HK1183791A - Ectodomains of influenza matrix 2 protein, expression system, and uses thereof - Google Patents

Description

Extracellular domain of influenza virus matrix 2 protein, expression system and application thereof

Technical Field

Influenza viruses are negative-sense RNA members of the Orthomyxoviridae (Orthomyxoviridae) family, causing human and animal disease. Influenza virus infections are common and can be epidemic or seasonal. Although influenza virus infection does not often cause death in infected individuals, the incidence is severe. Thus, influenza virus epidemics can result in significant economic losses. Furthermore, influenza virus infection may be more dangerous for certain groups of individuals, such as heart disease patients, c.a.r.a. patients or elderly.

Influenza viruses cause disease in a recurrent manner due to a number of complex factors, including: 1) established reservoirs of different subtypes of influenza A virus exist in waterfowls and waterfowls, 2) the ability of avian influenza viruses to recombine with influenza viruses in other animals, such as swine, a process known as 'antigen transfer', 3) mutations that accumulate in the viral gene product due to the lack of proofreading activity of the viral RNA polymerase, a process known as 'antigen drift' (Tollis, M. and L.Di Trani, vet.J.164:202-215 (2002)). Antigen transfer, antigen drift and the ability of avian influenza viruses to infect other hosts such as pigs and humans produce new viruses that cause severe disease in humans. These reassortment and mutation events in combination give rise to well-characterized antigenic variability in the two surface glycoproteins Hemagglutinin (HA) and Neuraminidase (NA) of the virus, which provides a mechanism for the virus to escape from the immune response (especially neutralizing antibodies) generated by previous infection or vaccination induction. (palette, P. and J.F. Young, Science 215: 1468-.

To predict a particular influenza subtype that may have a global impact on human health, influenza vaccines must be produced relying on a supervisory program. The time to produce a subtype-matched vaccine (consisting of inactivated or 'split' virus particles) typically takes a minimum of 6-8 months. In the face of severe influenza epidemics caused by viral subtypes, this time delay can lead to national or global spread with extremely high morbidity and mortality. Therefore, there is a great need for influenza vaccines that are effective against different subtypes of influenza virus.

Influenza virus contains 8 segments of single-stranded RNA-genetic instructions that form the virus. Its surface is covered with a layer of two different glycoproteins: one consisting of Hemagglutinin (HA) molecules and the other consisting of Neuraminidase (NA). The viral capsid is composed of viral ribonucleic acids and several so-called "internal" proteins (polymerase (PB1, PB2 and PA), matrix protein (M1) and Nucleoprotein (NP)). Since antibodies against HA and NA have traditionally proven to be most effective in combating infection, much research HAs focused on the structural, functional, and genetic variations of these molecules.

Unlike HA and NA, the outer domain of the transmembrane virus M2 protein (M2 e) is highly conserved and antibodies against this epitope are protective in mice (Treano, J.J., et al, J.Virol.64: 1375-. The M2 protein is an integral membrane protein of influenza a virus, expressed on the plasma membrane of virus-infected cells. Because of the low abundance of this protein in the virus, the mechanism of protection of the antibody response to this epitope is not mediated by viral neutralization, but rather by antibody-dependent, cell-mediated cytotoxicity. (Jegrerlehner, A., et al, J.Immunol.172: 5598-.

A major obstacle to the development of vaccines for inducing immune responses is the selection of suitable delivery forms. DNA plasmid vaccines and viral vectors, as well as recombinant proteins or peptides, used alone or together, are reasonable vaccine delivery forms, however, each form has its advantages and disadvantages. For example, DNA vaccines are easy to produce and administer safely, but are not effective enough, especially in clinical trials, to administer large (milligrams) doses. Liu, M.A.J.Intern.Med.253:402-410 (2003). The use of viral vectors to deliver vaccines has raised concerns, generally related to safety and pre-existing immunity to the vector. Therefore, the development of a safe and effective new influenza vaccine is highly desired.

Disclosure of Invention

The present invention provides an isolated polynucleotide comprising a coding region that encodes a polypeptide, wherein the polypeptide comprises at least 5 influenza matrix 2 protein (M2) ectodomain peptides. In one embodiment, the polypeptide comprises any 5 or more of the following amino acid sequences in any order relative to each other: (i) SEQ ID NO 1(M2e #1_ C), (ii) SEQ ID NO 2(M2e #2_ C), (iii) SEQ ID NO 3(M2e #3_ C), (iv) SEQ ID NO 4(M2e #4_ C), (v) SEQ ID NO 5(M2e #5_ C), (vi) SEQ ID NO 6(M2e #6_ C), (vii) SEQ ID NO 7(M2e #1_ S), (viii) SEQ ID NO 8(M2e #2_ S), (ix) SEQ ID NO 9(M2e #3_ S), (x) SEQ ID NO 10(M2e #4_ S), (xi) SEQ ID NO 11(M2e #5_ S) and (xi) SEQ ID NO 12 # S2 e.

Also provided are isolated polynucleotides comprising a coding region that encodes a polypeptide, wherein the polypeptide comprises any 3 or more of the following M2 ectodomain peptides arranged in any order relative to each other: (i) SEQ ID NO:1(M2e #1_ C), (ii) SEQ ID NO:2(M2e #2_ C), (iii) SEQ ID NO:3(M2e #3_ C), (iv) SEQ ID NO:4(M2e #4_ C), (v) SEQ ID NO:5(M2e #5_ C), (vi) SEQ ID NO:6(M2e #6_ C), (vii) SEQID NO:7(M2e #1_ S), (viii) SEQ ID NO:8(M2e #2_ S), (ix) SEQ ID NO:9(M2e #3_ S), (x) SEQ ID NO:10(M2e #4_ S), (xi) SEQ ID NO:11(M2e #5_ S), and (xii) SEQ ID NO:12(M2e #6_ S) in one embodiment, the polynucleotide comprises a coding region encoding a polypeptide comprising the following 6M 2 ectodomain peptides arranged in any order relative to each other: (i) 1(M2e #1_ C) SEQ ID NO, (ii) 2(M2e #2_ C) SEQ ID NO, (iii) 3(M2e #3_ C) SEQ ID NO, (iv) 4(M2e #4_ C) SEQ ID NO, (v) 5(M2e #5_ C) SEQ ID NO, and (vi) 6(M2e #6_ C) SEQ ID NO, or (i) 7(M2e #1-S) SEQ ID NO, (ii) 8(M2e #2_ S) SEQ ID NO, (iii) 9(M2e #3_ S) SEQ ID NO, (iv) 10(M2e #4_ S) SEQ ID NO, (v) 11(M2e #5_ S) SEQ ID NO, and (vi) 12(M2 e) SEQ ID NO.

In certain embodiments, the polynucleotides of the invention encode polypeptides comprising 6M 2 ectodomain peptides arranged in any order relative to each other with at least one linker peptide interposed between at least two M2 ectodomain peptides, and optionally with an epitope interposed between at least two M2 ectodomain peptides. The epitope may be a T cell epitope or a B cell epitope.

In some embodiments, the invention is directed to a vector comprising a polynucleotide encoding a polypeptide comprising multiple copies of the M2 ectodomain peptide. The vector may be a viral vector, such as a vaccinia virus vector, such as modified vaccinia virus ankara (MVA). The vectors of the invention may also express other polypeptides, such as influenza proteins or fragments thereof. The additional polypeptides may be selected from the group consisting of: hemagglutinin (HA), Neuraminidase (NA), Nucleoprotein (NP), matrix 1 protein (M1), matrix 2 protein (M2), non-structural protein (NS), RNA polymerase PA subunit (PA), RNA polymerase PB1 subunit (PB1), RNA polymerase PB2 subunit (PB2), or a combination of two or more thereof. In other embodiments, the invention is a host cell comprising the vector and an isolated polypeptide encoded by the polynucleotide.

Also included are compositions comprising the polynucleotide, the vector, such as MVA, the host cell, or the polynucleotide, and a pharmaceutically acceptable carrier. In one embodiment, the vaccine composition of the invention further comprises other influenza vaccine compositions. Other influenza vaccines can include MVA expressing influenza virus or fragments thereof.

In other embodiments, the invention includes a method of inducing an immune response against an influenza virus in a subject in need thereof, the method comprising administering to the subject an effective amount of the polynucleotide, the vector, the host cell, the polypeptide, or the composition, or any combination thereof, simultaneously or in any order. Also provided is a method of treating, preventing, or reducing symptoms of or a condition associated with an influenza virus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the polynucleotide, the vector, the host cell, the polypeptide, or the composition, or any combination thereof, simultaneously or in any order. The present invention also relates to a method of attenuating or ameliorating symptoms caused by or associated with an influenza virus infection in a subject in need thereof, said method comprising administering to said subject an effective amount of said polynucleotide, said vector, said host cell, said polypeptide or said composition, or any combination thereof, simultaneously or in any order. In other embodiments, the invention includes a method of vaccinating a subject in need thereof against an influenza virus infection, said method comprising administering to said subject an effective amount of said polynucleotide, said vector, said host cell, said polypeptide, or said composition, or any combination thereof, simultaneously or in any order.

The identification sequences used herein are as follows:

1 amino acid sequence of M2 ectodomain #1 with cysteine (M2e #1_ C)

2 amino acid sequence of M2 ectodomain #2 with cysteine (M2e #2_ C)

3 amino acid sequence of M2 ectodomain #3 with cysteine (M2e #3_ C)

4 amino acid sequence of M2 ectodomain #4 with cysteine (M2e #4_ C)

5 amino acid sequence of M2 ectodomain #5 with cysteine (M2e #5_ C)

6 amino acid sequence of M2 ectodomain #6 with cysteine (M2e #6_ C)

7 amino acid sequence of M2 ectodomain #1 with serine substitution (M2e #1_ S)

SEQ ID NO 8 amino acid sequence of M2 ectodomain #2 with serine substitution (M2e #2_ S)

SEQ ID NO 9 amino acid sequence of M2 ectodomain #3 with serine substitution (M2e #3_ S)

10 amino acid sequence of M2 ectodomain #4 with serine substitution (M2e #4_ S)

11 amino acid sequence of M2 ectodomain #5 with serine substitution (M2e #5_ S)

12 amino acid sequence of M2 ectodomain #6 with serine substitution (M2e #6_ S)

13 nucleic acid sequence encoding matrix 2(M2) protein of influenza A/Puerto Rico/8/34(H1N1)

14 amino acid sequence of matrix 2(M2) protein of influenza A/Puerto Rico/8/34(H1N1)

15, SEQ ID NO: nucleic acid sequences encoding METR _ C polypeptides

16 in SEQ ID NO: amino acid sequence of METR _ C polypeptide

17 in SEQ ID NO: nucleic acid sequences encoding METR _ S polypeptides

18, SEQ ID NO: amino acid sequence of METR _ S polypeptide

19, SEQ ID NO: nucleic acid sequences encoding NP consensus sequences

20, SEQ ID NO: amino acid sequence of NP consensus sequence

21-25 linker peptides of SEQ ID NO

26-27T cell epitopes of SEQ ID NOs

28, SEQ ID NO: artificial sequences

29-30 promoter of SEQ ID NO

31, SEQ ID NO: transcription termination signal

32-50 primers of SEQ ID NO

51 nucleic acid sequence encoding the HA protein of influenza A/Puerto Rico/8/34(H1N1)

52 amino acid sequence of HA protein encoding influenza A/Puerto Rico/8/34(H1N1)

53 nucleic acid sequence encoding the transmembrane domain of the M2 protein of the influenza A/Puerto Rico/8/34(H1N1)

54 amino acid sequence of the transmembrane domain of the M2 protein of influenza A/Puerto Rico/8/34(H1N1)

55 in SEQ ID NO: amino acid sequence of METR _ C polypeptide

56 in SEQ ID NO: amino acid sequence of METR _ S polypeptide

Brief description of the drawings

FIG. 1 is a schematic vector diagram of recombinant vector vEM 011. Abbreviations are as follows: the term "AmpR" represents an ampicillin resistance gene for selection in bacteria; the term "flanking (Flank) 1/flanking 2Del III" denotes a sequence homologous to the flanking region of the deletion site III in the MVA genome, the term "Ps" denotes a strongly synthetic promoter, BsdR denotes a gene coding for blasticidin resistance, the term "GFP" denotes a gene coding for green fluorescent protein, the term "F1 Del3 rpt" denotes a posterior repeat flanking 1Del III, and the term "LacZ" denotes the E.coli (E.coli) Lac Z gene for the detection of bacteria.

FIG. 2 shows a schematic vector diagram of a recombinant vector containing an influenza A gene: (A) vEM47 encodes an NP consensus sequence, (B) vEM57 encodes an M2 ectodomain tandem repeat (METR _ C) peptide, (C) vEM58 encodes an M2 ectodomain tandem repeat-serine substituted (METR _ S) peptide, (D) vEM61 encodes the full-length matrix 2 domain of influenza A/Puerto Rico/8/34 (Pr8M2), (E) vEM62 encodes the transmembrane domain derived from the matrix 2 domain of influenza A/Puerto Rico/8/34 (Pr8M2E-TML), and (F) vEM65 encodes the hemagglutinin protein of influenza A/Puerto Rico/8/34 (Pr8 HA).

FIG. 3 shows the PCR products of various MVAtors (A) MVAtor-NP consensus (mEM10), (B) MVAtor-METR _ C (mEM18), (C) MVAtor-METR _ S (mEM19), and (D) MVAtor-Pr8M2(mEM22), MVAtor-Pr8M2e _ TML (mEM23) and MVAtor-Pr8HA (mEM 17).

FIG. 4 shows a schematic of an exemplary PCR fragment. Abbreviations are as follows: the term "flanking 1Del 3" denotes flanking sequence 1 of insertion site deletion 3; the term "Ps" denotes the strong synthetic promoter of vaccinia virus, the term "flu gene" denotes the gene encoding the gene of interest, namely HA, NP, M2e, M2e _ TML and METR, respectively, and the term "flanking 2Del 3" denotes the flanking sequence 2 of deletion 3.

FIG. 5 represents a Western blot analysis of the following various MVAtor expressed influenza virus proteins: MVAtor-NP consensus (mEM10), MVAtor-METR _ C (mEM18), and MVAtor-METR _ S (mEM 19).

FIG. 6 represents the Hemadsorption Assay (HAD) of CEF cells infected with MVAtor-Pr8HA (MVAtor-Pr8), mock-infected CEF cells, and CEF cells infected with MVAtor (MVAtor).

Fig.7 represents an immunoassay for the detection of Pr8M2 in CEF cells: (A) cells infected with MVAtor-Pr8M2, and (B) cells infected with MVAtor-Pr8M2e _ TML.

Figure 8 represents the percent change in body weight after MVAtor immunization with the following expressed influenza virus proteins: pr8M2 (Gray, big solid circle), Pr8M2e-TML (Black, asterisk), METR-C (Gray, Small open circle), METR-S (Gray, solid diamond), NP-common (Black, solid triangle), MVAtor (Black, big open circle), and PBS (Black, Small solid circle).

Figure 9 shows viral load in immunized mice. Lung weight was taken for each of four mice per group to represent 50% tissue culture infectious dose (TCID 50)/lung weight.

FIG. 10 shows ELISA of sera from mice immunized with MVA vaccine using mouse IgG anti-M2 e antibody (14C 2). The M2e peptides used in this experiment (M2e #1 (row 4), M2e #4 (row 3), M2e #5 (row 2), and M2e #6 (row 1)) are shown in table 1.

Fig. 11 shows the weight loss IN MVA vaccine immunized mice delivered Intranasally (IN) (fig. 11A) and Intramuscularly (IM) (fig. 11B).

Fig. 12 represents the viral load of MVA vaccine immunized mice delivered intranasally (left column) and intramuscularly (right column).

Figure 13 shows the percentage change in body weight of mice immunized with MVAtor expressing influenza virus proteins: HA (grey, filled squares), NP (grey, filled triangles), M2 (grey, filled diamonds), and M2+ NP (grey, asterisks) as well as controls MVAtor (black, filled diamonds) and non-lethal H1N1PR8 (black, filled circles).

The ELISA results of fig. 14 show anti-NP (IgG anti-NP) immune responses of mice immunized with 1d21MVA, 2d21 MVA, 1d21MVA + NP, 2d21 MVA + NP, 1d21 MVA-M2eA + MVA-NP, 2d21 MVA-M2eA + MVA-NP, 1d21 non-lethal H1N1PR8 and 2d21 non-lethal H1N1PR 8MVA, using day 42 sera (before challenge).

FIG. 15 shows viral load of mice immunized with MVA, MVA-HA, MVA-NP, MVA-M2eA, MVA-M2e + NP, and A/PR/8/34 at day 2 (left column) and day 4 (right column) after H1N1PR8 viral challenge. The lung weight of each of the individual mice was taken to represent 50% tissue culture infectious dose (TCID 50)/lung weight.

Figure 16A shows the percent change in body weight following immunization with MVAtor expressing influenza virus proteins: MVA-PR8HA (black, small solid diamonds), MVA-PR8-M2+ NP (black, solid squares), and MVA-PR8-C + NP (grey, large solid diamonds) and control PBS (black, triangles).

Figure 16B represents the percent body weight change following immunization with MVAtor expressing all of the influenza virus proteins shown in table 9, as well as the negative control.

The ELISA results in FIG. 17 show that mice immunized with ConsNP, PR9M2+ ConsNP, PR8M2e-TML + ConsNP, METR-C + ConsNP, and METR-S + ConsNP were immunized with anti-NP (IgG anti-NP) immune responses using day 42 serum (before challenge).

The ELISA results of FIG. 18 show that mice immunized with M2, M2-TML, METR-C, METR-S, M2+ NP, M2-TML + NP, METR-C + NP, and METR-S + NP are immune responses against M2(IgG anti-M2 e peptide) using day 42 serum (pre-challenge).

FIG. 19 shows viral load IN the lungs of mice immunized intranasally (left column) or intramuscularly (right column) with PBS (IN), MVAtor, NP, M2, M2e-TML, METR-C, METR-S, HA and sublethally PR8(IN) 3 days after challenge with H1N1PR8 virus. The lung weight of each mouse was taken to represent 50% tissue culture infectious dose (TCID 50)/lung weight.

Figure 20A represents the percent change in body weight following immunization with MVAtor expressing influenza virus proteins: m1+ NP + METR-C (black, filled squares), M1+ NP + M2 (grey, open triangles), M2+ NP (grey, X), M1 (black, asterisk), and M1+ NP (grey, filled circles) as well as controls: Flavival (black, filled diamonds), MVAtor (black, small filled squares), and PBS (black, filled triangles).

Figure 20B shows the percentage of survival data for mice immunized with MVAtor expressing influenza virus proteins: m1+ NP + METR-C (grey, filled squares), M1+ NP + M2 (grey, open triangles), M2+ NP (grey, X), M1 (black, asterisk), and M1+ NP (grey, filled circles) as well as controls: Flavival (black, open diamonds), MVAtor (black, filled squares), and PBS (black, filled diamonds).

The ELISA results of figure 21 show anti-NP (left half) and anti-M2 (right half) immune responses in mice immunized with PBS, MVAtor, M1+ NP + METRC, M1+ NP + M2, M2+ NP, M1, M1+ NP and fluvall using day 39 serum (before challenge). The horizontal line represents the group mean. IgG anti-M2 levels were not assessed for the M1 treatment group.

The ELISA results of figure 22A show anti-M1 immune responses of mice immunized with PBS, MVAtor, M1+ NP + METRC, M1+ M2+ NP, M2+ NP, M1, M1+ NP, and fluvall, using day 39 sera (before challenge). The horizontal line represents the group mean.

The ELISA results of fig. 22B show anti-MVA immune responses in mice immunized with MVAtor, M1+ M2+ NP, M2+ NP, M1 and M1+ NP using day 21 (post-prime) (left half) and day 39 (pre-challenge) (right half) sera. The horizontal line represents the group mean.

FIG. 23 shows viral loads in lungs of mice immunized with PBS, MVAtor, M1+ NP + METRC, M1+ NP + M2, M2+ NP, M1, M1+ NP, and Flulaval at day 2 (left column) or day 4 (right column) after challenge with sH1N 1A/Mx/4108/09 virus.

FIG. 24 shows viral loads of turbinates of mice immunized with PBS, MVAtor, M1+ NP + METRC, M1+ NP + M2, M2+ NP, M1, M1+ NP, and Flulaval at day 2 (left bar) or day 4 (right bar) after challenge with sH1N 1A/Mx/4108/09 virus.

Detailed Description

The present invention provides polynucleotides encoding an ectodomain of influenza virus matrix 2 protein, vectors comprising said polynucleotides (e.g., MVA), polypeptides encoded by said polynucleotides and related compositions and methods of administering said polynucleotides, vectors (e.g., MVA), polypeptides or compositions for preventing or treating influenza virus infection.

