of any one of claims 22 to 29, comprising at least one influenza C virus or influenza C virus antigen.

31. 31. The vaccine of any one of claims 22 to 30, comprising one or more viruses or viral antigens, including human influenza A viruses and pandemic influenza viruses from non-human species.

32. The vaccine of any one of claims 22 to 31, wherein the human influenza A virus is selected from the group comprising H1N1, H2N2 and H3N2.

33. 1. A method of propagating a recombinant influenza virus comprising contacting a host cell with recombinant influenza virus SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, incubating the host cell under conditions suitable for viral replication for a sufficient period of time, and isolating progeny viral particles.

34. 1. A method of preparing a vaccine comprising: placing host cells in a bioreactor; contacting the host cells with recombinant virus SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; incubating the host cells under conditions suitable for virus growth for a sufficient period of time; isolating progeny virus particles; and formulating the progeny virus particles for administration as a vaccine.

35. 1. A method of immunizing a subject, comprising administering a composition comprising a recombinant influenza virus comprising a mutation in the M gene, wherein the mutation in the M gene renders the virus incapable of expressing an M2 protein or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:

4.

36. 1. A method of reducing the likelihood or severity of infection by influenza A virus in a subject, comprising administering a composition comprising a recombinant influenza virus comprising a mutation in the M gene, wherein the mutation in the M gene renders the virus incapable of expressing an M2 protein or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:

4.

37. 37. The method of claim 35 or 36, wherein the recombinant influenza virus comprises SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

38. 38. The method of any one of claims 35-37, comprising providing at least one booster dose of the composition, wherein the at least one booster dose is provided three weeks after the initial administration.

39. 39. The method of any one of claims 35 to 38, comprising administering the composition intranasally, intramuscularly, or intradermally.

40. 40. The method of claim 39, wherein the administration is performed intradermally.

41. 41. The method of claim 40, wherein the administering is performed using a microneedle delivery device.

42. 1. A method for intradermal administration of an immunogenic composition comprising: (a) providing a microneedle delivery device comprising: (i) a puncturing mechanism; and (ii) an immunogenic composition layer comprising a plurality of microneedles capable of puncturing the skin, the microneedle delivery device being capable of administering the immunogenic composition intradermally; and (b) depressing the puncturing mechanism, the immunogenic composition comprising a recombinant influenza virus comprising a mutation in an M gene, the mutation in the M gene rendering the virus unable to express an M2 protein or causing the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:

4.

43. 43. The method of claim 42, wherein the recombinant influenza virus comprises SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:

3.

44. 43. The method of claim 42, wherein the microneedle array is initially positioned inside a device housing, and actuation of a lever causes the microneedles to penetrate the bottom of the device and into the skin, thereby injecting the vaccine liquid into the skin.

45. A recombinant influenza virus comprising a mutation in the M gene, wherein the virus does not replicate in a non-altered host cell selected from the group consisting of Chinese hamster ovary (CHO) cells, Vero cells, or Madin-Darby canine kidney cells.

46. 46. ​​The recombinant virus of claim 45, wherein a mutation in the M gene renders the virus incapable of expressing an M2 protein or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:

4.

47. A recombinant cell comprising a nucleic acid encoding an influenza virus M2 ion channel gene, said nucleic acid being expressed within said cell.

48. A recombinant cell containing a 2,6-sialic acid receptor gene.

49. A recombinant cell comprising a cellular genome or an expression vector expressing (i) a viral M2 ion channel gene, and (ii) a 2,6-sialic acid receptor gene.

50. The recombinant cell of any one of claims 47 to 49, wherein the cell is a eukaryotic cell.

51. 51. The recombinant cell of any one of claims 47 to 50, wherein the eukaryotic cell is a Chinese hamster ovary cell or a Vero cell.

52. 52. The recombinant cell of any one of claims 47 to 51, further comprising a human influenza virus that does not express a functional M2 protein.

53. 53. A method of producing recombinant influenza virus particles comprising: (A) infecting a cell of any one of claims 47 to 52, which cell (i) constitutively expresses a functional M2 ion channel protein, or (ii) is introduced after viral infection to express said functional M2 ion channel protein, and which replicates successfully only in the presence of said functional M2 ion channel protein expressed by said cell, with human influenza virus particles; and (B) isolating progeny virus particles.

54. 54. The method of claim 53, further comprising incorporating the isolated viral particles into a vaccine.

55. 55. The method of claim 53 or 54, wherein the virus comprises a human influenza virus, and the virus does not express a functional M2 protein.

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