Methods of making and using the present invention include all conventional techniques of molecular biology, microbiology, immunology and vaccination. Such techniques are shown in documents including, but not limited to: sambrook Molecular Cloning, A Laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (1989) and third edition (2001), Genetic Engineering: Principles and Methods, Vol.1-25 (eds. J.Setlow, 1988), DNA Cloning (DNA Cloning), Vol.I and II (D.N Glover, eds. 1985), Oligonucleotide Synthesis (M.J.Gait, 1984), Nucleic Acid Hybridization (Nucleic Acid Hybridization) (B.D.Hames and S.J.Higgins, eds. 1984), transformation and Translation (Transcription and Translation) (B.D.Ha.J.J.J.J.J.1984), Cell Culture (1986, Cell Culture, pH., Cell Culture, 1986, Cell Culture, pH, Cell Culture, Cell Culture, inc.) and, in particular, volumes 154 and 155, Gene Transfer Vectors for Mammalian Cells (Gene Transfer Vectors for Mammalian Cells) (J.H.Miller and M.P.Calos eds. 1987, Cold spring Harbor Laboratory (Cold spring Harbor Laboratory), Mayer and Walker, eds. (1987), Immunochemical methods Cell and Molecular Biology (Immunochemical methods in Cell and Molecular Biology) (academic Press in London), Scopes, (1987) Protein Purification: Principles and Practice, Second Edition: principle and practice) second edition (Schpringer Press (Springer-Verlag, New York), and Handbook of Experimental Immunology (A laboratory Immunology Manual) volumes I-IV, (D.M. Weir and C.C. Blackwell eds 1986) (Sambrook et al (1989) Molecular Cloning: Alabortory Manual 2nd ed., ("Molecular Cloning: A laboratory Manual") second edition, Cold spring harbor laboratory Press and Ausubel et al eds. (1997) Current Protocols in Molecular Biology, ("New Molecular Biology laboratory Manual) (John Wiley & Sons, Inc.).

Definition of

It should be noted that the term "a" or "an" substance refers to one or more of that substance; for example, "a polynucleotide" is understood to represent one or more polynucleotides. Thus, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

The term "isolated" as used herein means that the polynucleotide, polypeptide or fragment, variant or derivative thereof, as well as the modified vaccinia virus ankara (MVA), have been removed from other biological material to which it is naturally associated. An example of an isolated polynucleotide is a recombinant polynucleotide contained in a vector. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially purified) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the invention. Isolated polynucleotides or nucleic acids according to the invention also include synthetically produced such molecules.

The term "isolated virus" as used herein means a virus, derivative or variant thereof that is removed from other biological material to which it is naturally associated, or that is recombinantly manipulated to include non-naturally occurring material. An example of an isolated virus is a virus containing polynucleotides from different species that are recombinantly inserted into the viral genome. Other examples of isolating viruses include viruses that contain heterologous polynucleotides and are maintained in host cells or purified (partially or substantially purified) viruses in solution.

The term "purified" as used herein means that the polynucleotide, polypeptide, virus, or fragment, variant, or derivative thereof is substantially free of other biological material with which it is naturally associated, or free of other biological material derived from, for example, a recombinant host cell genetically engineered to replicate the virus of the invention. For example, the purified virus of the invention comprises virus that is at least 70-100% pure, i.e., the virus present in the composition comprises 70-100% by weight of the total composition. In some embodiments, the purified virus of the invention is 75% to 99% pure by weight, 80% to 99% pure by weight, 90% to 99% pure by weight, or 95% to 99% pure by weight. The relative purity of the viruses of the invention can be readily determined by well-known methods.

The term "nucleic acid", "nucleotide" or "nucleic acid fragment" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide or construct. Two or more nucleic acids of the invention can be present in a single polynucleotide construct, e.g., on a single plasmid, or in separate (non-identical) polynucleotide constructs, e.g., on separate plasmids. In addition, any nucleic acid or nucleic acid fragment may encode a single polypeptide, such as a single antigen, cytokine, or regulatory polypeptide, or may encode more than one polypeptide, e.g., a nucleic acid may encode two or more polypeptides. In addition, the nucleic acid may encode a regulatory element such as a promoter or transcription terminator, or may encode a specialized element or motif of a polypeptide or protein such as a secretion signal peptide or functional domain. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al (1991), Nucleic Acid Res.19: 5081; Ohtsuka et al (1985), J.biol.chem.260: 2605-. The term nucleic acid encompasses polynucleotides, genes, cDNA, messenger rna (mRNA), plasmid DNA (pDNA) or derivatives of pDNA (e.g. (Darquet, a-M et al, GeneTherapy 4: 1341-.

The term "polynucleotide" is intended to encompass both a single nucleic acid or nucleic acid fragment and a plurality of nucleic acids or nucleic acid fragments and refers to isolated molecules or constructs such as viral genomes (e.g., non-infectious viral genomes), messenger RNA (mRNA), plasmid DNA (pDNA) or pDNA derivatives (e.g., polynucleotide-containing small loops as described in (Darqet, A-M et al, Gene Therapy4:1341-1349 (1997)).

The term "polypeptide" as used herein is intended to encompass both the singular "polypeptide" and the plural "polypeptide" and includes any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to, "peptide," "dipeptide," "tripeptide," "protein," "amino acid chain," or any other term used to refer to one or more chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used interchangeably with any of these terms. The term also includes polypeptides that have been post-translationally modified, such as glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage of known protecting/blocking groups, or modification of non-naturally occurring amino acids.

"codon optimization" is defined herein as modifying a nucleic acid sequence to increase its expression in a particular host cell by replacing at least one, more than one, or a number of codons of the native sequence with more frequent or most frequent codons used in the host gene. Each species exhibits a particular preference for certain codons for a particular amino acid.

The term "other" as used herein refers to any biological component that is different from the biological component of the subject. The additional component may be a host cell, virus, polypeptide, polynucleotide, gene or regulatory region such as a promoter. It is to be understood that the subject components may be derived from influenza virus, MVA, M2 ectodomain peptide or a polynucleotide appropriately encoding the M2 ectodomain peptide. For example, an "other polynucleotide" or "other nucleic acid" or "other gene" or "other sequence" or "exogenous DNA segment" of an M2 ectodomain gene of an influenza virus may be a promoter from a different virus, such as a cytomegalovirus, or a hemagglutinin of the same influenza virus. The term "other polypeptide", "other amino acid sequence", "other antigen" or "other protein" of the M2 ectodomain of influenza virus may be a His-tag or any influenza polypeptide, fragment, variant, derivative or analog thereof. In certain embodiments, the other polypeptide may be an influenza polypeptide, fragment, variant, derivative, or analog thereof. In other embodiments, the other polypeptide may be an M2 ectodomain polypeptide, fragment, variant, derivative, or analog thereof.

The term "influenza polypeptide" or "influenza antigen" as used herein encompasses any full length or mature polypeptide present within influenza virus, and other variants of full length or mature polypeptides present within influenza virus, fragments of full length or mature polypeptides present within influenza virus, serotypes, alleles and other variants of full length or mature polypeptide fragments present within influenza virus, derivatives of full length or mature polypeptides present within influenza virus, derivatives of full length or mature polypeptide fragments present within influenza virus, analogs of full length or mature polypeptides present within influenza virus, analogs of full length or mature polypeptide fragments present within influenza virus, and chimeric and fusion polypeptides comprising one or more full length or mature polypeptides present within influenza virus or full length or mature polypeptide fragments present within influenza virus. Non-limiting examples of influenza virus polypeptides are Hemagglutinin (HA), Neuraminidase (NA), Nucleoprotein (NP), matrix 1 protein (M1), matrix 2 protein (M2), non-structural protein (NS), or one or more RNA polymerase subunits, i.e., PA, PB1 and PB 2.

In one embodiment of the invention, the influenza polypeptide is an influenza HA protein, fragment, variant, derivative or analog thereof, such as a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a known HA sequence, such as SEQ ID No. 52, wherein the polypeptide is recognized by an antibody that specifically binds to the HA sequence. The HA sequence may be a full-length HA protein consisting essentially of HA or extracellular domain (ECD) (HA 1 and HA 2), Transmembrane (TM) domain, and Cytoplasmic (CYT) domain; or a fragment of said intact HA protein consisting essentially of the HA1 domain and the HA2 domain; or a fragment of said intact HA protein consisting essentially of HA1, HA2 and TM domains; or a fragment of said intact HA protein consisting essentially of the CYT domain; or a fragment of said intact HA protein consisting essentially of a TM domain; or a fragment of said intact HA protein consisting essentially of the HA1 domain; or a fragment of said intact HA protein, which consists essentially of the HA2 domain. The HA sequence may also include an HA1/HA2 cleavage site. The HA1/HA2 cleavage site is preferably located between the HA1 and HA2 sequences, but may be arranged in any order relative to the other sequences of the polynucleotide or polypeptide construct. The influenza virus HA sequence may be from a pathogenic virus strain.

In another embodiment, the influenza polypeptide is an influenza Nucleoprotein (NP) sequence, fragment, variant, derivative or analog thereof, e.g., comprising, consisting essentially of, or consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a known NP polypeptide, wherein the polypeptide is recognized by an antibody that specifically binds the NP polypeptide. In other embodiments, the influenza NP sequence comprises, consists essentially of, or consists of an NP consensus sequence such as SEQ ID NO: 20.

The influenza polypeptide can be a Neuraminidase (NA) protein, a fragment, variant, derivative or analog thereof. It is known that NA proteins localized on the influenza virus envelope catalyze the removal of terminal sialic acid residues from viral and cellular glycoconjugates. The NA protein of influenza A/Puerto Rico/8/1934(H1N1) has 454 amino acids, Genbank accession number AAM75160.1, which is incorporated herein by reference in its entirety. The NA protein consists of a cytoplasmic domain (amino acids 1-6), a transmembrane domain (amino acids 7-35) and an extracellular domain (amino acids 36-454). Non-limiting examples of influenza polypeptides comprise, consist essentially of, or consist of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a known influenza virus Neuraminidase (NA) sequence, wherein the polypeptide is recognized by an antibody that specifically binds to the NA protein. The NA sequence may be a full-length NA protein, which consists essentially of NA. The NA sequence can be a polypeptide comprising, consisting essentially of, or consisting of an extracellular domain, a Transmembrane (TM) domain, or a Cytoplasmic (CYT) domain of the NA sequence. The influenza NA sequence may be from a pathogenic viral strain.

The influenza polypeptide may be a matrix 1 (M1) protein, fragment, variant, derivative or analog thereof. The matrix 1 protein plays a key role in viral replication. M1 of influenza A/Puerto Rico/8/1934(H1N1) has 252 amino acids, Genbank accession number AAM75161.1, which is incorporated herein by reference in its entirety. Non-limiting examples of influenza polypeptides comprise, consist essentially of, or consist of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a known influenza virus M1 sequence, wherein the polypeptide is recognized by an antibody that specifically binds the M1 protein. The M1 sequence may be a polypeptide comprising, consisting essentially of, or consisting of a fragment of a NA sequence.

In other embodiments, the influenza polypeptide can be a non-structural (NS) protein, a fragment, variant, derivative, or analog thereof. The nonstructural protein (NS) inhibits post-transcriptional processing of cellular precursor mrnas by binding to and inhibiting two cellular proteins required for 3' -terminal processing of cellular precursor mrnas: 30kDa cleavage and polyadenylation specific factor (CPSF 4) and poly (A) -binding protein 2 (PABPN 1). The NS protein of influenza A/puerto Rico/8/1934(H1N1) has 230 amino acids, Genbank accession number AAM75163.1, which is incorporated herein by reference in its entirety. Non-limiting examples of influenza virus polypeptides comprise, consist essentially of, or consist of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a known influenza virus NS sequence, wherein the polypeptide is recognized by an antibody that specifically binds to the NS protein. The NS sequence can be a polypeptide that includes, consists essentially of, or consists of a fragment of the NS sequence.

In some embodiments, the influenza polypeptide is an RNA polymerase PA (polymerase acid protein) subunit, fragment, variant, derivative, or analog thereof. The PA polypeptide exhibits elongation factor activity in viral RNA synthesis. The PA protein of influenza A/Puerto Rico/8/1934(H1N1) has 716 amino acids, Genbank accession number AAM75157.1, which is incorporated herein by reference in its entirety. Non-limiting examples of influenza virus polypeptides comprise, consist essentially of, or consist of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a known influenza virus PA sequence, wherein the polypeptide is recognized by an antibody that specifically binds to the PA protein. The PA sequence can be a polypeptide comprising, consisting essentially of, or consisting of a fragment of a PA sequence.

In certain embodiments, an influenza polypeptide as used herein is an RNA polymerase PB1 (polymerase basic protein 1) subunit, fragment, variant, derivative, or analog thereof. The PB1 protein is responsible for replication and transcription of viral segments. The PB1 protein of influenza A/Puerto Rico/8/1934(H1N1) has 757 amino acids, Genbank accession No. AAM75156.1, which is incorporated herein by reference in its entirety. Non-limiting examples of influenza polypeptides comprise, consist essentially of, or consist of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a known influenza PB1 sequence, wherein the polypeptide is recognized by an antibody that specifically binds the PB1 protein. The PB1 sequence can be a polypeptide comprising, consisting essentially of, or consisting of a fragment of the PB1 sequence.

In other embodiments, the influenza polypeptide can be an RNA polymerase PB2 (polymerase basic protein 2) subunit, fragment, variant, derivative, or analog thereof. The PB2 protein is involved in the transcriptional initiation and cap-stealing (cap-stealing) mechanism, in which cell-capped precursor mRNA is used to generate primers for viral transcription. PB2 of influenza A/Puerto Rico/8/1934(H1N1) has 759 amino acids, Genbank accession No. AAM75155.1, which is incorporated herein by reference in its entirety. Non-limiting examples of influenza polypeptides comprise, consist essentially of, or consist of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a known influenza PB2 sequence, wherein the polypeptide is recognized by an antibody that specifically binds the PB2 protein. The PB2 sequence can be a polypeptide comprising, consisting essentially of, or consisting of a fragment of the PB2 sequence.

The term "coding region" as used herein refers to a nucleic acid portion consisting of codons that are translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it can be considered part of the coding region, and any flanking sequences, such as promoter, ribosome binding site, transcription terminator and the like, are outside the coding region.

The terms "fragment," "analog," "derivative," or "variant" when referring to an influenza polypeptide include any polypeptide that retains at least some of the immunogenicity or antigenicity of a naturally occurring influenza protein. Influenza polypeptide fragments of the invention include proteolytic fragments, deletion fragments, and in particular influenza polypeptide fragments that exhibit increased solubility during expression, purification, and or administration to an animal. Fragments of influenza polypeptides also include proteolytic fragments or deletion fragments that exhibit reduced pathogenicity when delivered to a subject. Polypeptide fragments also include any polypeptide portion that comprises an antigenic or immunogenic epitope of the native polypeptide, including linear and three-dimensional epitopes.

An "epitope fragment" of a polypeptide antigen is an epitope-containing antigenic portion. An "epitope fragment" can, but need not, comprise an amino acid sequence other than one or more epitopes.

The term "variant" as used herein refers to a polypeptide that differs from the polypeptide in question by amino acid substitutions, deletions, insertions, and/or modifications. Variants may occur naturally, such as subtype variants. The term "subtype variant" refers to a polypeptide or polynucleotide present in different influenza virus subtypes, including, but not limited to, human A/puerto Rico/8/34(H1N1), human A/Viet Nam/1203/2004(H5N1), human A/Hong Kong/156/97(H5N1), human A/Hong Kong/483/97(H5N1), human A/Hong Kong/1073/99(H9N2), avian A/chicken/HK/G9/97 (H9N2), porcine A/porcine/Hong Kong/10/98(H9N2), avian A/FPV/Rostock/34 (H7N), avian A/turkey/Italy/4620/99 (H7N), avian A/FPV/hybrid/34 (H7N7), human A/Neona/20/99 (Calonia N1) H1), Human A/Hong Kong/1/68(H3N2), human A/Shiga/25/97(H3N2), human A/Singapore/1/57(H2N2), human A/Leningrad/134/57(H2N2), human A/Annrbor/6/60 (H2N2), human A/Brev Mission/1/18(H1N1), porcine A/porcine/Wisconsin/464/98 (H1N1), human A/Netherlands/219/03(H7N7) and human A/Wyoming/3/2003(H3N 2). Subtype variants are naturally occurring variants, but they may also be produced using mutagenesis techniques known in the art.

Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. In one embodiment, the variant polypeptide differs from the identified sequence by substitution, deletion, or addition of 5 or fewer amino acids. Such variants can generally be identified by modifying the polypeptide sequence and evaluating the antigenic properties of the modified polypeptide using, for example, the representative procedures described herein.

Polypeptide variants have at least about 60-70%, e.g., 75%, 80%, 85%, 90%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity to the identified polypeptide. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Polypeptide derivatives are polypeptides that have been altered to exhibit other characteristics not found on the native polypeptide. Examples include fusion proteins. Analogs are other forms of the polypeptides of the invention. One example is a preprotein that can be activated by cleavage of the preprotein to produce an active mature polypeptide.

Variants may also or additionally comprise other modifications, e.g., the polypeptide may be coupled or coupled, e.g., fused to other polypeptides, e.g., a signal (or leader) sequence at the N-terminus of the protein, which co-or post-translationally directs protein transfer. The polypeptide may also be coupled or generated to a linker or other sequence to facilitate synthesis, purification, or identification of the polypeptide (e.g., 6-His) or to enhance binding of the polypeptide to a solid support. For example, the polypeptide may be coupled or coupled to an immunoglobulin Fc region. The polypeptide may also be coupled or conjugated to a sequence that confers or modulates an immune response to the polypeptide (e.g., T cell epitopes, B cell epitopes, cytokines, chemokines, etc.) and/or enhances uptake and/or processing of the polypeptide by antigen presenting cells or other immune system cells. The polypeptides may also be coupled or coupled to other polypeptides/epitopes from influenza viruses and/or from other bacteria and/or other viruses to generate hybrid immunogenic proteins that alone or in combination with various adjuvants can elicit protective immunity to other pathogenic organisms.

The term "sequence identity" as used herein refers to the relationship between two or more polynucleotide sequences or two or more polypeptide sequences. Sequences are said to be "identical" at a position in one sequence when that position is occupied by the same nucleic acid base or amino acid residue at the corresponding position in the compared sequence. The percentage of "sequence identity" is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences, giving the number of "identical" positions. The number of "identical" positions is then divided by the total number of positions in the window of comparison and multiplied by 100 to give the percentage of "sequence identity". Percent "sequence identity" is determined by comparing two optimally aligned sequences over a window of alignment. In order to optimize alignment of sequences for comparison, the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions known as gaps, while the reference sequence remains unchanged. An optimal alignment is one that, even if gapped, produces the greatest possible number of "identical" positions between the reference and compared sequences. The terms "sequence identity" and "identical" are used interchangeably herein. Thus, sequences sharing a percentage of "sequence identity" are understood to be the same as a percentage of "identity". The percent "sequence identity" between two sequences can be determined using the version of the program "BLAST 2 sequences" available from the National Center for Biotechnology Information from 9.1.2004, including the programs BLASTN (for nucleotide sequence comparisons) and BLASTP (for polypeptide sequence comparisons), based on the algorithms of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12): 5873-. When using the "BLAST 2 sequence", standard parameters from 9.1.2004 can be used for word size (3), gap opening penalty (11), extension gap penalty (1), gap reduction (50), expectation (10), and any other desired parameters including, but not limited to, substrate selection.

The term "epitope" as used herein refers to a portion of a polypeptide having antigenic or immunogenic activity in an animal, e.g., a mammal, e.g., a human. As used herein, an "immunogenic epitope" is defined as the portion of a protein that elicits an immune response in an animal, as measured by any method known in the art. The term "antigenic epitope" as used herein is defined as that portion of a protein to which an antibody or T cell receptor immunospecifically binds its antigen, as measured by any method known in the art. Immunospecific binding does not include non-specific binding but does not necessarily preclude cross-reactivity with other antigens. While all immunogenic epitopes are antigenic, an antigenic epitope need not be immunogenic.

An "effective amount" is an amount that is effective to treat or prevent a disease when administered to an individual as a single dose or as part of a series of doses. For example, an amount is effective when the amount administered reduces the incidence of influenza virus infection relative to untreated individuals, as measured 2 weeks after challenge with infectious influenza virus. This amount will vary depending on the health and physical condition of the individual being treated, the taxonomic group of individuals being treated (e.g., human, non-human primate, etc.), the responsiveness of the individual's immune system, the degree of protection desired, the formulation of the vaccine, the expert's assessment of medical condition, and other relevant factors. It is expected that the effective amount will fall within a relatively broad range that can be determined by routine experimentation. Typically a single dose is about 10 μ g to 10mg MVA/kg body weight or amount of modified carrier organism or host cell sufficient to provide a comparable amount of recombinantly expressed influenza polypeptide.

The term "subject" means any subject, particularly a mammalian subject, in need of diagnosis, prognosis, immunization or therapy. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals such as bears, sport animals, pets such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows; primates such as apes, monkeys, orangutans, and gorillas; canines such as dogs and wolves; felines such as cats, lions, and tigers; equine such as horse, donkey and zebra; food animals such as cows, pigs and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. In certain embodiments, the animal is a human subject.

The term "animal" is intended to encompass both the singular "animal" and the plural "animal" and includes mammals and birds, as well as fish, reptiles, and amphibians. The term animal also encompasses model animals such as disease model animals. In some embodiments, the term animal includes animals of economic or other value, such as economically important farm animals, sport animals, performance animals, ancestor animals, rare or endangered animals, or companion animals. In particular, the mammal may be a human subject, a food animal or a companion animal.

As used herein, "subject in need thereof" refers to an individual in need of treatment, i.e., prevention, cure, prevention, or reduction of severity of influenza virus infection, and/or to not aggravate symptoms of influenza virus infection over a specified period of time.

The terms "primary" or "first time" or "prime" and "boost" or "boost" as used herein refer to the first and subsequent immunizations, respectively, i.e. according to the common definition of these terms in immunology. However, in certain embodiments, e.g., where the priming component and the boosting component are in a single formulation, first and subsequent immunizations may not be necessary because the "priming" and "boosting" compositions are administered simultaneously.

The term "passive immunization" refers to the development of immunity to an antigen by a host animal that is administered to an antibody produced by another animal rather than producing its own antibodies to the antigen. The term "active immunization" refers to the production of antibodies by a host animal due to the presence of a target antigen.

As used herein, "immune response" refers to the response of a receptor to the introduction of a polynucleotide, polypeptide, attenuated poxvirus such as MVA, or composition of the invention, typically characterized by, but not limited to, the production of antibodies and/or T cells. Typically, the immune response may be a cellular response such as induction or activation of CD4+ T cells or CD8+ T cells or both specific for influenza virus M2 ectodomain or M2 Ectodomain Tandem Repeat (METR), an increased humoral response to influenza virus M2 ectodomain-specific or METR-specific antibody production, or both cellular and humoral responses. Immune responses established by vaccines containing MVA of the present invention include, but are not limited to, responses to proteins expressed by host cells following entry of MVA into the host cells. In the present invention, the immune response prevents the formation or development of influenza virions following subsequent challenge by an infectious agent (e.g., influenza a virus). The immune response may also include a mucosal response, such as a mucosal antibody response, e.g., S-IgA production, or a mucosal cell-mediated response, e.g., a T cell response.

As used herein, a "vaccine" is a composition comprising an immunogenic agent and a pharmaceutically acceptable diluent in combination with excipients, adjuvants, additives and/or protective agents. The immunogen may be composed of the entire infectious agent or a subset of molecules of said infectious agent (synthesized or recombinantly produced by said infectious agent, including but not limited to polypeptides or polynucleotides). For example, a "MVA vaccine" as used herein means a composition comprising an isolated MVA comprising at least one polynucleotide encoding multiple copies of the M2 ectodomain peptide (e.g., M2e tandem repeats) and a pharmaceutically acceptable diluent in combination with excipients, adjuvants, additives, and/or protective agents. When the vaccine is administered to a subject, the immunogen stimulates an immune response that, upon subsequent challenge with an infectious agent, protects the subject from disease or alleviates the pathology, symptoms, or clinical manifestations caused by the agent. The vaccine according to the invention may be therapeutic or prophylactic. Therapeutic (therapeutic) vaccines are administered after infection, with the aim of reducing or inhibiting disease progression. Prophylactic vaccines aim to prevent the first infection or reduce the infection load. The agent used in the vaccine against influenza-associated disease may be an attenuated influenza virus, or a purified or artificially processed molecule associated with an influenza virus, such as a recombinant protein, a synthetic peptide, a DNA plasmid, and a recombinant virus or bacterium that expresses an influenza virus protein. One skilled in the art will readily appreciate that the vaccine may also include other components such as excipients, diluents, carriers, preservatives, adjuvants or other immunopotentiators, or combinations thereof.

By multivalent vaccine is meant any vaccine prepared from two or more poxviruses, such as MVA, each expressing a different antigen, such as a different influenza virus antigen. Alternatively, a multivalent vaccine includes a single isolated poxvirus, such as MVA, that includes polynucleotides encoding two or more antigens (e.g., different influenza antigens). The two or more antigens, such as influenza antigens, may be derived from the same polypeptide, but contain different epitopes that induce non-cross-reactive immune responses.

The term "immunogenic carrier" as used herein refers to a first polypeptide, or a fragment, variant or derivative thereof, which enhances the immunogenicity of a second polypeptide, such as an antigenic epitope, fragment, variant or derivative thereof.

The term "adjuvant" refers to any material having the ability to: (1) alter or increase the immune response to a particular antigen or (2) increase or assist the effect of a pharmaceutical agent. As used herein, any compound that may increase the expression, antigenicity or immunogenicity of the MVA of the present invention is a potential adjuvant. In some embodiments, the term adjuvant refers to a TLR-stimulating adjuvant, wherein the TLR adjuvant includes a compound that stimulates a TLR receptor (e.g., TLR1-TLR 13) such that the immune system response to the vaccine composition of the invention is enhanced. TLR adjuvants include, but are not limited to, CpG and MPL.

The term "attenuated" as used herein includes the inability of an infectious agent, e.g. a poxvirus such as MVA, to replicate in at least one host cell, e.g. any mammalian cell, such as any human cell. Attenuated MVA may still have limited replication capacity in certain mammalian cells, such as BS-C-1 and CV-1 cells. Although incapable of replication in certain mammalian cells, MVA may retain the full capacity to replicate in other mammalian cells (e.g., BHK-21 cells) as well as avian cells, e.g., primary or immortalized chicken or duck cells such as AGE1cr, AGE1crThe cell replication cycle of an attenuated MVA can be blocked at any stage of its life cycle. For example, the cell replication cycle of attenuated MVA is blocked at a later stage, preventing the generation and release of new viruses. The term "replication" or "replicating" as used herein refers to the ability to progress through certain parts of the viral life cycle, such as transcription and translation of viral gene products and nucleic acid replication, and in some cases the ability to produce or develop mature infectious viral particles.

Polynucleotide

The present invention provides isolated polynucleotides comprising coding regions that encode polypeptides comprising multiple copies of the ectodomain of influenza virus matrix 2 protein to induce an immune response against influenza virus.

The influenza matrix 2 protein ("M2") is a proton-selective ion channel protein that is integrated into the viral envelope of influenza a virus. The channel itself is a homotetramer in which the units form a helix stabilized by two disulfide bonds. The M2 protein unit consists of three protein domains: an extracellular domain exposed to the external environment having 24 amino acids at the N-terminus, a transmembrane region having 19 hydrophobic amino acids, and a cytoplasmic domain having 54 amino acids at the C-terminus. The M2 protein plays an important role in the life cycle of influenza A virus. The proteins localize in the viral envelope, allowing hydrogen ions to enter the viral particle (virion) from the endosome, thereby lowering the pH within the virus, which separates the viral matrix protein M1 from the nucleoprotein RNP. This is a critical step in the de-coating of the virus and in the exposure of its contents to the host cell cytoplasm.

The present invention provides an isolated polynucleotide comprising a coding region encoding a polypeptide, wherein the polypeptide comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 influenza virus matrix 2 protein (M2) ectodomain peptides, variants, fragments, derivatives, or analogs thereof. Wherein the M2 ectodomain peptides may be arranged or combined in any order relative to each other. In one embodiment, the polynucleotides of the invention comprise a coding region encoding a polypeptide comprising at least 6M 2 ectodomain peptides, fragments, variants, fragments, derivatives or analogs thereof, wherein the at least 6M 2 ectodomain peptides are arranged or combined in any order relative to each other. The term "multiple copies of M2 ectodomain peptide" is also used herein interchangeably with "M2e tandem", "M2 ectodomain tandem", or "METR".

The matrix 2(M2) protein used in the present invention can be obtained from influenza A hepatoviruses including, but not limited to, human A/Puerto Rico/8/34(H1N1), human A/Viet Nam/1203/2004(H5N1), human A/Viet Nam/DT-036/2005(H5N1), human A-Novoslbrsk/29/2005 (H5N1), avian A/anser/Mongolia/1/05 (H5N1), A/cat/Thailand/KU-02/04 (H5N1), human A/Hong Kong/213/03(H5N1), avian A/chicken/Guiandong/174/04 (H5N1), human A/Hong Kong/156/97(H5N1), human A/Hong Kong/483/97(H5N1), avian A/quail/Hong Kong/G1/97(H9N2), avian A/duck/Hong Kong/Y260/97(H9N2), avian A/chicken HK/FY23/03(H9N2), avian A/turkey/Germany/3/91 (H1N1), human A/Hong Kong/H9N 2 (H5N 1073/99), human A/Hong Kong/3635 (H5N 3877), Avian A/chicken/HK/G9/97 (H9N2), porcine A/pig/Hong Kong/10/98(H9N2), porcine A/pig/Saskatchewan/18789/02 (H1N1), avian A/wild duck/Alberta/1302003 (H1N1), avian A/wild duck/NY/6750/78 (H2N2), avian A/wild duck/Potsdam/177-4/83 (H2N2), avian/A/duck/Hokkaldo/95/2001 (H2N2), avian A/duck/Korea/S9/2003 (H3N2), porcine A/pig/Shandong/2/03 (H9N 3542)H5N1), avian A/chicken/California/0139/2001 (H6N2), avian A/peacock/Sweden/3/2000 (H6N2), avian A/goose/Hong Kong/W217/97 (H6N9), avian A/chicken/British Columbia/04(H7N3), avian A/waterfowl/Delaware/9/96 (H9N2), avian A/duck/Hong Kong/Y439/97(H9N2), avian A/short-necked wild duck/Hong Kong/W312/97(H6N1), porcine A/pig/Korea/S452/2004 (H9N2), avian A/chicken/Netherlands/1/2003 (H7N 48), avian A/39393939a/Alberta/1 (H1N1), avian A/Hunan/114/05 (H6N 582), Pig A/pig/Cotes d' Armor/1482/99(H1N1), pig A/pig Belzig/2/2001(H1N1), avian A/turkey/Italy/220158/2002 (H7N3), avian A/HK/2108/2003(H9N2), avian A/FPV/Rostock/34(H7N1), avian A/turkey/Italy/4620/99 (H7N1), avian A/FPV/Weybrid/34 (H7N7), avian A/FPV/Dobson/27(H7N7), human A/New Calonia/20/99 (H1N1), human A/Hong Kong/1/68(H3N2), human A/Chartetesville/03/2004 (H3N2), human A/Canterbury/H3/2), human A/Canng Kong/2 (H3N2) and human A/HK 599N 599, Human A/Singapore/1/57(H2N2), human A/Leningrad/134/57(H2N2), human A/Ann Arbor/6/60(H2N2), human A/Brevib Session/1/18 (H1N1), human A/Canada/720/05(H2N2), porcine A/porcine/Wisconsin/464/98 (H1N1), porcine A/porcine/Texas/4199-2/98 (H3N2), avian A/turkey/Ohio/313053/2004 (H3N2), A/turkey/North Carolina/avian (H3N2), A/goose/Guingdong/1/98 (H5N1), human A/Netlands/219/03 (H7N7), human A/Wilson-68633 (Smith H874H 1), human A/Ranigold/36874 (H58/1), Human A/Japan/305/57(H2N2), avian A/chicken/Iran/16/2000 (H9N2), avian A/mallard/MN/1/2000 (H5N2), A/Leiden/01272/2006(H3N2), A/Tilburg/45223/2005(H3N2), A/Pennsylvania/PIT25/2008(H3N2), A/NYMC X-171A (PuertoRico/8/1934-Brisbane/10/2007) (H3N2), A/Managua/26/2007(H3N2), A/hongKong/1-1-MA-20D/1968(H3N2), A/Czeublic/1/1966 (H2N2), A/chicken/nghai/2/1999 (H9N2)]A/Myanmar/M187/2007(H3N2), A/guinea fowl/New York/101276-1/2005(H7N2), avian A/American duck/New York/87493-3/2005(H7N2), avian A/turkey/New York/122501-2/2005(H7N2), avian A/wild duck/Italy/4223-2/2006 (H5N2) and human A/Wyoming/3/2003(H3N 2).

In one embodiment, the extracellular domain of the matrix 2 protein of the invention is derived from influenza virus A/puerto Rico/8/34(H1N 1). Full-length M2 protein of human A/Puerto Rico/8/34(H1N1) of influenza virus contains 97 amino acids as shown in SEQ ID NO: 14. The nucleic acid encoding the M2 protein (SEQ ID NO: 14) is set forth herein as SEQ ID NO: 13. The influenza virus surface exposed N-terminal sequence was 23 amino acids without an N-terminal methionine or 24 amino acids with an N-terminal methionine (underlined sequence of SEQ ID NO:14 below) and identified as the ectodomain ("M2 e").

Matrix 2 protein sequence of influenza A/Puerto Rico/8/34(H1N1)

(SEQ ID NO:14)

0 MSLLTEVETP IRNEWGCRCN GSSDPLTIAA NIIGILHLTL WILDRLFFKC

50 IYRRFKYGLK GGPSTEGVPK SMREEYRKEQ QSAVDADDGH FVSIELE

As used herein, an M2 ectodomain peptide, variant, derivative, fragment or analog thereof can comprise, consist essentially of, or consist of an antibody epitope located in an M2 ectodomain peptide, wherein the M2 ectodomain peptide comprises about 8-39 amino acids, about 9-38 amino acids, about 10-37 amino acids, about 11-36 amino acids, about 12-35 amino acids, about 13-34 amino acids, about 14-33 amino acids, about 15-32 amino acids, about 16-31 amino acids, about 17-30 amino acids, about 18-29 amino acids, about 19-28 amino acids, about 20-27 amino acids, about 21-26 amino acids, about 22-25 amino acids, or about 23-24 amino acids. In one embodiment, the M2 ectodomain peptide consists essentially of or consists of 23 amino acids. Non-limiting examples of antibody epitopes include, consist essentially of, or consist of an amino acid sequence selected from the group consisting of EVETPTRN (amino acids 5-12 of SEQ ID NO: 1), SLLTEVETPT (amino acids 1-10 of SEQ ID NO: 1), ETPTRNEWECK (amino acids 7-17 of SEQ ID NO: 2), EVETPIRNEW (amino acids 5-14 of SEQ ID NO: 3), and LTEVETPIRNEWGCRCN (amino acids 3-19 of SEQ ID NO: 3).

Alternatively, the M2 ectodomain peptide may be a variant, derivative or analog thereof that is recognized by an antibody that specifically binds to a peptide consisting of SEQ id nos 1, 2, 3, 4, 5 or 6. For example, a variant, derivative or analog of the M2 ectodomain peptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 1, 2, 3, 4, 5 or 6(M2e #3_ C), wherein the variant, derivative or analog is recognized by an antibody that specifically binds to a peptide consisting of SEQ ID No. 1, 2, 3, 4, 5 or 6. An example of an antibody that specifically binds to a peptide consisting of SEQ ID NO. 3 is monoclonal antibody 14C2, as described in U.S. Pat. No. 5,290,686, which is incorporated herein by reference in its entirety. Non-limiting examples of M2 ectodomain peptides are listed in table 1.

Table 1 list of 6M 2e peptide (C) forms used in METR vaccine

Furthermore, M2 ectodomain peptides, variants, fragments, derivatives or analogs thereof as used herein may include, but are not limited to, M2 ectodomain peptide variants in which cysteine (C) in the M2 ectodomain peptide is substituted with serine (S). This substitution prevents disulfide bond formation between the two cysteines, but does not affect the immunogenicity of the M2 ectodomain peptide. Non-limiting examples of serine substituted M2 ectodomain peptides are shown in table 2.

Table 2 list of 6M 2e peptide (S) forms used in METR vaccine

Amino acid sequence of the M2e peptide	Peptide name	SEQ ID NO:
			SLLTEVETPTRNEWESRSSDSSD	M2e#1_S	SEQ ID NO:7
SLLTEVETPTRNEWESKSIDSSD	M2e#2_S	SEQ ID NO:8
			SLLTEVETPIRNEWGSRSNGSSD	M2e#3_S	SEQ ID NO:9
SLLTEVETPIRNEWGSRSNDSSD	M2e#4_S	SEQ ID NO:10
			SLLTEVETLTRNGWESRSSDSSD	M2e#5_S	SEQ ID NO:11
SLLTEVETPTRNGWESKSSDSSD	M2e#6_S	SEQ ID NO:12

In one embodiment, the polynucleotide of the invention encodes a protein comprising an amino acid sequence selected from the group consisting of: SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 11, SEQ ID NO 12 and combinations of SEQ ID NO 7, 8, 11 and 12.

The present invention relates to an isolated polynucleotide comprising a coding region encoding a polypeptide, wherein said polypeptide comprises at least 3 of the following M2 ectodomain peptides, arranged in any order relative to each other: (i) SEQ ID NO 1(M2e #1_ C), (ii) SEQ ID NO 2(M2e #2_ C), (iii) SEQ ID NO 3(M2e #3_ C), (iv) SEQ ID NO 4(M2e #4_ C), (v) SEQ ID NO 5(M2e #5_ C), (vi) SEQ ID NO 6(M2e #6_ C), (vii) SEQ ID NO 7(M2e #1_ S), (viii) SEQ ID NO 8(M2e #2_ S), (ix) SEQ ID NO 9(M2e #3_ S), (x) SEQ ID NO 10(M2e #4_ S), (xi) SEQ ID NO 11(M2e #5_ S), and (xii) SEQ ID NO 12(M2 # 12 _ S2 e). In one embodiment, the polynucleotide comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12M 2 ectodomain peptides arranged in any order relative to each other.

In another embodiment, a polynucleotide of the invention comprises a nucleic acid sequence encoding a polypeptide, wherein the polypeptide comprises at least 6 of the following M2 ectodomain peptides arranged in any order relative to each other: (i) SEQ ID NO 1(M2e #1_ C), (ii) SEQ ID NO 2(M2e #2_ C), (iii) SEQ ID NO 3(M2e #3_ C), (iv) SEQ ID NO 4(M2e #4_ C), (v) SEQ ID NO 5(M2e #5_ C), (vi) SEQ ID NO 6(M2e #6_ C), (vii) SEQ ID NO 7(M2e #1_ S), (viii) SEQ ID NO 8(M2e #2_ S), (ix) SEQ ID NO 9(M2e #3_ S), (x) SEQ ID NO 10(M2e #4_ S), (xi) SEQ ID NO 11(M2e #5_ S), and (xii) SEQ ID NO 12(M2 # 12 _ S2 e). In one embodiment, the M2 ectodomain peptide of the invention comprises the following amino acid sequences in any order combined or arranged relative to each other: (i) 1(M2e #1_ C) SEQ ID NO 2(M2e #2_ C), (iii) SEQ ID NO 3(M2e #3_ C), (iv) SEQ ID NO 4(M2e #4_ C), (v) SEQ ID NO 5(M2e #5_ C), and (vi) SEQ ID NO 6(M2e #6_ C) or an amino acid sequence selected from the group consisting of (i) SEQ ID NO 7(M2e #1-S), (ii) SEQ ID NO 8(M2e #2_ S), (iii) SEQ ID NO 9(M2e #3_ S), (iv) SEQ ID NO 10(M2e #4_ S), (v) SEQ ID NO 11(M2e # S), and (vi) SEQ ID NO 5812 # S). In a particular embodiment, the isolated polynucleotide of the invention comprises a coding region encoding a polypeptide comprising the following amino acid sequences in any order relative to each other: (a) 1, 2, 3, 4, 5, and 6 or (b) 7, 8, 9, 10, 11, and 12. In another particular embodiment, the polypeptide of the invention comprising multiple copies of the M2 extracellular domain peptide (METR) sequence comprises, consists essentially of, or consists of SEQ ID NOs 1-6. In another specific embodiment, the METR sequence comprises, consists essentially of, or consists of SEQ ID NO 16 or SEQ ID NO 55.

METR _ C sequence (SEQ ID NO:16)

SLLTEVETPTRNEWECRCSDSSD GSASG

SLLTEVETPTRNEWECKCIDSSD SGSGA

SLLTEVETPIRNEWGCRCNGSSD SAGSG

SLLTEVETPIRNEWGCRCNDSSD QVHFQPLPPAVVKL

SLLTEVETLTRNGWECRCSDSSD QFIKANSKFIGITE

ALLTEVETPTRNGWECKCSDSSD

METR _ C sequence (SEQ ID NO:55)

SLLTEVETPTRNEWECRCSDSSD GSASG

SLLTEVETPTRNEWECKCIDSSD SGSGA

SLLTEVETPIRNEWGCRCNGSSD SAGSG

SLLTEVETPIRNEWGCRCNDSSD GSASG

SLLTEVETLTRNGWECRCSDSSD SGSGA

SLLTEVETPTRNGWECKCSDSSD

In other embodiments, the polypeptide comprising a METR sequence comprises, consists essentially of, or consists of SEQ ID NOs 7-12. In a specific embodiment, the METR sequence comprises, consists essentially of, or consists of SEQ ID No. 18 or SEQ ID No. 56.

METR _ S sequence (SEQ ID NO:18)

SLLTEVETPTRNEWESRSSDSSD GSASG

SLLTEVETPTRNEWESKSIDSSD SGSGA

SLLTEVETPIRNEWGSRSNGSSD SAGSG

SLLTEVETPIRNEWGSRSNDSSD QVHFQPLPPAVVKL

SLLTEVETLTRNGWESRSSDSSD QFIKANSKFIGITE

SLLTEVETPTRNGWESKSSDSSD

METR _ S sequence (SEQ ID NO:56)

SLLTEVETPTRNEWESRSSDSSD GSASG

SLLTEVETPTRNEWESKSIDSSD SGSGA

SLLTEVETPIRNEWGSRSNGSSD SAGSG

SLLTEVETPIRNEWGSRSNDSSD GSASG

SLLTEVETLTRNGWESRSSDSSD SGSGA

SLLTEVETPTRNGWESKSSDSSD

Also provided are isolated polynucleotides comprising coding regions that encode influenza virus Nucleoprotein (NP) consensus sequences. The influenza NP protein is structurally related to an influenza gene (RNA) segment and has a length of 498 amino acids. The primary function of the NP is to encapsidate the viral genome for RNA transcription, replication and packaging. The NP gene is relatively well conserved with a maximum amino acid difference of less than 11% (Shu, L.L., et al, Nucleic Acids Res.22:5047-5053 (1993)). The influenza NP consensus sequences of the present invention were obtained by comparing 700 most common NP influenza A sequences to an alignment of recently (2004-) 2007 viruses published in the influenza sequence database (www.flu.lanl.gov /). The NP consensus sequence can induce an immune response against influenza virus. Exemplary sequences of the NP consensus sequence include, consist essentially of, or consist of SEQ ID NO: 20.

NP consensus sequence (SEQ ID NO:20)

0 MASQGTKRSY EQMETDGDRQ NATEIRASVG KMIDGIGRFY IQMCTELKLS

50 DHEGRLIQNS LTIEKMVLSA FDERRNKYLE EHPSAGKDPK KTGGPIYRRV

100 DGKWMRELVL YDKEEIRRIW RQANNGEDAT AGLTHIMIWH SNLNDATYQR

150 TRALVRTGMD PRMCSLMQGS TLPRRSGAAG AAVKGIGTMV MELIRMVKRG

200 INDRNFWRGE NGRKTRSAYE RMCNILKGKF QTAAQRAMVD QVRESRNPGN

250 AEIEDLIFLA RSALILRGSV AHKSCLPACA YGPAVSSGYD FEKEGYSLVG

300 IDPFKLLQNS QIYSLIRPNE NPAHKSQLVW MACHSAAFED LRLLSFIRGT

350 KVSPRGKLST RGVQIASNEN MDNMGSSTLE LRSGYWAIRT RSGGNTNQQR

400 ASAGQTSVQP TFSVQRNLPF EKSTIMAAFT GNTEGRTSDM RAEIIRMMEG

450 AKPEEVSFRG RGVFELSDEK ATNPIVPSFD MSNEGSYFFG DNAEEYDN

In certain embodiments, the polypeptide encoded by a polynucleotide of the invention further comprises a linker inserted between any two of the M2 ectodomain peptides. In one embodiment, any M2 ectodomain peptide present in the polypeptide has a linker peptide interposed between it and the adjacent M2 ectodomain peptide. Generally, any given polypeptide may have no linker, one linker, or multiple linker peptides. When more than one linker peptide is present in the polypeptide, the linkers may be the same or different. The polypeptide may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 linker peptides. The linker peptide may be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 amino acids. In one embodiment, the linker peptide is 5 amino acids in length. In other embodiments, the linker peptide has low or minimal immunogenicity, hydrophobicity, or hydrophilicity to the host.

In other embodiments, the linker peptide is homologous to an amino acid sequence found in a lower organism. In other embodiments, the linker peptide is not homologous to an amino acid sequence found in a primate. If these linker peptides are immunogenic and cause production of antibodies, these antibodies will circulate in the host and have the opportunity to bind any cognate antigen. If the possible antigen is a self-protein, the immunogenicity is not required. The immunogenicity may be beneficial if the possible antigen is homologous to the pathogen or other microorganism.

In certain embodiments, the linker peptide used in the present invention may be homologous to an amino acid sequence found in the following species: burkholderia (Burkholderia sp.) H160, Arthrospira maxima CS-328, Heliothis armigera (Helicoverpa armigera) SNPV, Francisella noveri (Francisella noveriana) FTG, California albilineans (Peromyculus californica), Helicobacter pylori (Helicobacter pylori) G27, Vibrio salmonicida (Alivibrio salmonida) I1238, Lycopersicon pannicum (Solanium pennellii), Saccharomyces cerevisiae (Saccharomyces cerevisiae) AWRI 1, Cryptosporium hominis (Cryptosporium hominis), Gyrosoma sp (Bordossarkanensis) Nians, Nitrosococcus niloticus (Bacillus subtilis) C-27, Corynebacterium parvum (Cryptococcus sp) H2, Corynebacterium parvum, Clostridium sp-11, Clostridium sp.sp.3632, Clostridium sp.sp.3611, Clostridium sp.faecalis, Clostridium sp.c-11, Clostridium sp.sp.sp.3632, Bacillus subtilis, Clostridium sp.c-11, Bacillus subtilis, Bacillus coli, Bacillus subtilis H3, Bacillus subtilis sp.c.c-11, Bacillus subtilis sp.c.c.c.c.c.c.c.c.c.c.c.c.c.c.3, Bacillus sp.c.c.c.3, Bacillus sp.3, Bacillus sp.c.c.c.c.c.3, Bacillus sp.c.3, Bacillus sp.c.sp.sp.3, Bacillus sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.3, Bacillus sp.sp.sp.sp.sp.sp.sp, Clostridium pasteurianum (Clostridium bartlettii) DSM 16795, Claviceps purpurea (Claviceps purpurea), globefish (Tetraodon nigroviris), essential Polybacter (Polynucleobacter nereus) STIR1, rumen-stimulating fungus (Piromyces rhizinflatus), neuraminidase [ influenza A virus (A/chicken/Iran/16/2000 (H9N2)) ], neuraminidase [ influenza A virus (A/wild duck/MN/1/2000 (H5N2)) ], Escherichia coli (Escherichia coli) O157: H7 strain EC4045, S1 glycoprotein [ infectious bronchitis virus ], neuraminidase [ influenza A virus (A/Leiden/01272/2006(H3N2)) ], neuraminidase [ influenza A virus (A/Tilburg/45223/2005(H3N2)) ], neuraminidase [ influenza A virus A/influenza A virus (MClvo 63171/PIV A/685A) No. A/6858 (MClvn 638/influenza A638)) ] 1934-Brisbane/10/2007) (H3N2)) ], neuraminidase [ influenza A virus (A/Managua/26/2007(H3N2)) ], neuraminidase [ influenza A virus (A/HongKong/1-MA-20D/1968 (H3N2)) ], neuraminidase [ influenza A virus (A/CzechRepublic/1/1966(H2N2)) ], neuraminidase [ influenza A virus (A/chicken/Shanghai/2/1999 (H9N2)) ], neuraminidase [ influenza B virus (B/Myanmar/M170/2007) ], neuraminidase [ influenza A virus (A/Myanmar/M187/2007(H3N2) ], neuraminidase [ influenza A virus (A/Yomban/101276/N1/2005)) ], neuraminidase [ influenza A virus (A/Yomban/101276/397 (H3N2)) ], neuraminidase [ influenza A virus (A/Managana/M3957) ]), Neuraminidase [ influenza a virus (a/american domestic duck/New York/87493-3/2005(H7N2)) ], neuraminidase [ influenza a virus (a/turkey/New York/122501-2/2005(H7N2)) ], and/or neuraminidase [ influenza a virus (a/wild duck/Italy/4223-2/2006 (H5N2)) ]. In one embodiment, the linker peptide used in the present invention is Gly-Ser-Ala-Ser-Gly (GSASG) (SEQ ID NO: 21).

In other embodiments, the linker peptide is homologous to an amino acid sequence found in Staphylococcus aureus (Staphylococcus aureus) bacteriophage phi2958PVL, oxytetracycline (Streptomyces rimosus), gyrus jumps (Bodo saltans), proteoliposphilus thermophilus (coprotherobacter proteoliticus) DSM 5265, and/or Leptospirillum (Leptospirillum sp.) group II '5-way CG'. In a specific embodiment, the linker peptide used in the present invention is Ser-Gly-Ser-Gly-Ala (SGSGSGA) (SEQ ID NO: 22).

In other embodiments, the linker peptide is homologous to an amino acid sequence found in Drosophila montana (Drosophila montana), a polyprotein [ tomato torrado virus ], an immunoglobulin heavy chain variable region [ canine (Canis lupusfamiliaris) ], a polyprotein [ dengue virus 1], or an oxysterone (oxysterol) binding protein [ mus musculus (mususculus) ]. In one embodiment, the linker peptide used in the present invention is Ser-Ala-Gly-Ser-Gly (SAGSG) (SEQ ID NO: 23).

The linker peptide used in the present invention may be any linker known in the art, for example, an scFv linker for a single chain protein such as an scFv. In one embodiment, the linker peptide is the sequence (Gly)_n. In another embodiment, the linker peptide comprises the sequence (GlyAla)_n. In other embodiments, the linker peptide comprises the sequence (GGS)_n、(GGGS)_n(SEQ ID NO:24) or (GGS) n (GGGGS) n (SEQ ID NO:25), wherein n is an integer of 1 to 10,5 to 20, 10 to 30, 20 to 50, 40 to 80, or 50 to 100.

The invention also provides an isolated polynucleotide encoding a fusion protein comprising a coding region encoding a polypeptide comprising multiple copies of M2 extracellular domain peptide (METR), wherein the polypeptide further comprises one or more epitopes. In one embodiment, the polypeptide further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 epitopes. In another embodiment, the epitope is at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 amino acids in length. In one embodiment, any M2 ectodomain peptide present in the polypeptide has an epitope interposed between it and the adjacent M2 ectodomain peptide. In another embodiment, the polypeptide further comprises one epitope or more than one epitope interposed between the M2 extracellular domain peptides. When more than one epitope is present, the epitopes may be the same or different.

The epitope may be a B cell epitope or a T cell epitope. The B cell epitopes used in the present invention may be derived from M2 protein, for example an antibody epitope located within the N-terminal 19-20 amino acids of the extracellular domain of M2. Non-limiting examples of B-cell epitopes are amino acids 5-12 of SEQ ID NO. 1, amino acids 1-10 of SEQ ID NO. 1, amino acids 7-17 of SEQ ID NO. 2, amino acids 5-14 of SEQ ID NO. 3 or amino acids 3-19 of SEQ ID NO. 3. The B cell epitope may be derived from other domains of the M2 protein, such as a transmembrane domain or a cytoplasmic domain. Alternatively, the B cell epitope may be obtained from any influenza virus protein or fragment thereof, such as Hemagglutinin (HA), Neuraminidase (NA), Nucleoprotein (NP), matrix 1 protein (M1), matrix 2 protein (M2), non-structural protein (NS), RNA polymerase PA subunit (PA), RNA polymerase PB1 subunit (PB1), RNA polymerase PB2 subunit (PB 2).

The T cell epitopes used in the present invention may comprise any number of amino acids and be derived from any known antigen or immunogen. In one embodiment, the T cell epitope may be derived from any influenza virus protein or fragment thereof, such as Hemagglutinin (HA), Neuraminidase (NA), Nucleoprotein (NP), matrix 1 protein (M1), matrix 2 protein (M2), non-structural protein (NS), RNA polymerase PA subunit (PA), RNA polymerase PB1 subunit (PB1), or RNA polymerase PB2 subunit (PB 2). In one embodiment, a T helper epitope may comprise 9 core amino acids flanked by 3 flanking amino acids each of which totals 15 amino acids. Its binding to the notch of the major histocompatibility complex (MHC in mice, HLA in humans) can be calculated by known methods. High scoring peptides are predicted to be ligands for those MHC of HLA molecules.

In one embodiment, the T cell epitope for use in the present invention comprises an amino acid sequence selected from SEQ ID NO:26, SEQ ID NO:27, or both, as described in U.S. Pat. No. 6,663,871, which is incorporated herein by reference in its entirety.

The present invention includes multiple copies of the M2 ectodomain peptide, one or more optional linker peptides interposed between two or more M2 ectodomain peptides, and/or one or more optional epitopes interposed between two or more M2 ectodomain peptides. In one embodiment, the polynucleotides of the invention encode a polypeptide comprising at least 6M 2 ectodomain peptides (M2e #1, M2e #2, M2e #3, M2e #4, M2e #5, and M2e #6), one or more linker peptides interposed between any two or more M2 ectodomain peptides (e.g., M2e #1-M2e #2, M2e #2-M2e #3, M2e #3-M2e #4, M2e #4-M2e #5, or M2e #5-M2e #6), and one or more epitopes interposed between any two or more M2 ectodomain peptides. In one embodiment, the polypeptide comprises 6M 2 ectodomain peptides, 3 linker peptides interposed therebetween, and two epitopes interposed therebetween, wherein:

(1) a first linker peptide interposed between the first M2 ectodomain peptide (M2e #1) and the first M2 ectodomain peptide (M2e # 2);

(2) a second linker peptide interposed between the second M2 ectodomain peptide (M2e #2) and the third M2 ectodomain peptide (M2e # 3);

(3) a third linker peptide interposed between the third M2 ectodomain peptide (M2e #3) and the fourth M2 ectodomain peptide (M2e # 4);

(4) a first epitope interposed between the fourth M2 ectodomain peptide (M2e #4) and the fifth M2 ectodomain peptide (M2e # 5); and

(5) a second epitope interposed between the fifth M2 ectodomain peptide (M2e #5) and the sixth M2 ectodomain peptide (M2e # 6).

In some embodiments, an isolated polynucleotide of the invention comprises a coding region that encodes a polypeptide comprising multiple copies of the M2 extracellular domain peptide, wherein the coding region further comprises additional nucleic acid sequences. In certain embodiments, the additional nucleic acid may encode an additional polypeptide, optionally fused to a polypeptide of the invention. The additional polypeptides may include at least one immunogenic epitope of an influenza virus, wherein the epitope elicits a B cell (antibody) response, a T cell response, or both.

Various other nucleic acids may be used to encode their respective other polypeptides. In one embodiment, the additional polypeptide is fused to a METR polypeptide of the invention. In other embodiments, the additional polypeptide is not fused to a METR polypeptide of the invention, but is produced in the same vector that expresses the METR polypeptide. Non-limiting examples of such other nucleic acid sequences are nucleic acid sequences encoding influenza proteins, variants, derivatives, analogues or fragments thereof. The influenza virus protein may be selected from the group consisting of: hemagglutinin (HA), Neuraminidase (NA), Nucleoprotein (NP), matrix 1 protein (M1), matrix 2 protein (M2), non-structural protein (NS), RNA polymerase PA subunit (PA), RNA polymerase PB1 subunit (PB1), or RNA polymerase PB2 subunit (PB 2).

In some embodiments, the additional polypeptide is selected from the group consisting of: n-or C-terminal peptides conferring stability, secretion or simple purification, i.e.His-tag, ubiquitin-tag, NusA-tag, chitin-binding domain, ompT, ompA, pelB, DsbA, DsbC, C-myc, KSI, polyaspartic acid, (Ala-Trp-Trp-Pro) N (SEQ ID NO:28), polyalanine, polycysteine, polyarginine, B-tag, HSB-tag, Green Fluorescent Protein (GFP), hemagglutinin influenza Virus (HAI), calmodulin-binding protein (CBP), galactose-binding protein, maltose-binding protein (MBP), cellulose-binding domain (CBD' S), dihydrofolate reductase (DHFR), glutathione S-transferase (GST), Streptococcus protein G, staphylococcal protein A, T7genelO, avidin/streptavidin/ep-tag, streptavidin/streptavidin-linked protein, or a mixture thereof, trpE, chloramphenicol acetyltransferase, lacZ (. beta. -galactosidase), His-patched (patch) thioredoxin, FLAG^TMPeptides (Sigma-Aldrich)), S-tags, and T7-tags. See, e.g., Stevens, R.C., Structure,8: R177-R185 (2000). The heterologous polypeptide can also include any pre-sequence and/or pre-sequence that facilitates transport, translocation, processing, and/or expression of the METR sequence or any useful immunogenic sequence, including but not limited to T cell epitopes or other immunogenic proteins and/or epitopes encoding microbial pathogens. Other suitable additional polypeptides may include leader or signal sequences.

Codon optimization

Also included within the scope of the invention are codon optimized polynucleotides encoding polypeptides comprising multiple copies of the M2 ectodomain peptide sequence. Modification of the nucleic acid encoding the polypeptide is readily accomplished by those skilled in the art, for example, by oligonucleotide-directed site-directed mutagenesis of a polynucleotide encoding the polypeptide. The modified polypeptide may be encoded by a codon-optimized nucleotide sequence. The modifications result in one or more amino acid substitutions, insertions, deletions and/or modifications to express the polypeptide, including fragments, variants and derivatives. Such modifications may increase the immunogenicity of the antigen, for example by increasing the cellular immune response compared to the unmodified polypeptide. The modifications may increase the solubility of the polypeptide. Alternatively, the modification may be ineffective. For example, the M2 extracellular domain peptide may be introduced, deleted, or modified by specific cleavage sites for proteolytic activity in antigen presenting cells to enhance the immune response to specific epitopes.

It will be appreciated by those of ordinary skill in the art that due to the redundancy of the genetic code, the various nucleic acid coding regions will encode the same polypeptide. Deviations in the nucleotide sequence, including codons encoding any polypeptide chain amino acid, allow for variations in the sequence of the encoding gene. Since each codon consists of three nucleotides and the nucleotides comprising the DNA are limited to four specific bases, there are 64 possible nucleotide combinations of which 61 encode amino acids (the remaining three codons encode the signal end translation). The "genetic code" showing which codon encodes which amino acid is shown in table 3 herein. Thus, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are encoded by 4 triplets, serine and arginine by 6, and tryptophan and methionine by only one triplet. This degeneracy allows the base composition of DNA to vary over a wide range without altering the amino acid sequence of the polypeptide encoded by the DNA.

Table 3: standard genetic code

It is understood that any polynucleotide encoding a polypeptide of the present invention falls within the scope of the present invention, regardless of the codons used.

Many organisms show a preference for the use of a particular codon for encoding an insertion of a particular amino acid in a growing polypeptide chain. Codon preference or codon bias, differences in codon usage between organisms, provided by the degeneracy of the genetic code, and have been well documented between many organisms. Codon bias is often correlated with the efficiency of translation of messenger rna (mrna), which in turn is believed to depend on, but not limited to, the nature of the codon being translated and the availability of a particular transfer rna (trna) molecule, among other things. The predominance of the selected tRNA in the cell typically reflects the most frequently used codon in peptide synthesis. Thus, genes can be regulated for optimal gene expression in a given organism based on codon optimization.

The present invention provides isolated polynucleotides, including polynucleotides comprising, consisting essentially of, or consisting of a coding region encoding an influenza virus protein, such as a METR, disclosed herein. In this embodiment, the codon usage is adapted for optimal expression in a cell of a given prokaryote or eukaryote.

Polynucleotides are prepared by incorporating codons preferred for a gene of a given species into a DNA sequence. Also provided are polynucleotide expression constructs, vectors, and host cells comprising nucleic acid segments of codon-optimized coding regions encoding influenza polypeptides, such as METR, and various methods of using the polynucleotide expression constructs, vectors, and host cells for treating or preventing influenza infection in an animal.

Given the large number of gene sequences available for various animal, plant and microbial species, the relative frequency of codon usage can be calculated. Codon usage tables are available, for example, on the "codon usage database" available from www.kazusa.or.jp/codon/(5/30.2006), and these tables are applicable in many ways. See Nakamura, Y., et al, "Codon use structured from the international DNA sequence databases: status for the year 2000 (Codon usage as tabulated by International DNA sequence databases)," nucleic acids Res.28:292 (2000). Codon usage calculated from GenBank release 151.0 is shown in Table 4 (from www.kazusa.or.jp/codon/supra). These tables use mRNA nomenclature, so uracil (U) found in RNA is used in the tables, rather than thymine (T) found in DNA. The table was adjusted so that the frequency was calculated for each amino acid, rather than all 64 codons.

Table 4: codon usage table of human gene (Homo sapiens)

By using these or similar tables, one of ordinary skill in the art can apply the frequency to any given polypeptide sequence and generate nucleic acid fragments of codon-optimized coding regions that encode the polypeptide but use codons optimized for a given species.

There are many options for designing codon-optimized coding regions for synthesis by any of the methods described above, using standard and routine molecular biology procedures well known to those of ordinary skill in the art. In one method, a series of complementary oligonucleotide pairs, each 80-90 nucleotide long and spanning the length of the desired sequence, are synthesized using standard methods. These oligonucleotide pairs are synthesized such that, upon annealing, they form a double-stranded fragment of 80-90 base pairs, comprising a sticky end, as each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6,7, 8, 9, 10 or more bases beyond the region of the pair that is complementary to the other oligonucleotide. The single stranded ends of each pair of oligonucleotides are designed to anneal to the single stranded ends of the other pair of oligonucleotides. The oligonucleotide pairs are annealed, then about 5-6 of these double-stranded fragments are annealed together by the sticky single-stranded ends, which are then ligated together and cloned into a standard bacterial cloning vector, such as the TOPO vector available from, for example, Invitrogen Corporation of calsbad, california. The construct was then sequenced by standard methods. Several of these constructs, i.e., about 500 base pair fragments, were prepared consisting of 5-6 fragments of 80-90 base pairs joined together so that the entire desired sequence is present in a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. The final construct was then cloned into a standard bacterial cloning vector and sequenced. Other methods will be immediately apparent to those skilled in the art. Furthermore, gene synthesis is readily available commercially.

Carrier

The present invention relates to vectors such as plasmids, cosmids, viruses, and bacteriophages conventionally used in genetic engineering comprising a polynucleotide encoding an influenza virus antigen or polypeptide comprising multiple copies of the M2 ectodomain peptide, such as METR, in any order relative to each other.

In one embodiment, the vector is an expression vector and/or a gene transfer or targeting vector. In another embodiment, the vector is a viral vector. Expression vectors derived from viruses such as retrovirus, vaccinia virus, adeno-associated virus, adenovirus, herpes virus, or bovine papilloma virus may be used to deliver the polynucleotides or vectors of the invention to a target cell population. Methods well known to those skilled in the art can be used to construct recombinant viral vectors; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual (A handbook of Molecular Cloning laboratories), Cold spring harbor Laboratory in New York (1989) and Ausubel, Current Protocols in Molecular Biology (New methods in Molecular Biology), Green publishing Co., Ltd. and Wiley Interscience (1994). Alternatively, the polynucleotides and vectors of the invention may be recombined into liposomes for delivery to target cells. Vectors containing a polynucleotide of the invention, e.g., multiple copies of the M2 extracellular domain sequence (METR), can be transferred into a host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation may be used for other cellular hosts, see Sambrook, supra. Generally, vectors compatible with the present invention comprise a selectable marker, suitable restriction sites to facilitate cloning of a desired gene, and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.

In certain embodiments, the invention relates to poxviruses, such as vaccinia viruses, such as modified vaccinia virus ankara (MVA), comprising a polynucleotide encoding a polypeptide comprising an influenza polypeptide, such as multiple copies of M2 Ectodomain (METR), NP, or both. MVA is a highly attenuated vaccinia virus strain, a member of the orthopoxvirus genus of the poxviridae family. Poxviruses include four genera of poxviruses, namely orthopoxvirus, parapoxvirus, yatapoxvirus and molluscum poxvirus. Orthopoxviruses include, but are not limited to, variola virus (a medium that causes smallpox), vaccinia virus, monkeypox virus, and raccoon poxvirus; parapoxviruses include, but are not limited to, capripoxvirus, pseudovaccinia and bovine influenza virus, yatapoxviruses include, but are not limited to, tanapo virus and yaba monkey tumor virus, and molluscum poxviruses include Molluscum Contagiosum Virus (MCV).

Vaccinia virus is used as a live vaccine to immunize against human smallpox disease or to engineer viral vectors for recombinant gene expression or potentially as a recombinant live vaccine (Mackett, M. et al, 1982 PNAS USA 79:7415-7419; Smith, G.L. et al, 1984 Biotech GenetEngin Rev 2: 383-407). Engineered viral vectors may contain DNA sequences (genes) encoding foreign antigens such as influenza polypeptides with the aid of DNA recombination techniques. If the gene is integrated at a position in the viral genome that is not essential for the viral life cycle, a newly generated recombinant vaccinia virus incorporating the foreign gene may infect the host cell and thereby induce expression of the foreign gene in the host cell. (U.S. Pat. Nos. 5,110,587;83,286; and 110,385, which are incorporated herein by reference in their entirety). The recombinant vaccinia virus (e.g., MVA) produced by this method is used according to the invention as a live vaccine against infectious influenza disease in vivo.

In one embodiment, a vaccinia virus strain used herein is exemplified by a highly attenuated modified vaccinia virus ankara (MVA). MVA is generated by long-term serial passages of Ankara strains of vaccinia virus (CVA) on chicken embryo fibroblasts (see Mayr, A. et al 1975 Infection 3:6-14; Switzerland patent No. 568,392). MVA viruses are publicly available, e.g., from the American Type culture Collection (ATCC number: VR-1508). MVA distinguishes between attenuated virulence and limited ability to regenerate infectious virions, as in certain mammalian cells, based on its degree of attenuation, while maintaining good immunogenicity and full ability to replicate and produce infectious virions in avian cells. MVA viruses have alterations in their genome that induce attenuation relative to the parental CVA strain. A total of 31,000 base pairs (about 10% of its genome) of 6 major deletions of genomic DNA (deletions I, II, III, IV, V and VI) have been identified (Meyer, H. et al 1991J Gen Virol 72: 1031-. The resulting MVA virus becomes extremely attenuated in mammalian cells. Due to its attenuation, the MVA of the present invention may be non-toxic even in immunosuppressed individuals and the side effects associated with the use of MVA in live vaccines against infectious influenza diseases are very small.

The vector, such as the MVA of the present invention, can undergo limited replication in human cells, since its replication is blocked at later stages of infection. Limited replication prevents assembly of mature infectious virions. However, vectors such as the MVA of the present invention can express viral and recombinant genes at high levels even in non-permissive cells and can serve as efficient and safe gene expression vectors.

In one embodiment of the invention, an isolated polynucleotide sequence encoding an influenza polypeptide, such as a METR fusion, is fused to the MVA-flanking sequence adjacent to a naturally occurring deletion, such as deletion I, deletion II, deletion III, deletion IV, deletion V or deletion VI, or other nonessential site present at the 5 'or 3' end of said polynucleotide in the MVA genome. Non-essential regions of the MVA genome include, but are not limited to, intergenic regions and naturally occurring deletion regions as well as other genes that are not required for replication, such as the tk gene. The DNA sequence carrying the polynucleotide sequence encoding one or more influenza virus antigens, such as METR, may be linear or circular, be a polymerase chain reaction product or a plasmid, and may further include a regulatory sequence such as a promoter operably linked to the coding region encoding at least one influenza virus polypeptide. Non-limiting examples of such regulatory elements include the vacciniｃA 11kｃA gene described in EP-A-198,328 and the 7.5kDｃA gene (EP-A-110,385), each of which is incorporated herein by reference in its entirety.

In some embodiments, the present invention provides a recombinant MVA comprising a polynucleotide comprising a promoter operably linked to a coding region encoding an influenza polypeptide antigen, such as METR. In a specific embodiment, the promoter is a viral promoter (e.g., a vaccinia virus or modified vaccinia virus ankara promoter). In other embodiments, the promoter is a synthetic promoter. In other embodiments, the promoter is a strong promoter. In a specific embodiment, the promoter is a strong synthetic promoter. In one embodiment, the promoter is the PS promoter having the sequence AAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATA (SEQ ID NO:29) (Chakrabarti, Sisler and Moss (1997). Biotechniques 23: 1094-. In other embodiments, the promoter is the modified H5 promoter having the sequence AAAAAATGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATT (SEQ ID NO:30) (Rosel et al (1986) J.Virol.60(2): 236-249).

Poxvirus transcriptional control regions include promoters and transcriptional termination signals. Gene expression in poxviruses is regulated over time and the promoters of early, mid and late genes contain different structures. Certain poxvirus genes are constitutively expressed and the promoters of these "early-late" genes carry hybrid constructs. Synthetic early-late promoters have also been developed. See Hammond J.M., et al, J.Virol. methods 66:135-8(1997), Chakrabarti S., et al, Biotechniques 23:1094-7 (1997). Thus, any poxvirus promoter such as an early, late or constitutive promoter may be used in the present invention.

Non-limiting examples of early promoters include the 7.5-kD promoter (also a late promoter), DNA pol promoter, tk promoter, RNA pol promoter, 19-kD promoter, 22-kD promoter, 42-kD promoter, 37-kD promoter, 87-kD promoter, H3' promoter, H6 promoter, D1 promoter, D4 promoter, D5 promoter, D9 promoter, D12 promoter, I3 promoter, M1 promoter, and N2 promoter. See, for example, Moss, B., "Poxviridae and the Replication of the Poxviridae" incorporated in Virology (Virology) second edition, ed.B.N.fields, D.M.Knipe et al eds., Lavender Press 2088 (1990). The early genes transcribed in vaccinia virus and other poxviruses recognize the transcription termination signal TTTTTNT (SEQ ID NO:31), where N can be any nucleotide. Transcription usually terminates about 50bp upstream of the signal. Thus, if a heterologous gene is expressed from the poxvirus early promoter, care must be taken to eliminate the presence of this signal in the coding region of the gene. See, e.g., Earl, p.l., et al, j.virol.64:2448-51 (1990).

Examples of late promoters include, but are not limited to, the 7.5-kD promoter, the MIL promoter, the 37-kD promoter, the 11L promoter, the 12L promoter, the 13L promoter, the 15L promoter, the 17L promoter, the 28-kD promoter, the H1L promoter, the H3L promoter, the H5L promoter, the H6L promoter, the H8L promoter, the D11L promoter, the D12L promoter, the D13L promoter, the A1L promoter, the A2L promoter, the A3L promoter, and the P4b promoter. See, for example, Moss, B., "Poxviridae and the Replication of the Poxviridae" incorporated in Virology (Virology) second edition, ed.B.N.fields, D.M.Knipe et al eds., Lavender Press (1990) p.2090. The late promoter does not appear to recognize the transcription termination signal recognized by the early promoter.

Non-limiting examples of constitutive promoters useful in the present invention include the synthetic early-late promoter described by Hammond and Chakrabarti, MH-5 early-late promoter, and the 7.5-kD or "p 7.5" promoter.

The invention also relates to vectors, such as MVA, and other nucleic acid sequences comprising the polynucleotides of the invention. The additional nucleic acid sequence may be inserted at the same or a different insertion site as the site at which the polynucleotide sequence encoding multiple copies of M2 extracellular domain peptide (METR) is inserted. The additional nucleic acid sequence may include coding regions encoding additional polypeptides. The additional nucleic acid sequence may also be linked to a promoter. In one embodiment, the vectors of the invention, such as MVA, encode two or more antigens or immunogens, one antigen being a METR or NP consensus sequence and the other antigen being another polypeptide. The additional polypeptide may be an influenza virus protein, variant, fragment, derivative or analog thereof, as used herein.

In a specific embodiment, the vectors of the invention, such as MVA, express at least two influenza virus antigens, such as METR and HA, METR and NP, HA and NP, and at least 3 influenza virus antigens, such as METR, HA and NP. In certain embodiments, the vectors of the invention express at least 4, at least 5, at least 6, at least 7, or at least 8 influenza virus antigens.

The invention also provides a method of producing a vector, such as MVA, comprising introducing into a host cell infected with the vector, such as MVA, an isolated polynucleotide (DNA) construct comprising a polynucleotide encoding multiple copies of M2 extracellular domain peptide (METR) to allow for homologous recombination. Once the DNA construct encoding the METR polypeptide is introduced into the host cell and the exogenous DNA sequence is recombined with viral DNA, the resulting recombinant MVA virus comprises a polynucleotide encoding a METR polypeptide. The invention also provides for the isolation of the resulting MVA using known methods, such as marker-assisted isolation. The DNA construct carrying the METR polypeptide gene can also be introduced into MVA-infected cells by transfection, for example by calcium phosphate precipitation (Graham et al 1973Virol 52:456-467; Wigler et al 1979Cell 16: 777-785), by electroporation (Neumann et al 1982EMBO J.1: 841-845), by microinjection (Graessmann et al 1983Meth Enzymol101: 482-492), by liposomes (Straubinger et al 1983Meth Enzymol101: 512-527), by protoplasts (Schaffher 1980 PNAS USA 77: 2163-2167) or by other methods known to the person skilled in the art. The references to the transfection methods listed herein are incorporated by reference in their entirety.

According to the present invention, recombinant MVA vaccinia viruses can be isolated by several well-known techniques, such as selection protocols based on the K1L gene. As a non-limiting example, the DNA construct may comprise a DNA sequence encoding vaccinia virus K1L protein or K1L-derived polypeptide as a marker, and a DNA sequence encoding a METR polypeptide flanked on both ends by DNA sequences flanking non-essential sites within the MVA genome, such as a naturally occurring deletion, e.g., deletion III.

Host cells for use in the present invention include, but are not limited to, eukaryotic cells, avian cells, mammalian cells, or human cells. Non-limiting examples of eukaryotic cells are BHK-21(ATCC CCL-10), BSC-1 (ATCCCL-26), CV-1(ECACC 87032605), or MA104(ECACC 85102918). In one embodiment, the host cell is an avian cell, including but not limited to a chicken cell, a duck cell, or a quail cell. Non-limiting examples of avian cells are chicken fibroblasts, quail fibroblasts, QT9 cells, QT6 cells, QT35 cells, Vero cells, MRC-5 cells, chick embryo-derived LSCC-H32 cells, chicken DF-1 cells, or primary Chick Embryo Fibroblasts (CEF) cells. In other embodiments, avian cells used as host cells in the present invention are immortalized. The immortalized avian cells can be immortalized duck cells including, but not limited to, AGE1cr cells and age1cr. pix cells as described in U.S. patent application No. US 2008/0227146a1 and international publication No. WO 2007/054516a1, which are incorporated herein by reference in their entirety. Other immortalized duck cells for use herein include embryonic stem cells such as those described in U.S. patent application No. US 2010/062489A1Cells, which are incorporated herein by reference in their entirety. Other useful avian cell lines are described in PCT application publication No. WO 2006/1088646A2, U.S. application publication No. 2006/0233834a1, and U.S. patent nos. 5,830,510 and 6,500,668, all of which are incorporated herein by reference in their entirety.

Polypeptides

The invention also includes polypeptides comprising multiple copies of M2 extracellular domain peptide (METR). In one embodiment, the invention is an isolated polypeptide comprising at least 5 of the following influenza virus matrix 2 protein ectodomain peptides arranged in any order relative to each other: (i) SEQ ID NO 1(M2e #1_ C), (ii) SEQ ID NO 2(M2e #2_ C), (iii) SEQ ID NO 3(M2e #3_ C), (iv) SEQ ID NO 4(M2e #4_ C), (v) SEQ ID NO 5(M2e #5_ C), (vi) SEQ ID NO 6(M2e #6_ C), (vii) SEQ ID NO 7(M2e #1_ S), (viii) SEQ ID NO 8(M2e #2_ S), (ix) SEQ ID NO 9(M2e #3_ S), (x) SEQ ID NO 10(M2e #4_ S), (xi) SEQ ID NO 11(M2e #5_ S), and (xii) SEQ ID NO 12 # S2 e).

In one embodiment, the present invention provides an isolated polypeptide comprising at least 3 of the following M2 ectodomain peptides arranged in any order relative to each other: (i) SEQ ID NO 1(M2e #1_ C), (ii) SEQ ID NO 2(M2e #2_ C), (iii) SEQ ID NO 3(M2e #3_ C), (iv) SEQ ID NO 4(M2e #4_ C), (v) SEQ ID NO 5(M2e #5_ C), (vi) SEQ ID NO 6(M2e #6_ C), (vii) SEQ ID NO 7(M2e #1_ S), (viii) SEQ ID NO 8(M2e #2_ S), (ix) SEQ ID NO 9(M2e #3_ S), (x) SEQ ID NO 10(M2e #4_ S), (xi) SEQ ID NO 11(M2e #5_ S), and (xii) SEQ ID NO 12(M2 # 12 _ S2 e). In another embodiment, the polypeptide comprises at least 4, 5, 6,7, 8, 9, 10, 11, or 12M 2 ectodomain peptides. In some embodiments, the polypeptides of the invention are fusion proteins and induce an immune response against influenza virus. In certain embodiments, the polypeptides of the invention comprise at least 3 of the following M2 ectodomain peptides arranged in any order relative to each other: (a) (i) SEQ ID NO:1(M2e #1_ C), (ii) SEQ ID NO:2(M2e #2_ C), (iii) SEQ ID NO:3(M2e #3_ C), (iv) SEQ ID NO:4(M2e #4_ C), (v) SEQ ID NO:5(M2e #5_ C), and (vi) SEQ ID NO:6(M2e #6_ C) or (b) (i) SEQ ID NO:7(M2e #1-S), (ii) SEQ ID NO:8(M2e #2_ S), (iii) SEQ ID NO:9(M2e #3_ S), (iv) SEQ ID NO:10(M2e #4_ S), (v) SEQ ID NO:11(M2e # S), and (vi) SEQ ID NO: 5812 # 25 (M2 # S).

In one embodiment, the polypeptide of the invention comprises the following 6 amino acid sequences in any order relative to each other: (a) (i) SEQ ID NO:1(M2e #1_ C), (ii) SEQ ID NO:2(M2e #2_ C), (iii) SEQ ID NO:3(M2e #3_ C), (iv) SEQ ID NO:4(M2e #4_ C), (v) SEQ ID NO:5(M2e #5_ C), (vi) SEQ ID NO:6(M2e #6_ C), (b) (i) SEQ ID NO:7(M2e #1-S), (ii) SEQ ID NO:8(M2e #2_ S), (iii) SEQ ID NO:9(M2e #3_ S), (iv) SEQ ID NO:10(M2e #4_ S), (v) SEQ ID NO:11(M2e # S), (vi) SEQ ID NO: 5812 # S (M2 # 25). In other embodiments, the polypeptide of the invention comprises the NP consensus sequence of SEQ ID NO 20.

The polypeptides of the invention may be fusion proteins, which also include other polypeptides. Non-limiting examples of such other polypeptides are other influenza polypeptides, variants, derivatives, analogs, or fragments thereof. The other polypeptide may be immunogenic or antigenic and may be any known antigen.

Composition comprising a metal oxide and a metal oxide

Compositions, such as pharmaceutical or vaccine compositions, comprising an immunologically effective amount of an isolated polynucleotide, polypeptide or vector, such as MVA, are further embodiments of the invention. Such compositions may include, for example, lipopeptides (e.g., Vitiello, A. et al, J. Clin. invest.95:341,1995), coated polypeptides such as in poly (DL-lactide-co-glycolide) ("PLG") microspheres (see, e.g., Eldridge, et al, Molec. Immunol.28:287-294,1991: Alonso et al, Vaccine 12:299-306,1994; Jones et al, Vaccine 13:675-681, 1995); polypeptide compositions contained in immunostimulatory complexes (ISCOMs) (see, e.g., Takahashi et al, Nature 344:873-875,1990; Hu, et al, Clin Exp Immunol.113:235-243,1998), multiple antigen peptide systems (MAP) (see, e.g., Tam, J.P., Proc.Natl.Acad.Sci.U.S.A.85:5409-5413,1988; Tam, J.P., J.Immunol.methods 196:17-32,1996), particles of viral or synthetic origin (e.g., Kofler, N.et al, J.Mumunol.methods.192: 25,1996; Eldridge, J.H. et al, Sem.Hematol.30:16,1993; Fahren, L.D., Immunol.J.R.et al, Nature Med.7:649,1995; adjuvants (e.g., Warren. H. et al, Warne.H. 483.J.H., Val.J.J.H. J.H. J.J.H. 9: 14; Liposome, R.S.S.J.S.S.J.J.J.J.Immunol.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.H. Immunol.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.. The composition may be a pharmaceutical, antigen, immunogen or vaccine composition.

The compositions of the invention, e.g. vaccine compositions, may be formulated in known manner. Suitable methods of preparation are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences), 16 th edition, compiled by A.Osol, Mack Publishing Co.Easton, Pa. (1980) and Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences), 16 th edition, compiled by A.R.Gennaro, Mack Publishing company, Iston, Pa. (1995), both of which are incorporated herein by reference in their entirety. Although the composition is administered as an aqueous solution, it may also be formulated as an emulsion, gel, solution, suspension, lyophilized form, or any other form known in the art. In addition, the composition may include pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives. Once formulated, the compositions of the present invention can be administered directly to a subject. The subject to be treated may be an animal; in particular a human.

The concentration of polynucleotide, polypeptide or vector, such as MVA, in the compositions of the invention may vary widely, i.e. from less than about 0.1%, typically 2% or at least about 2%, up to 20% -50% or more by weight; the choice will be made primarily by fluid volume, viscosity, etc., according to the particular mode of administration chosen.

In one embodiment, the compositions of the invention include an isolated polynucleotide comprising a coding sequence that encodes a polypeptide comprising multiple copies, e.g., at least 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 of the following M2 ectodomain peptides, arranged in any order relative to each other: (i) SEQ ID NO 1(M2e #1_ C), (ii) SEQ ID NO 2(M2e #2_ C), (iii) SEQ ID NO 3(M2e #3_ C), (iv) SEQ ID NO 4(M2e #4_ C), (v) SEQ ID NO 5(M2e #5_ C), (vi) SEQ ID NO 6(M2e #6_ C), (vii) SEQ ID NO 7(M2e #1_ S), (viii) SEQ ID NO 8(M2e #2_ S), (ix) SEQ ID NO 9(M2e #3_ S), (x) SEQ ID NO 10(M2e #4_ S), (xi) SEQ ID NO 11(M2e #5_ S), and (xii) SEQ ID NO 12(M2 # 12 _ S2 e). In another embodiment, a composition, such as a vaccine composition of the invention, comprises one or more vectors, such as MVA, comprising a polynucleotide encoding multiple copies of M2 extracellular domain peptide (METR) or NP consensus sequences. In some embodiments, a composition comprising a polypeptide of the invention comprises multiple copies of the M2 ectodomain peptide.

In some embodiments, host cells having vectors comprising polynucleotides of the invention incorporate compositions, such as those described in Eko, et al, J.Immunol.,173:3375-3382, 2004.

Certain compositions may also include one or more adjuvants before, after, or simultaneously with the polynucleotide, polypeptide, or vector, such as MVA. Various materials exhibit adjuvant activity by various mechanisms. Possible adjuvants screened for their ability to enhance the immune response according to the present invention include, but are not limited to: inert carriers such as aluminum, bentonite, latex and acrylic particles, pluronic block polymers such as(Block copolymer CRL-8941, squalene (a metabolizable oil) and microsilica stabilizers), storage formers such as Freund's adjuvant, surface active materials such as saponin, lysolecithin, retinal, Quil A, liposomes and Pluronic polymer formulations, macrophage stimulators such as bacterial lipopolysaccharide, polycationic polymers such as chitosan, alternative pathway supplementation activators such as insulin, zymosan, endotoxin and levamisole, and non-ionic surfactants such as poloxamers, poly (oxyethylene) -poly (oxypropylene) triblock copolymers, cytokines and growth factors, bacterial components (such as endotoxins, particularly superantigens; exotoxins and cell wall components), aluminum-based salts such as aluminum hydroxide, calcium-based salts, silica, polynucleotides, toxoids, serum proteins, materials of viral and viral origin, toxic agents, Venom, imidazoquinoline compounds, poloxamers, mLT and cationic lipids. International patent application PCT/US95/09005, which is incorporated herein by reference, describes the use of heat labile toxin mutant forms of enterotoxigenic e.coli ("mLT") as adjuvants. U.S. patent No. 5,057,540, incorporated herein by reference, describes the adjuvant Qs 21. In some embodiments, the adjuvant is a toll-like receptor (TL)R) a stimulating adjuvant. See, e.g., Science 312: 184-. TLR adjuvants include compounds that stimulate TLRs (e.g., TLR1-TLR 17) resulting in an increased immune system response to the vaccine compositions of the invention. An example of a TLR adjuvant includes, but is not limited to, CpG (coley pharmaceutical Group Inc.) and MPL (Corixa.) CpG7909 described in WO 98/018810, U.S. patent application No. 2002/0164341a, U.S. patent No. 6,727,230, and international publication No. WO98/32462, which are incorporated herein by reference in their entirety.

The adjuvant dosage may vary depending on the particular adjuvant. For example, in some aspects, a dosage range may include: for CpG, 10 μ g/dose-500 μ g/dose or 50 μ g/dose-200 μ g/dose. Dosage ranges may include, for MPL, 2. mu.g/dose to 100. mu.g/dose or 10. mu.g/dose to 30. mu.g/dose. The dosage range may include, for aluminum hydroxide, from 10 μ g/dose to 500 μ g/dose or from 50 μ g/dose to 100 μ g/dose. In a prime-boost regimen, an adjuvant may be used in combination with a prime, a boost, or both, as described elsewhere herein.

In certain adjuvant compositions, the adjuvant is a cytokine. Certain compositions of the invention include one or more cytokines, chemokines, or compounds that induce the production of cytokines and chemokines, or polynucleotides encoding one or more cytokines, chemokines, or compounds that induce the production of cytokines and chemokines. Examples of cytokines include, but are not limited to, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), Colony Stimulating Factor (CSF), Erythropoietin (EPO), interleukin 2(IL-2), interleukin-3 (IL-3), interleukin 4(IL-4), interleukin 5(IL-5), interleukin 6(IL-6), interleukin 7(IL-7), interleukin 8(IL-8), interleukin 9(IL-9), interleukin 10(IL-10), interleukin 11(IL-11), interleukin 12(IL-12), interleukin 13(IL-13), interleukin 14(IL-14), interleukin 15(IL-15), Interleukin 16(IL-16), interleukin 17(IL-17), interleukin 18(IL-18), interferon alpha (IFN), interferon beta (IFN), interferon gamma (IFN), interferon omega (IFN), interferon tau (IFN), interferon gamma inducing factor I (IGIF), transforming growth factor beta (TGF-), RANTES (post-activation regulation, normal T-cell expression and presumed secretion), macrophage inflammatory proteins (such as MIP-1 alpha and MIP-1 beta), Leishmania (Leishmania) elongation-initiating factor (LEIF), and Flt-3 ligand.

The ability of an adjuvant to increase the immune response to an antigen is often manifested by a significant increase in immune-mediated responses or reduction in disease symptoms. For example, an increase in humoral immunity is usually manifested by a significant increase in antibody titer produced against an antigen, and an increase in T cell activity is usually manifested by an increase in cell proliferation or cytotoxicity or cytokine secretion. Adjuvants may also alter the immune response, for example by changing the primary humoral or Th2 response to a primary cellular or Th1 response. The immune response of a given antigen can be tested by various immunoassays well known to those of ordinary skill in the art and/or described elsewhere herein.

In addition, multiple copies of the M2 extracellular domain peptide (METR) polypeptide may be conjugated to bacterial toxoids, such as toxins from diphtheria, tetanus, cholera, helicobacter pylori (H pylori), or other pathogens. Furthermore, the METR polypeptide may be coupled to a bacterial polysaccharide, such as a capsular polysaccharide from Neisseria (Neisseria spp.), streptococcus pneumoniae (streptococcus pneumoniae spp.) or Haemophilus influenzae type b (Haemophilus influenzae) bacteria.

For solid compositions, conventional non-toxic solid carriers can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable non-toxic composition is formed by incorporating any of the normally used excipients such as those listed above and typically 10-95% of the active ingredient, i.e., one or more polypeptides of the invention (typically at a concentration of 25% -75%).

In some embodiments, the present invention relates to multivalent vaccines. For example, a multivalent vaccine of the invention, when administered in an effective amount to a subject in need thereof, may comprise a polynucleotide, polypeptide or vector, such as MVA, wherein said polynucleotide or vector, such as MVA, encodes two or more influenza virus epitopes or said polypeptide comprises two or more influenza virus epitopes. The two or more influenza epitopes may be derived from the same or different antigens. In certain embodiments, the multivalent vaccine induces an immune response against influenza matrix 2 protein and other influenza virus proteins or fragments thereof. In a specific embodiment, the composition of the invention comprises two or more polynucleotides, polypeptides or vectors of the invention, such as MVA. As a specific example, the invention may include compositions comprising two or more populations of MVAs, wherein a first MVA comprises a polynucleotide comprising a coding region encoding multiple copies of the M2 ectodomain peptide (METR) of the invention, and a second MVA comprises a polynucleotide encoding other antigens such as other influenza proteins or fragments thereof. In one embodiment, the additional antigen is HA, NP consensus sequence, variants, derivatives, analogs, or fragments thereof. In another embodiment, the other antigen in the second MVA is a polypeptide comprising multiple copies of the M2e ectodomain, which ectodomain is different from the METR sequence expressed in the first MVA.

In certain embodiments, the multivalent vaccine compositions of the invention comprise the polynucleotide, polypeptide or vector, such as MVA, wherein the polynucleotide, polypeptide or vector, such as MVA, induces an immune response against influenza virus and a polypeptide that elicits an immune response against one or more other organisms and/or viruses, such as haemophilus influenzae type b (haemophilus flujunction), hepatitis b virus, hepatitis a virus, hepatitis c virus, corynebacterium diphtheriae (corynebacterium diphteriae), Clostridium tetani (Clostridium tetani), poliovirus (Polio virus), measles virus (Rubeola virus), Rubella virus (Rubella virus), myxovirus (myxovirus), Neisseria (Neisseria) such as gonococcus (n.gonorrhoean), haemophilus duchenii (haemophilus), bacillus capsulatus (bacillus capsulatus), or Granuloma (Granuloma), when administered in an effective amount to a patient in need thereof, Human papillomaviruses of type I and type II (HPV)), Ureaplasma urealyticum (Ureaplasma urealyticum), mycoplasma hominis (Mycoplasmahominis), Treponema pallidum (Treponema pallidum), poxviruses of the genus molluscum, Human Immunodeficiency Virus (HIV), Epstein Barr Virus (EBV), herpes simplex virus or varicella-zoster virus.

The multivalent vaccines of the invention may comprise a polynucleotide, polypeptide or vector such as MVA and a compatible vaccine, wherein both the vaccine of the invention and the compatible vaccine are targeted to a similar patient population such as an immunocompromised population, a child, an infant or an elderly human.

Methods and regimens for treatment/prevention

Also provided is a method of treating or preventing an influenza virus infection or a condition associated with an influenza virus infection in a subject, the method comprising: administering to a subject in need thereof a composition comprising a polynucleotide, polypeptide or vector of the invention, such as MVA. In certain embodiments, the subject is a vertebrate such as a mammal, e.g., a primate such as a human. In some embodiments, the present invention relates to a method of inducing an immune response against influenza virus in a subject, such as a host animal, comprising administering an effective amount of a composition comprising any one or more of the polynucleotides, polypeptides or vectors, such as MVA, of the present invention.

In some embodiments, an animal can be prophylactically treated with the polynucleotide, polypeptide, or vector, such as MVA, or composition, such as a prophylactic vaccine to establish or enhance its immunity against one or more influenza virus species prior to exposure of a healthy animal to influenza virus or infection with influenza virus symptoms, thereby preventing disease or reducing the severity of disease symptoms. One or more polynucleotides, polypeptides, vectors such as MVA or compositions of the invention may also be used to treat an animal that has been exposed to or has suffered from symptoms associated with influenza virus to further stimulate the animal's immune system to reduce or eliminate symptoms associated with the exposure. As defined herein, "treating an animal" refers to preventing, curing, delaying or reducing the severity of and/or not aggravating symptoms in an animal caused by infection with an influenza virus, such as flu, with one or more polynucleotides, polypeptides or vectors, such as MVA, or compositions comprising said polynucleotides, polypeptides or vectors, such as MVA. It is not necessary that any of the polynucleotides, polypeptides, vectors such as MVA or compositions of the present invention provide complete protection against influenza virus infection or complete cure or elimination of all symptoms associated with influenza virus infection. As used herein, "an animal in need of treatment and/or prevention of immunity" refers to an animal in need of treatment, i.e., prevention, cure, delay or lessening of severity of symptoms associated with influenza virus infection and/or that the symptoms do not worsen within a specified period of time.

Treatment with a pharmaceutical composition comprising said polynucleotide, polypeptide or vector, such as MVA, may be carried out separately or, where appropriate, in combination with other treatments.

In therapeutic applications, the polynucleotides, polypeptides, vectors such as MVA or compositions of the invention are administered to a patient in an amount sufficient to elicit an effective CTL response against an influenza virus-derived polypeptide, thereby curing or at least partially preventing symptoms and/or complications. Amounts suitable for accomplishing this are defined as "therapeutically effective doses" or "unit doses". An effective amount for such use will depend, for example, on the polynucleotide, polypeptide, vector such as MVA or composition of the invention, the mode of administration, the stage and severity of the disease being treated, the weight and general health of the patient, and the judgment of the prescribing physician. Typically, MVA vaccine priming ranges from (for therapeutic or prophylactic administration) about 100pfu to about 1 × 10¹⁵MVA of pfu, in some embodiments about 10⁵pfu-about 10⁹pfu MVA, then booster dose of about 10³pfu-about 10⁸pfu, in some embodiments 10⁶pfu-about 10⁹pfu MVA, following a booster regimen, lasting from weeks to months, depending on the patient response and condition obtained by measuring specific CTL activity in the patient's blood. In other embodiments, the dose range for human priming is generally from about 10pfu to about 1x10 (for therapeutic or prophylactic administration) for a 70kg patient²⁰MVA of pfu, in some embodiments 1000pfu, 5x10⁴pfu、10⁵pfu、5x10⁵pfu、10⁶pfu、5x10⁶pfu、10⁷pfu、5x10⁷pfu、10⁸pfu、5x10⁸pfu、10⁹pfu or 10¹⁰pfu, then boosted at the same dose range for weeks to months according to a boosting regimen, depending on the patient's response and condition by measuring specific CTL (cytotoxic T lymphocyte) activity in the patient's blood. In a specific non-limiting embodiment of the invention, the host is administered from about 10pfu to about 1x10¹⁵pfu of the MVA of the invention, or a fragment, derivative, variant or analogue thereof, or in some embodiments 10⁴pfu-about 1X10¹⁰pfu or 10⁷pfu-10⁹pfu。

In a non-limiting embodiment of the invention, the effective amount of the composition of the invention increases the antibody titer to at least 2 or 3 times the antibody titer prior to administration.

It is noted that the polynucleotides, polypeptides, vectors such as MVA and compositions of the present invention are generally useful in severe disease states, i.e., life-threatening or potentially life-threatening situations. In this case, the treating physician may and will consider appropriate administration of large excesses of these polypeptide compositions, given the minimization of foreign matter and the relative non-toxicity of the polypeptide.

For therapeutic applications, administration is initiated at the first sign of an influenza virus infection. This is followed by a booster dose until at least the symptoms are substantially eliminated and continued for a period of time thereafter. In chronic infections, booster doses may be required after a loading dose.

Treatment of infected individuals with the compositions of the present invention can accelerate resolution of infection in acutely infected individuals. For those individuals who are prone to develop (or have a predisposition for) a chronic infection, the composition is particularly useful in a method of preventing acute development of a chronic infection. As described herein, when a susceptible individual is identified prior to or during infection, the composition can be targeted to minimize the need for administration to a larger population.

More specifically, the compositions of the present invention can be administered to any tissue of an animal, including, but not limited to, muscle, skin, brain tissue, lung tissue, liver tissue, spleen tissue, bone marrow tissue, thymus tissue, heart tissue such as myocardium, endocardium, and pericardium, lymphatic tissue, blood tissue, bone tissue, pancreatic tissue, kidney tissue, gall bladder tissue, stomach tissue, intestinal tissue, testicular tissue, ovarian tissue, uterine tissue, vaginal tissue, rectal tissue, nervous system tissue, ocular tissue, glandular tissue, tongue tissue, or connective tissue such as cartilage.

In addition, the compositions of the present invention can be administered to any internal cavity of a vertebrate, including, but not limited to, the lungs, mouth, nasal cavity, stomach, abdominal cavity, intestine, any ventricle, vein, artery, capillary, lymphatic cavity, uterine cavity, vaginal cavity, rectal cavity, joint cavity, cerebral cavity, spinal canal, eye cavity, salivary duct cavity, or liver. When the compositions of the present invention are administered to the salivary gland duct lumen or liver, the desired polypeptide is encoded in each salivary gland and liver such that the polypeptide is delivered from each salivary gland and liver into the blood stream of the spinal animal. Certain methods of using salivary glands, liver, and pancreas to administer to secretory organs of the gastrointestinal system to release desired polypeptides into the blood stream are disclosed in U.S. patent nos. 5,837,693 and 6,004,944, both of which are incorporated herein by reference in their entirety.

In certain embodiments, one or more compositions of the invention are delivered to an animal by the methods described herein to achieve an effective immune response and/or to achieve an effective therapeutic or prophylactic immune response. Any mode of administration may be used so long as the mode results in delivery or expression of the desired polypeptide in a sufficient amount to produce an immune response to influenza virus in an animal in need of such response and/or to produce a prophylactic or therapeutic immune response to influenza virus. In accordance with the disclosed methods, the compositions of the present invention can be administered by: mucosal delivery, transdermal delivery, subcutaneous injection, intravenous injection, oral administration, pulmonary administration, intramuscular (i.m.) administration, or by epidural injection. Other suitable routes of administration include, but are not limited to, intratracheal, transdermal, intraocular, intranasal, inhalation, intracavity, intratracheal (e.g., in the pancreas), and intraparenchymal (i.e., in any tissue). Transdermal delivery includes, but is not limited to, intradermal (e.g., dermal or intradermal), transdermal (e.g., transdermal), and transmucosal delivery (i.e., into or through skin or mucosal tissue). Intraluminal administration includes, but is not limited to, oral, vaginal, rectal, nasal, peritoneal, or intestinal lumens as well as intrathecal (i.e., in the spinal canal), intraventricular (i.e., in the brain or ventricle), intracameral (i.e., in the atrium), and subarachnoid (i.e., in the subarachnoid space of the brain).

Any mode of administration may be used so long as the mode results in delivery or expression of the desired polypeptide in a sufficient amount to produce an immune response to influenza virus in an animal in need of such response and/or to produce a prophylactic or therapeutic immune response to influenza virus. The methods of administration of the invention include needle injection, catheter infusion, biolistic syringes, particle accelerators (e.g. "gene gun" or air-filled "needleless" syringes) Med-E-Jet (Vahlsing, H., et al, J.Immunol. methods 171,11-22(1994)), Pigjet (Schrijver, R., et al, Vaccine 15, 1908. quadrature. 1916(1997)), Biojector (Bio-injector) (Davis, H., et al, Vaccine 12, 1503. quadrature. 1509(1994); Gramzinski, R., et al, mol.Med.4,109-118(1998)), Advantajet (Linmayer, I., et al, diabets Care 9: Pha 297(1986)), available Mars-Jet (Mars, J.AndRoedl, E.J.Oct. Oc. 821. J., 21. J.821), topical surgical skin depot (Biocide, et al, Biocide, Card. J.9: 297, 1986)), topical depot (gelatin) or other therapeutic agents such as topical depot for example, topical depot, transdermal delivery, or topical depot, or topical delivery of drugs (gelatin) for example, such as a depot, for example, for use of a pharmaceutical preparations, y., et al, Life sciences 65,2193-2203(1999)) or topical application. Some methods of administration are intramuscular needle-based injections and pulmonary application by catheter infusion. Each reference cited in this paragraph is incorporated herein by reference in its entirety.

Following immunization with a polynucleotide, polypeptide, vector such as MVA or composition of the invention, the immune system of the host responds to the vaccine by generating large amounts of HTL (helper T lymphocytes) and/or CTL (cytotoxic T lymphocytes) specific for the desired antigen. Thus, the host becomes at least partially immune to late stage infection or at least partially resistant to developing chronic infection.

In some embodiments, a polynucleotide, polypeptide, vector, such as MVA, or composition of the invention stimulates a cell-mediated immune response sufficient to protect an animal against influenza virus infection. In other embodiments, the polynucleotides, polypeptides, vectors, such as MVA, or compositions of the invention induce a humoral immune response. In certain embodiments, the polynucleotides, polypeptides, vectors, such as MVA, or compositions of the invention stimulate humoral and cell-mediated responses, the combination of which is sufficient to protect an animal against influenza virus infection.

In other embodiments, the component that induces a T cell response is combined with a component that induces an antibody response against a target antigen of interest. Thus, in certain embodiments of the invention, the vaccine compositions of the invention are combined with polypeptides or polynucleotides that induce or assist in neutralizing an antibody response against an antigen of interest. One embodiment of the composition comprises a class I epitope of the invention, and(Epimmunee, san Diego, Calif.) molecules (e.g., as described in U.S. Pat. No. 5,736,142, which is incorporated herein by reference in its entirety.)

The polynucleotides, polypeptides, vectors such as MVA or compositions comprising the same can be incorporated into animal cells in vivo and produce antigenic amounts of influenza M2-derived polypeptides, fragments, variants or derivatives thereof in vivo. After administration of the compositions according to the methods, the METR polypeptide is expressed in the animal in an amount sufficient to elicit an immune response. The immune response can be used, for example, to generate antibodies to influenza virus for use in diagnostic tests or as a laboratory reagent.

The invention also provides methods of generating, enhancing or modulating a prophylactic and/or therapeutic immune response against an influenza virus in an animal comprising administering to an animal in need of therapeutic and/or prophylactic immunization one or more compositions described herein. In some embodiments, the compositions comprise recombinant MVAs comprising a polynucleotide comprising a codon-optimized coding region encoding a polypeptide of the present invention, which is optimized for expression in a given host organism, such as a human, or a fragment comprising the coding region, encoding a fragment, variant or derivative thereof. The recombinant MVA is internalized into animal cells in vivo and produces an immunologically effective amount of influenza polypeptide or fragment or variant in vivo. Following administration of the composition according to the method, the influenza virus-derived polypeptide is expressed in the animal in a therapeutically or prophylactically effective amount.

The compositions of the present invention can be administered to an animal at any time during the life cycle of the animal to which they are administered. For example, the composition may be administered shortly after birth. In humans, the compositions of the invention may be administered at the time of administration of the other vaccine, e.g., at birth, 2 months, 4 months, 6 months, 9 months, 1 year, 5 years, or adolescence. In some embodiments, administration of a composition of the invention can be performed prior to the start of immunosuppressive therapy.

In addition, the compositions of the invention may be used in any desired immunization or administration regimen, such as a single administration or as part of a scheduled vaccination, such as a yearly vaccination, or in a prime-boost regimen, wherein a polypeptide or polynucleotide of the invention is administered before or after administration of the same or a different polypeptide or polynucleotide.

Recent studies have shown that prime-boost regimens are generally suitable methods of administering vaccines. In a prime-boost regimen, one or more compositions of the invention may be used in a "prime boost" regimen. Examples of "prime-boost" schemes can be found in Yang, Z, et al J.Virol.77: 799-. In a non-limiting example, one or more vaccine compositions comprising a polynucleotide, polypeptide or vector of the invention, such as MVA, are delivered to an animal, thereby eliciting an immune response in the animal to an influenza virus M2 polypeptide, and then a second immunogenic composition is used as a booster vaccine.

In another non-limiting example, the priming composition and the boosting composition are combined in a single composition or a single formulation. For example, a single composition may comprise an isolated polynucleotide or vector such as a polynucleotide comprising an encoding influenza virus protein, fragment, variant, derivative or analogue thereof or an isolated polypeptide comprising an influenza virus protein, fragment, variant, derivative or analogue thereof as a priming component and a polynucleotide, polypeptide or vector of the invention such as MVA as a boosting component. In this embodiment, the composition may be contained in a single vial with the priming component and the boosting component mixed together. Typically, the polynucleotide fraction provides an enhancement to the isolated polypeptide fraction, since peaks in the expression level of the polypeptide from the polynucleotide do not occur later in the administration (e.g., 7-10 days). A composition comprising a priming component and a boosting component is referred to herein as a "combination vaccine composition" or a "single-formulation heterologous prime-boost vaccine composition". Furthermore, the priming composition may be administered prior to the boosting composition, or even after the boosting composition if it is expected that the boosting composition will take a longer time to act.

In another embodiment, the priming composition may be administered simultaneously with the boosting composition, but in a separate formulation, wherein the priming component and the boosting component are separated.

Medicine box

The polynucleotides, polypeptides or vectors of the invention, such as MVA or compositions, can be provided in kit form together with methods of administering recombinant MVA or compositions of the invention. In some embodiments, the kit can further include instructions for vaccine administration.

Typically, the kit will include in a container, such as a unit dosage form, a desired composition of the invention and instructions for administration. Methods of administering the compositions of the present invention may include, for example, sterile syringes, nebulizers (e.g., inhalation or any other nasal or pulmonary method of administration), gels, creams, transdermal patches, transmucosal patches (or any other buccal or sublingual method of administration), or oral tablets. In some embodiments, a kit of the invention comprises two or more methods of administering a polynucleotide, polypeptide, vector or composition of the invention, such as two or more syringes.

In some embodiments, the kit further comprises more than one container containing a polynucleotide, polypeptide, or composition of the invention. For example, in some embodiments, the kit can include a container containing a priming component of the invention and a separate container containing a boosting component of the invention.

The container is optionally accompanied by notes or printed instructions. For example, the printed instructions may be in the form of a government agency regulating the manufacture, use or sale of a pharmaceutical or biological product, the instructions reflecting approval by the manufacturing, use or sale authority for administration to a human. The "printed instruction book" may be, for example, one of a book, a booklet, a brochure, or a leaflet.

The kit can further comprise a storage unit for storing the kit components (e.g., methods of administration, containers containing the recombinant MVA or compositions of the invention, printed instructions, etc.). The storage unit may be, for example, a bag, a box, an envelope or any other container suitable for use in the present invention. For example, the storage unit is large enough to accommodate the components required to administer the methods of the invention.

The invention can also include a method of delivering a recombinant MVA or composition of the invention to an animal, such as a human, in need thereof, said method comprising (a) registering on a computer readable medium the identity of a human administrator (e.g., physician's assistant, medical nurse, pharmacist, veterinarian) allowed to administer a polynucleotide, polypeptide, vector or composition of the invention, (b) providing the human with counseling information regarding the concomitant risk of a polynucleotide, polypeptide, vector or composition of the invention, (c) obtaining informed consent of the human to receive a polynucleotide, polypeptide, vector or composition of the invention despite the concomitant risk; and (e) allowing it to obtain a polynucleotide, polypeptide, vector or composition of the invention.

Examples

Example 1 recombinant vector construction

Influenza A virus gene Pr8HA sequence (SEQ ID NO:51-52), NP consensus sequence (SEQ ID NO:19-20), Pr8M2(SEQ ID NO:13-14), Pr8M2e _ TML (SEQ ID NO:53-54), METR _ S (SEQ ID NO:17-18 and 56) and METR _ C (SEQ ID NO:15-16 and 55) were cloned into the vEM11 recombinant vector (FIG. 1). The resulting recombinant vectors vEM47 (encoding NP consensus), vEM58 (encoding the METR _ S peptide), vEM57 (encoding the METR _ C peptide), vEM61 (encoding Pr8M2), vEM62 (encoding Pr8M2e-TML) and vEM65 (encoding Pr8HA) are shown in fig. 2A-F.

Example 2 homologous recombination and recombinant virus isolation

For the modified MVA viral vector MVAtor^TM(EBS company (Emergent Biosolutions)) was inserted with an influenza virus gene, CEF cells were infected with MVAtor and then transfected with the recombinant vectors shown in FIGS. 2A-F. 2-3 sets of parallel arrangement were carried out in each vector. First, each well of a 6-well plate was seeded with 5X10⁵CEF cells in combination at 37 ℃ and 5% CO₂Incubate for 24 hours. The next day, cell density was assumed to be 10 per well⁶A cell. MVAtor standards were diluted in Opti-Pro SFM containing 4mM L glutamine and 2.5. mu.g gentamicin per ml, so that 500. mu.l contained 5X10⁴TCID₅₀I.e. working concentration of 1X10⁵TCID₅₀Ml and gave a moi of 0.05. For infection, growth medium was removed from the cells, 500 μ l of diluted MVAtor standard was added per well and incubated for one hour at room temperature with shaking. The virus inoculum was removed and the cells were washed with Opti-Pro SFM. The transfected cells were left in 2.0ml Opti-Pro SFM at the time of establishment of the transfection reaction.

The transfection reaction was set up in sterile 5ml PS tubes. Approximately 1.0-2.0. mu.g of recombinant vector of DNA and 6. mu.l of transfection reagent (FuGene HD) were used for transfection, according to the FuGENE HD standard protocol supplied by the supplier. The cells were incubated at 37 ℃ and 5% CO in an incubator₂Incubate for 48 hours.

After incubation, the cells were screened on fluorescent cells indicating the presence of MVAtor and recombinant vector within the cells. Cells were screened on foci and used for 2 or 3 passages of recombinant MVAtor under selection conditions with the setting for most efficient gfp expression. For this passage, infected transfected cells were scraped into the medium by using a cell spatula and transferred into 1.5ml vials. Virus was released by sonication according to EPDD-SOP-EQU-033. 1/10-1/2 transfection set was placed on fresh CEF cells seeded in 12-well plates, filled to 1ml and 5. mu.g blasticidin added per ml.

For plaque purification of recombinant MVAtors, recombinant MVAtors were inoculated in serial dilutions in 6-well and 96-well plates, respectively, under selective conditions (5. mu.g/ml blasticidin per ml Opti-ProSFM). Single fluorescent plaques were separated with a 100 μ l pipette. The isolated virus was transferred into 1.5ml vials and released by sonication. The virus was analyzed by PCR on empty vector (as described below) and the isolate showing the weakest empty vector signal was passaged on fresh CEF cells seeded in 12-well plates. Plaque purification was repeated until the isolate was free of empty vector. Once pure clones were isolated, the virus was passaged without blasticidin and non-fluorescent virus without selection/reporter cassette was isolated.

To delete the selection cassette, the pure recombinant virus was passaged without blasticidin. Plaques with no fluorescence were isolated and tested on the expected insertion of influenza a virus genes in the genome. The recombinant MVAtor was then amplified to 3xT175 and subjected to detailed preliminary tests (PCR, sequencing, expression, titer) in the following virus stock: (1) MVAtor-NP consensus sequence (mEM10), P21pp8, (2) MVAtor-METR _ C (mEM18), P14pp8, (3) MVAtor-METR _ S (mEM19), P18pp13, (4) MVAtor-Pr8M2(mEM22), P13pp3, (5) MVAtor-Pr8M2e _ TML (mEM23), P14pp3, and (6) MVAtor-Pr8HA (mEM 17). The virus bulk was shown to have 100% correct sequence read and no residual empty vector and function.

After isolation of pure recombinant MVAtor without selection/reporter cassette, the virus was amplified in T225 flasks. Primary CEF cells in 10T 225 flasks 1:4 were distributed into a total of 40T225 flasks for amplification of each individual recombinant MVAtor. After 2 days of incubation, cells in one flask were trypsinized and counted. 39T 225 flasks were infected with 0.1 moi at 37 ℃ and 5% CO₂Incubate for 72 hours. The flasks were harvested and the contents of all flasks were collected. By ultrasonic treatmentHomogenization of the collected virus stock is performed using flow cell equipment. The flask containing the virus stock that must be purified is placed on ice and connected to a flow cell. The ultrasonic flow cell device is arranged as follows: amplitude 100%, cycle 1, pump speed 50 ml/min, i.e. 0.8 and on. The stock solution was frozen after sonication until purification.

The purified stock solution was diluted and filled to a final fill volume of 600. mu.l per bottle containing 109TCID per ml, taking into account the titer₅₀(nominal titer). The filling resulted in vial contents of (1)91xMVAtor-NP consensus sequence (mEM10), (2)35xMVAtor-METR _ C (mEM18), (3)29xMVAtor-METR _ S (mEM19), (4)56xMVAtor-Pr8M2(mEM22), (5)38xMVAtor-Pr8M2e _ TML (mEM23), and (6)30xMVAtor-Pr8HA (mEM17) for each virus. All vials were stored at-70 ℃ until further use, i.e., testing or shipping. One vial of each virus was stored in a-70 ℃ sample archive.

Example 3 PCR for exclusion of empty vector contamination

To confirm that residual non-recombinant MVAtor was completely excluded from the filled vial of purified virus stock, DNA from purified and filled MVAtor-NP for mEM10, MVAtor-METR _ C for mEM18, MVAtor-METR _ S for mEM19, MVAtor-Pr8M2 for mEM22, or MVAtor-Pr8M2e _ TML for mEM23 was isolated and used for PCR. The recombinant vector vEM47 was used as a positive control for the recombinant virus (vEM 47). DNA isolated from MVAtor was used as a positive control (MVA) for the empty vector. Using H₂O and CEF cells were used as negative controls.

Example 4 sequencing of influenza Virus genes and flanking sequences of insertion sites

The DNA of each virus was isolated and the entire insertion site was amplified by PCR using primers 5'- -ggagctccactatttagttggtggtcgcc-3' (SEQ ID NO:32) (oVIV47) and 5'-cgggtaccctagtttccggtgaatgtg-3' (SEQ ID NO:33) (oVIV 89). Using these primers, the inserted influenza virus genes as well as flanking sequences for homologous recombination were amplified (fig. 4).

The PCR fragment was purified and sent to GATC for sequencing. For sequencing, the primers were selected to cover the entire PCR fragment as follows:

TABLE 5 primers

Example 5 expression analysis of influenza Polypeptides

Westerm blots were performed to analyze the expression of MVAtor-NP as well as the two-METR constructs (SEQ ID NO:16 for the METR _ C polypeptide and SEQ ID NO:18 for the METR _ S polypeptide). In 6-well plates, 6 × 10 was seeded per well in the appropriate number of wells⁵A cell. The number of wells was determined as follows: number of recombinant MVAtor samples + MVAtor control + CEF control. Cells were infected with MVAtor and MVAtor-NP/-METR _ S, -METR _ C, respectively, using moi of 1 according to standard protocols. After 24 hours of infection, 300 μ l RIPA buffer (pre-chilled on ice) was added per well and the plates were incubated on ice for 5 minutes. Cells were scraped into RIPA buffer and cell suspensions were transferred each into 1.5ml vials and placed on ice. Each vial was charged with a 0.5. mu.l amount of protease inhibitor cocktail. A volume of 60. mu.l per sample was transferred to a new 1.5ml vial and 22. mu.l of loading dye and 8. mu.l of 2-mercaptoethanol were added. Mu.l of inactivated influenza virus was used as positive control for influenza virus. A volume of 8. mu.l RIPA buffer was added. All samples were incubated on ice for 5 minutes. All samples were then heated in a hot mixer at 95 ℃ for 10 minutes.

A blood cell adsorption assay (HAD) was performed to analyze the expression of MVAtor-Pr8 HA. To this end, CEF cells were infected with MVAtor and MVAtor-HA (mEM17), respectively. Another set of cells was mock infected. 24 hours post infection, the cells were incubated with a 1% human erythrocyte dilution (FIG. 6).

An immunoassay was performed to analyze the expression of MVAtor-M2 and MVAtor-M2e-TML (FIG. 7). Cells were infected with MVAtor-Pr8M2(FIG.7A) and MVAtor-Pr8M2e-TML (FIG. 7B). In parallel, cells were infected with MVAtor or incubated without infection.

Example 6 in vivo efficacy of MVA vaccine-body weight and viral load

MVAtors expressing METR-C (SEQ ID NO:16) or METR-S (SEQ ID NO:18) were tested for their in vivo efficacy. The efficacy of the MVA vaccine was observed by the following evaluations: 1) weight loss or mortality of mice in the challenge group compared to the control group, and 2) viral load in the lungs. Groups of mice (8 mice per group) were immunized intramuscularly twice with MVA vaccine: (1) MVA-Pr8M2 (MVA construct expressing full length M2 of influenza A virus Puerto Rico 1934H1N 1(Pr 8)), (2) MVA-Pr8M2e-TML (MVA construct expressing the native transmembrane region of M2 (TML)), (3) MVA-METR-C (MVA construct expressing METR polypeptides with cysteine), (4) MVA-METR-S (MVA construct expressing METR polypeptides with serine substituted cysteine), (5) MVA-ConsNP (MVA construct expressing NP consensus sequence), (6) MVAtor (MVA vector alone), and (7) PBS as a negative control.

3 weeks after immunization, mice were intrapulmonary infected with 50mcL of influenza A virus (A/PR/8/34, H1N1) at 629TCID50 per mouse. Mice were monitored daily for weight loss. Data are mean ± SEM (percent) compared to body weight before challenge. Mice were sacrificed if BW decreased to 25%. As a result, the negative control mice lost weight and died. The weight loss in mice receiving the MVA-M2eTML construct (14%) was measurable, but only 7% of the weight loss in the METR construct was similar to full-length M2(Pr8M 2).

Influenza virus was quantitatively recovered from infected mouse lungs and virus loads were compared to determine the immune benefit of prior vaccinations.

Groups 4 cryotubes containing individual mouse lungs were removed from-80 ℃ and thawed on ice. Each lung was weighed. L-15-2X PSK medium (Leibovitz +4mM L-glutamine +2X antibiotic-antimycotic) was transferred into each tube so that the lung weight was equal to 10% of the total volume. Each lung was homogenized with Power Gen 125 and a disposable homogenizer until complete (30 seconds-1 minute). The lung homogenate was centrifuged at 30000rpm at 4 ℃ for 20 minutes. Aliquoting the lung homogenate supernatant; the 200mcL and two 400mcL aliquots for TCID50 were frozen directly in dry ice. The aliquots were stored at-80 ℃.

Vortex lung homogenates, add 20mcL to 4 wells containing MDCK cells seeded 18-24 hours ago at 4X 10e5 cells in 180mcL serum-free MEM containing Trypsin (TPCK) and 4mM glutamine and 2X antibiotic-antimycotic. Serial dilutions 10-fold were performed down the plate by transferring 20mcL from column a into 180mcL medium in column B and continuing to column H. Stock influenza A/PR/8/34 ATCC VR-9 was used to infect MDCK cells in 4 wells on each of three test platesAs a positive control. The source of the positive control A/PR/8/34 was three vial sources in a temperature-failure-80 ℃ freezer. Infected MDCK cells at 37 ℃ in 5% CO₂Incubate for 4 days. The plates were washed once with PBS and MDCK cells and 0.1% turkey red blood cells were incubated at 4 ℃ for 30 minutes. Unbound turkey red blood cells were washed vigorously four times with PBS and infected MDCK cells were visualized by adherent red blood cells. Log 10TCID50 titers were calculated from infected wells counting MDCK cells. The results are expressed as TCID 50/gram of lung tissue weight by calculating the inverse Log 10TCID50 divided by the gram of lung tissue weight for each mouse. The mean, standard deviation and coefficient of variation were determined for each group and expressed as%.

Example 7 antibody response against M2e in immunized mice

To determine the antibody titer of the immunized animals, mice were immunized intramuscularly 1, 2 or three times with the following MVA vaccines: (1) MVA-Pr8M2, (2) MVA-Pr8M2e-TML, (3) MVA-METR-C, (4) MVA-METR-S, (5) MVA-Pr8M2_ MVA-ConsNP, (6) MVA-Pr8M2e-TML + MVA-ConsNP, (7) MVA-Pr8M2e-TML + MVA-ConsNP, (8) MVA-ConsNP, (9) control group, (10) with inserted MVA virus (not expressing influenza virus antigen), (11) infection with PR8 infectious influenza virus sublethal (no MVA vaccine), and (12) PBS as positive control. After final immunization, the mice were challenged with infectious influenza virus (A/Pr/8/34). For M2ELISA, biotinylated peptides consisting of the M2e region were immobilized on ELISA plates by avidin format. Of the 6 peptides presented within the M2 Ectodomain Tandem Repeat (METR), representative anti-M2 e titers were obtained by ELISA plate coating using peptide 4, representing influenza a Puerto Rico 1934H1N 1. The serum titers identified by peptide #4(SEQ ID NO:4) are summarized in Table 6.

TABLE 6 anti-M2 e peptide #4 antibody response (μ g/mL, geometric mean. + -. geometric SD)

As depicted in fig. 10, MVA-METR vaccines expressing multiple M2e regions produced higher IgG anti-M2 peptide serum levels than MVA-M2 vaccine titers.

IN another study, BALB/c mice were immunized twice with MVA vaccine from intranasally to the lung (IN) or Intramucosally (IM): (1) sublethal doses of influenza a virus Puerto Rico 1934H1N1 (positive control), (2) MVA-HA (MVA construct expressing full-length HA), (3) MVA-METR-S (MVA construct expressing a serine substituted METR), (4) MVA-M2 (MVA construct expressing full-length M2), (5) MVA-METR-C (MVA construct expressing a METR with a native cysteine), (6) MVA-Pr8M2e-TML (MVA construct containing the native transmembrane domain of M2(TML), (7) PBS (negative control), (8) MVA-ConsNP (MVA construct expressing the NP consensus sequence), and (9) MVAtor alone (negative control). Immune sera were obtained 21 days after the second vaccine immunization and tested by ELISA, coated with one of four peptides, each representing a different strain of influenza a virus protein: m2e # 1H 51999-2008 (second column from the back of the figure), M2e # 4H1 and H3 (column from the back of the figure), M2e # 5H 9 and H6 (second column from the front of the figure), M2e # 6H 7 and H3, H8, H10, H2, H6, H9 (column from the front of the figure).

Table 7: anti-M2 e peptide #4 antibody response (μ g/mL, geometric mean. + -. geometric SD)

Changes IN body weight of Intranasal (IN) or Intramuscular (IM) immunized mice are shown IN FIGS. 11A and 11B.

Viral load was measured IN Intranasal (IN) or Intramuscular (IM) immunized mice. Viral load in lung tissue of mice immunized twice with MVA vaccine was measured 3 days after influenza a virus challenge. The data are shown in FIG. 12.

Example 8 immune response to Low dose H1N1PR8 challenge in NP and M2 immunized mice

The efficacy of NP + M2 vaccination against homologous low dose influenza virus infection (H1N 1Pr 8) was tested in mice. Mice in this study were immunized intramuscularly on days 0 and 21 with a MVAtor-based vaccine containing a conserved influenza virus antigen, as summarized in table 8.

Table 8: treatment group

Mice were challenged with intranasally administered H1N1PR8 (< 2LD 50) on day 42. Body weight, survival and viral load were analyzed (day 2 and day 4).

As shown in figure 13, HA, NP, M2+ NP and the non-lethal immune group were protected from weight loss. In the M2 immunized group, 6 out of 10 mice lost <20% of body weight on day 8. In the MVAtor immunization group, 10 of 10 mice lost <20% of body weight on day 8. All treatment and control groups had 100% survival.

In another study, anti-NP immune responses were tested by ELISA using recombinant NP (ImGenex) coated plates. The ELISA results are shown in figure 14: 1d21MVA, 2d21 MVA, 1d21MVA + NP, 2d21 MVA + NP, 1d21 MVA-M2eA + MVA-NP, 2d21 MVA-M2eA + MVA-NP, 1d21 non-lethal H1N1PR8 and 2d21 non-lethal H1N1PR 8. "1d 21" refers to the immune response measured after a single vaccination and "2d 21" refers to the immune response measured after 2 doses of vaccine construct. These results show that an anti-NP immune response was observed after two immunizations.

Viral load in lung tissue of mice immunized with MVA, MVA-HA, MVA-NP, MVA-M2eA, MVA-M2e + NP or a/PR/8/34 Intramuscular (IM) was measured at day 2 and day 4 post H1N1PR8 virus challenge. The viral load results are shown in figure 15. Animals immunized with MVA-HA had 2-3 orders of magnitude lung load reduction on both day 2 and day 4. The results show that animals immunized with NP + M2 showed a 2-order reduction in pneumoviral load on day 2. However, any of the groups receiving NP or M2 antigens showed no significant decrease in pneumovirus replication at day 4.

Example 9 immune response to lethal dose of H1N1PR8 challenge in NP and M2 immunized mice

The efficacy of NP + M2 vaccination against a homolethal dose of influenza virus infection (H1N 1Pr 8) was tested in mice. Mice IN this study were immunized Intranasally (IN) or Intramuscularly (IM) with MVAtor-based vaccines containing conserved influenza virus antigens on days 0 and 21, as summarized IN table 9.

Table 9: treatment group

Mice were challenged with intranasally administered H1N1PR8 (3 LD 50) on day 42. Hemagglutinin inhibition (HAI) (pre-challenge), body weight, survival and viral load were analyzed (day 3).

Influenza a virus H1N1 Puerto Rico 8/1934, abbreviated as "PR 8", is a mouse-adapted influenza virus used in the HAI test of this study. Prior to HAI, PR8 was assayed for hemagglutinin potency using turkey red blood cells and then diluted to an appropriate concentration of 8 units hemagglutinin per ml (dilution factor 1024) for HAI. For HAI, 25mcL mouse serum was incubated with 75mcL receptor-disrupting enzyme (RDE) for 30 minutes at 37 ℃, then diluted to 250mcL in saline (1/10 serum dilution) and stored at 4 ℃ prior to use.

Sera were tested in 3 or 4 serum pools using the HAI method. Serum (25 mcL) was serially diluted 2-fold in 25mcL PBS, to which 25mcL of the diluted influenza virus was added and incubated at room temperature for 30 minutes. Turkey red blood cells (50 mcL, washed and diluted) were added and incubated for 60 minutes at room temperature before assessing hemagglutinin inhibition. The HAI results are shown in table 10.

Table 10: HAI results

Group of	Animals immunized with MVAtor vectors indicating antigens	Pre-attack (GMT) HAI
			1	Sublethal dose-H1N 1	119±528
2	MVA-HA	1092±740
			11	PBS	<20
13	MVAtor alone	<20

N =8 mice/group, <20= below the limit of detection

As shown in fig. 16A and 16B, NP + M2 immunization groups were compromised in vivo. All immunized groups were 100% free from death, while MVAtor and PBS control group survived 0% (0/10).

In another study, anti-NP and anti-M2 immune responses were tested by ELISA using plates coated with recombinant NP (Ehrgen Co.) and M2 peptide #4(SEQ ID NO:4), respectively. The anti-NP ELISA results for ConsNP, Pr8M2+ ConsNP, Pr8M2e-TML + ConsNP, METR-C + ConsNP, and METR-S + ConsNP are shown in FIG. 17. The anti-M2 ELISA results for M2, M2-TML, METR-C, METR-S, M2+ NP, M2-TML + NP, METR-C + NP, and METR-S + NP are shown in FIG. 18. These results show that anti-NP and anti-M2 immune responses were generated in vaccinated animals.

Viral load IN lung tissue of mice immunized with Intranasal (IN) or Intramuscular (IM) PBS (IN), MVAtor, NP, M2, M2e-TML, METR-C, METR-S, HA and sublethal PR8(IN) was measured 3 days after challenge with H1N1PR8 virus. The viral load results are shown in figure 19. These results show that animals immunized intranasally with NP, M2, M2e-TML, METR-C, and METR-S are partially protected from viral replication in the lungs. There was no significant decrease in viral replication in the lungs of the group receiving the intramuscular NP or M2 antigen.

Example 10 immune response to lethal dose of porcine H1N1 challenge in NP, M2, and M1 immunized mice

The efficacy of NP + M2+ M1 vaccination against a homolethal dose of influenza virus infection (sH 1N1 a/Mx/4108/09) was tested in mice. Mice in this study were immunized intramuscularly on days 0 and 21 with a MVAtor-based vaccine containing a conserved influenza virus antigen, as summarized in table 11.

Table 11: treatment group

Mice were challenged with intranasal administration of sH1N 1A/Mx/4108/09 (-20 LD 50) on day 42. Hemagglutinin inhibition (HAI) (pre-challenge), body weight, survival and viral load were analyzed (days 2 and 4).

The HAI test was performed as described in example 9 above. The HAI results are shown in table 12.

Table 12: HAI results

The mice were given a fluvalal dose: 150 μ L, IM (4.5 μ g HA for each of the 3 viruses); human dose: 500 μ L, IM (15 μ g HA for each of 3 viruses). All pre-exsanguination samples of group 1 (fluvalal) and group 8(PBS) were below detection limit (< 20). Group 8(PBS) was below the detection limit (< 20) for all 39-day samples of both H1N1 pandemic viruses (before challenge).

As shown in fig. 20A, the NP + M2 vaccine did protect against weight loss. However, no significant additional benefit was observed with the addition of M1 to NP + M2. Fig. 20B shows the survival results of vaccinated mice. The survival results of this study are shown in table 13 below.

Table 13: survival results

Immunization group	% survival
		Flulaval	100%(8/8)
M1+NP+METRC	100%(8/8)
		M1+NP+M2	100%(8/8)
M2+NP	100%(8/8)
		M1	38%(3/8)
M1+NP	88%(7/8)
		MVAtor	13%(1/8)
PBS	25%(2/8)

These results show that the lethality was significantly lower (p =0.0035) in the groups receiving M1+ NP + M2 and M1+ NP + METR-C than in the PBS group.

In another study, anti-NP and anti-M2 immune responses were tested by ELISA using recombinant NP (EPDU, Inc.) and M2 peptide #4(SEQ ID NO:4) coated plates, respectively. The anti-NP and anti-M2 ELISA results for PBS, MVAtor, M1+ NP + METRC, M1+ NP + M2, M2+ NP, M1, M1+ NP, and Fluvalal are shown in FIG. 21. These results show that vaccination induced strong anti-NP and anti-M2 immune responses.

anti-M1 and anti-MVA immune responses were tested by ELISA using recombinant M1 and MVA CT84 coated plates, respectively. The anti-M1 and anti-MVA ELISA results for PBS, MVAtor, M1+ NP + METRC, M1+ M2+ NP, M2+ NP, M1, M1+ NP, and Flulanval are shown in FIGS. 22A-B. These results show that vaccination did not induce an immune response against-M1.

Viral load of lung tissues of mice immunized with PBS, MVAtor, M1+ NP + METRC, M1+ NP + M2, M2+ NP, M1, M1+ NP, and Flulaval Intramuscular (IM) was tested on days 2 and 4 after challenge with sH1N 1A/Mx/4108/09 virus. The results of viral replication in the lungs are shown in figure 23. The results show that the tested vaccines did not induce viral replication in the lung.

Viral loads in nasal turbinates of mice immunized with PBS, MVAtor, M1+ NP + METRC, M1+ NP + M2, M2+ NP, M1, M1+ NP, and Flulaval Intramuscular (IM) were tested on days 2 and 4 after challenge with sH1N 1A/Mx/4108/09 virus. The results of viral replication in the lungs are shown in figure 24. The results show that the viral load of the turbinates was partially reduced in all vaccinated groups. Addition of M1 in the vaccine did not enhance the reduction of viral load in the turbinate.

Claims (98)

1. An isolated polynucleotide comprising a coding region that encodes a polypeptide, wherein said polypeptide comprises at least 5 influenza matrix 2 protein (M2) ectodomain peptides.

2. The polynucleotide of claim 1, wherein said polypeptide comprises any 5 or more of the following amino acid sequences in any order relative to each other:

i.SEQ ID NO:1(M2e#1_C);

ii SEQ ID NO:2(M2e#2_C);

iii.SEQ ID NO:3(M2e#3_C);

iv.SEQ ID NO:4(M2e#4_C);

v.SEQ ID NO:5(M2e#5_C);

vi.SEQ ID NO:6(M2e#6_C);

vii SEQ ID NO:7(M2e#1_S);

viii.SEQ ID NO:8(M2e#2_S);

ix.SEQ ID NO:9(M2e#3_S);

x.SEQ ID NO:10(M2e#4_S);

SEQ ID NO:11(M2e #5_ S), and

xii.SEQ ID NO:12(M2e#6_S)。

3. an isolated polynucleotide comprising a coding region that encodes a polypeptide comprising any 3 or more of the following M2 ectodomain peptides arranged in any order relative to each other:

i.SEQ ID NO:1(M2e#1_C);

ii.SEQ ID NO:2(M2e#2_C);

iii.SEQ ID NO:3(M2e#3_C);

iv.SEQ ID NO:4(M2e#4_C);

v.SEQ ID NO:5(M2e#5_C);

vi.SEQ ID NO:6(M2e#6_C);

vii SEQ ID NO:7(M2e#1_S);

viii.SEQ ID NO:8(M2e#2_S);

ix.SEQ ID NO:9(M2e#3_S);

x.SEQ ID NO:10(M2e#4_S);

SEQ ID NO:11(M2e #5_ S), and

xii SEQ ID NO:12(M2e#6_S)。

4. the polynucleotide of claim 3, wherein said polypeptide comprises any 3 or more of the following M2 ectodomain peptides, arranged in any order relative to each other:

i.SEQ ID NO:1(M2e#1_C);

ii SEQ ID NO:2(M2e#2_C);

iii.SEQ ID NO:3(M2e#3_C);

iv.SEQ ID NO:4(M2e#4_C);

SEQ ID NO:5(M2e #5_ C), and

vi.SEQ ID NO:6(M2e#6_C)。

5. the polynucleotide of claim 3, wherein said polypeptide comprises any 3 or more of the following M2 ectodomain peptides, arranged in any order relative to each other:

i.SEQ ID NO:1(M2e#1_S);

ii.SEQ ID NO:2(M2e#2_S);

iii.SEQ ID NO:3(M2e#3_S);

iv.SEQ ID NO:4(M2e#4_S);

SEQ ID NO:5(M2e #5_ S), and

vi.SEQ ID NO:6(M2e#6_S)。

6. the polynucleotide of any one of claims 3 to 5, wherein said polypeptide comprises any 4 or more of said M2 ectodomain peptides arranged in any order relative to one another.

7. The polynucleotide of any one of claims 3 to 6, wherein said polypeptide comprises any 5 or more of said M2 ectodomain peptides arranged in any order relative to one another.

8. The polynucleotide of any one of claims 1 to 7, wherein said polypeptide comprises any 6 or more of said M2 ectodomain peptides arranged in any order relative to one another.

9. The polynucleotide of any one of claims 1-5, 7 and 8, wherein said polypeptide comprises the following M2 ectodomain peptides arranged in any order relative to each other: SEQ ID NO:1(M2e #1_ C), SEQ ID NO:2M2e #2_ C), SEQ ID NO:3(M2e #3_ C), SEQ ID NO:4(M2e #4_ C), SEQ ID NO:5(M2e #5_ C), and SEQ ID NO:6(M2e #6_ C).

10. The polynucleotide of any one of claims 1-4, 5,7 and 8, wherein said polypeptide comprises the following M2 ectodomain peptides in any order relative to each other: SEQ ID NO 7(M2e #1_ S), SEQ ID NO 8(M2e #2_ S), SEQ ID NO 9(M2e #3_ S), SEQ ID NO 10(M2e #4_ S), SEQ ID NO 11(M2e #5_ S), and SEQ ID NO 12(M2e #6_ S).

11. The polynucleotide of any one of claims 1-10, wherein said polypeptide induces an immune response against influenza virus.

12. The polynucleotide of any one of claims 1 to 11, wherein said at least 2 matrix 2 protein (M2) extracellular domain peptides are fused together without an intervening linker peptide therebetween.

13. The polynucleotide of any one of claims 1 to 12, wherein a linker peptide is inserted between said at least 2M2 ectodomain peptides.

14. The polynucleotide of claim 13, wherein said linker peptide comprises at least 5 amino acids.

15. The polynucleotide of claim 13 or 14, wherein said linker peptide has homology to an amino acid sequence found in a lower organism.

16. The polynucleotide of any one of claims 13-15, wherein said linker peptide has no homology to an amino acid sequence found in a primate.

17. The polynucleotide of claim 16, wherein said one or more linker peptides comprise an amino acid sequence selected from the group consisting of SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, and any combination thereof.

18. The polynucleotide of any one of claims 1-17, wherein said polypeptide further comprises at least one T cell epitope.

19. The polynucleotide of claim 18, wherein said at least one T cell epitope comprises one or more of SEQ ID No. 26 and SEQ ID No. 27.

20. The polynucleotide of any one of claims 1 to 19, wherein said polypeptide comprises the amino acid sequence of seq id No. 16 [ METR _ C form-full length ].

21. The polynucleotide of any one of claims 1 to 19, wherein said polypeptide comprises the amino acid sequence of seq id No. 18 [ METR _ S form-full length ].

22. The polynucleotide of any one of claims 1-21, wherein said coding region is codon optimized for expression in humans.

23. The polynucleotide of any one of claims 1 to 22, wherein said coding region further comprises a promoter operably linked to said influenza virus matrix 2 protein (M2) ectodomain peptide coding region.

24. The polynucleotide of claim 23, wherein said promoter comprises the nucleotide sequence of SEQ ID No. 29.

25. The polynucleotide of any one of claims 1 to 24, wherein said coding region further comprises an additional polypeptide.

26. The polynucleotide of claim 25, wherein said other polypeptide is Hemagglutinin (HA), Neuraminidase (NA), Nucleoprotein (NP), matrix 1 protein (M1), matrix 2 protein (M2), non-structural protein (NS), RNA polymerase PA subunit (PA), RNA polymerase PB1 subunit (PB1), RNA polymerase PB2 subunit (PB2), or a combination of two or more of said influenza polypeptides or fragments thereof.

27. The polynucleotide of claim 26, wherein said other polypeptide is HA or a fragment thereof.

28. The polynucleotide of claim 27, wherein said HA comprises the amino acid sequence of SEQ ID No. 52.

29. The polynucleotide of claim 26, wherein said additional polypeptide is NP or a fragment thereof.

30. The polynucleotide of claim 29, wherein said NP comprises the amino acid sequence of SEQ ID No. 20.

31. The polynucleotide of claim 25, wherein the additional polypeptide is a His-tag, ubiquitin tag, NusA tag, chitin-binding domain, ompT, ompA, pelB, DsbA, DsbC, c-myc, KSI, polyaspartic acid, (AIa-Trp-Pro) n (SEQ ID NO:28), polyalanine, polycysteine, polyarginine, B-tag, HSB-tag, Green Fluorescent Protein (GFP), calmodulin-binding protein (CBP), galactose-binding protein, maltose-binding protein (MBP), cellulose-binding protein (CBD' S), dihydrofolate reductase (DHFR), glutathione S-transferase (GST), streptococcal protein G, staphylococcal protein A, T7genelO, avidin/streptavidin/iStrep-tag, trpE, chloroacetyltransferase, chloramphenicol, pesa, lacZ (D-galactosidase), His-repair thioredoxin, FLAGTM peptide, S-tag, T7-tag, or a combination of two or more of said polypeptides.

32. A vector comprising the polynucleotide of any one of claims 1-31.

33. The vector of claim 32, wherein the vector is a viral vector.

34. The vector of claim 33, wherein the viral vector is selected from the group consisting of: vaccinia virus vectors, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors.

35. The vector of claim 34, wherein said vaccinia virus vector comprises a modified vaccinia virus ankara (MVA).

36. The vector of claim 35, wherein said vector further expresses an additional polypeptide.

37. The vector of claim 36, wherein said additional polypeptide is Hemagglutinin (HA), Neuraminidase (NA), Nucleoprotein (NP), matrix 1 protein (M1), matrix 2 protein (M2), nonstructural protein (NS), RNA polymerase PA subunit (PA), RNA polymerase PB1 subunit (PB1), RNA polymerase PB2 subunit (PB2), or a combination of two or more of said influenza polypeptides or fragments thereof.

38. The vector of claim 37, wherein said additional polypeptide is HA or a fragment thereof.

39. The vector of claim 38, wherein said HA comprises the amino acid sequence of SEQ ID NO 52.

40. The vector of claim 37, wherein said additional polypeptide is NP or a fragment thereof.

41. The vector of claim 40, wherein said NP comprises the amino acid sequence of SEQ ID NO. 20.

42. The vector of any one of claims 35-41, wherein MVA is capable of replication in avian cells.

43. The vector of claim 42, wherein the avian cell is immortalized.

44. The vector of claim 42 or 43, wherein the avian cell is a duck cell.

45. The vector of claim 44, wherein said duck cell is a Duck retina cell line or an embryonic derived cell line.

46. The vector of claim 44 or 45, wherein the duck cell is AGE1cr, AGE. pIX or EB 66.

47. The vector of any one of claims 35 to 46, wherein the polynucleotide is inserted into a naturally occurring deletion site in the MVA genome.

48. The vector of claim 47, wherein said naturally occurring deletion site is deletion site 3.

49. The vector of any one of claims 35 to 48, wherein the polynucleotide is inserted into the Open Reading Frame (ORF) of the MVA genome.

50. The vector of any one of claims 32-49, wherein said vector induces an immune response against influenza virus when administered to a subject in need thereof.

51. The vector of claim 50, wherein the immune response is a humoral immune response, a cell-mediated immune response, or a combination of both humoral and cell-mediated immune responses.

52. The vector of any one of claims 32 to 51, wherein the vector is capable of preventing, ameliorating or treating a disease or disorder associated with influenza virus.

53. A host cell comprising the polynucleotide of any one of claims 1-31 or the vector of any one of claims 32-52.

54. The host cell of claim 53, further comprising an additional recombinant MVA comprising a polynucleotide encoding at least one influenza virus protein or fragment thereof.

55. The host cell of claim 54, wherein the host cell is an avian cell.

56. The host cell of any one of claims 56-55, wherein the host cell is immortalized.

57. The host cell of any one of claims 56-56, wherein the host cell is a duck cell.

58. The host cell of claim 57, wherein the duck cell is a duck-dwelling retinal cell or an embryonic-derived stem cell.

59. The host cell of claim 58, wherein the cell is AGE1cr, AGE1cr. pIX or EB 66.

60. A polypeptide encoded by the polynucleotide of any one of claims 1-31 or the vector of any one of claims 32-52 or the host cell of any one of claims 53-59.

61. A method of producing an influenza polypeptide, the method comprising culturing the host cell of any one of claims 53-59 and recovering the polypeptide.

62. A composition comprising the polynucleotide of any one of claims 1-31, the vector of any one of claims 32-52, the host cell of any one of claims 53-59, or the polypeptide of claim 60, and a pharmaceutically acceptable carrier.

63. The composition of claim 62, wherein the composition further comprises an adjuvant.

64. The composition of claim 63, wherein the adjuvant is selected from the group consisting of: alum, bentonite, latex and acrylic particles, pluronic block polymers, squalene, storage formers, surface active materials, lysolecithin, retinal, Quil a, liposomes, and pluronic polymer formulations, macrophage stimulators, alternative pathway supplement activators, nonionic surface bacterial components, aluminum-based salts, calcium-based salts, silica, polynucleotides, toxoids, serum proteins, viral and virus-derived materials, toxic agents, venoms, imidazoquinoline compounds, poloxamers, toll-like receptor (TLR) agonists, mLT, CpG, MPL, cationic lipids, Qs21, and combinations of two or more of these adjuvants.

65. The composition of any one of claims 62 to 64, further comprising an additional influenza vaccine.

66. The composition of claim 65, wherein the additional influenza vaccine comprises MVA encoding one or more of the following proteins, or fragments thereof, in any order relative to each other: hemagglutinin (HA), Neuraminidase (NA), Nucleoprotein (NP), matrix 1 protein (M1), matrix 2 protein (M2), non-structural protein (NS), RNA polymerase PA subunit (PA), RNA polymerase PB1 subunit (PB1), RNA polymerase PB2 subunit (PB 2).

67. The composition of claim 66, wherein the protein is HA or a fragment thereof.

68. The composition of claim 67, wherein said HA comprises the amino acid sequence of SEQ ID NO 52.

69. The composition of claim 68, wherein the protein is NP or a fragment thereof.

70. The composition of claim 69, wherein the NP comprises the amino acid sequence of SEQ ID NO 20.

71. A kit, comprising: (a) the polynucleotide of any one of claims 1-31, the vector of any one of claims 32-52, the host cell of any one of claims 53-59, the polypeptide of claim 60, or the composition of any one of claims 62-70; and (b) administering the polynucleotide, vector, MVA, host cell, polypeptide composition, or any combination thereof.

72. A method of inducing an immune response against an influenza virus in a subject in need thereof, the method comprising administering to the subject an effective amount of the polynucleotide of any one of claims 1-31, the vector of any one of claims 32-52, the host cell of any one of claims 53-59, the polypeptide of claim 60, or the composition of any one of claims 62-70, or any combination thereof, simultaneously or in any order.

73. The method of claim 72, wherein the immune response comprises an antibody response.

74. The method of claim 72, wherein the immune response comprises a cell-mediated immune response.

75. The method of claim 72, wherein the immune response comprises a cell-mediated immune response and an antibody response.

76. The method of any one of claims 72-75, wherein the immune response is a mucosal immune response.

77. A method of treating, preventing, or reducing symptoms of an influenza virus infection or a condition associated with an influenza virus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the polynucleotide of any one of claims 1-31, the vector of any one of claims 32-52, the host cell of any one of claims 53-59, the polypeptide of claim 60, or the composition of any one of claims 62-70, or any combination thereof, simultaneously or in any order.

78. A method of attenuating or ameliorating symptoms caused by an influenza virus infection or a condition associated with an influenza virus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the polynucleotide of any one of claims 1-31, the vector of any one of claims 32-52, the host cell of any one of claims 53-59, the polypeptide of claim 60, or the composition of any one of claims 62-70, or any combination thereof, simultaneously or in any order.

79. A method of vaccinating a subject in need thereof against an influenza virus infection, the method comprising administering to the subject an effective amount of the polynucleotide of any one of claims 1-31, the vector of any one of claims 32-52, the host cell of any one of claims 53-59, the polypeptide of claim 60, or the composition of any one of claims 62-70, or any combination thereof, simultaneously or in any order.

80. The method of any one of claims 72 to 79, wherein the influenza virus is an influenza A virus.

81. The method of claim 80, wherein the influenza A virus comprises hemagglutinin of type H1, H2, H3, H4, H5, H6, H7, or H9.

82. The method of any one of claims 72 to 81, wherein the influenza virus is selected from a bird or a mammal.

83. The method of claim 82, wherein the mammal is a human.

84. The method of any one of claims 72-83, wherein the subject is an animal.

85. The method of any one of claims 72-84, wherein the subject is a vertebrate.

86. The method of any one of claims 72-85, wherein the subject is a mammal.

87. The method of claim 86, wherein the mammal is a human.

88. The method of any one of claims 72-87, wherein the administering is by intradural injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration.

89. The method of any one of claims 72-88, wherein the subject is at risk of infection with an influenza virus.

90. The method of any one of claims 72-89, further comprising the step of administering at least one priming.

91. The method of claim 90, wherein priming comprises administering a vaccine composition.

92. The method of claim 91, wherein the vaccine composition is a DNA vaccine or a polypeptide vaccine.

93. The method of any one of claims 72-89, further comprising the step of administering at least one booster.

94. The method of claim 93, wherein the boosting comprises administering a vaccine composition.

95. The method of claim 94, wherein the vaccine composition is a DNA vaccine or a polypeptide vaccine.

96. The method of claim 90, 91, 93, or 94, wherein the vaccine composition comprises the polynucleotide of any one of claims 1-31, the vector of any one of claims 32-52, the host cell of any one of claims 53-59, the polypeptide of claim 60, the composition of any one of claims 62-70, or any combination thereof.

97. A method of producing a vaccine against influenza virus, the method comprising: (a) culturing the host cell of any one of claims 53-59 and (b) isolating the MVA from the host cell.

98. The vector of claim 13, wherein a linker peptide is inserted between the M2 ectodomain peptides.